CN114317463A - An oncolytic adenovirus recombinant carrying TMTP1 and tBid, its construction method and application - Google Patents
An oncolytic adenovirus recombinant carrying TMTP1 and tBid, its construction method and application Download PDFInfo
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Abstract
本发明公开了一种携带TMTP1和tBid的溶瘤腺病毒重组体、其构建方法及应用，属于医学基因工程技术领域。本发明是在人类5型腺病毒基因的E1A保守序列2区缺失第920nt‑946nt区27个碱基，再在Hexon高变区5的第19641nt‑19655nt区域插入编码肿瘤靶向肽TMTP1的基因序列，同时缺失E3区位于ADP基因第29477nt‑29714nt区域，构成缺失区，再在所述缺失区插入线粒体凋亡肽tBid的基因序列并引入Cla1酶切位点。本发明还公开了上述溶瘤腺病毒重组体的构建方法及应用。本发明的携带TMTP1和tBid的溶瘤腺病毒重组体兼具理想的靶向作用和强效杀伤作用。
The invention discloses an oncolytic adenovirus recombinant carrying TMTP1 and tBid, a construction method and application thereof, and belongs to the technical field of medical genetic engineering. The present invention is to delete 27 bases in the 920nt-946nt region of the E1A conserved sequence 2 of the human type 5 adenovirus gene, and then insert the gene sequence encoding the tumor targeting peptide TMTP1 into the 19641nt-19655nt region of the Hexon hypervariable region 5 At the same time, the E3 region is deleted in the 29477nt-29714nt region of the ADP gene to form a deletion region, and the gene sequence of the mitochondrial apoptosis peptide tBid is inserted into the deletion region and the Cla1 restriction site is introduced. The invention also discloses the construction method and application of the above oncolytic adenovirus recombinant. The oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention has both ideal targeting effect and potent killing effect.
Description
技术领域technical field
本发明涉及一种携带TMTP1和tBid的溶瘤腺病毒重组体、其构建方法及应用，属于医学基因工程技术领域。The invention relates to an oncolytic adenovirus recombinant carrying TMTP1 and tBid, a construction method and application thereof, and belongs to the technical field of medical genetic engineering.
背景技术Background technique
基因治疗(gene therapy)是指将外源基因导入靶细胞，以纠正或补偿因基因缺陷或基因表达异常引起的疾病。作为基因治疗载体，溶瘤病毒治疗恶性肿瘤的发展前景良好。在众多溶瘤病毒疗法的载体中，重组腺病毒载体是应用最为广泛的，其临床可行性和安全性已获公认。腺病毒基因治疗载体具有以下生物学优势和临床应用优势：(1)感染谱广，对多种组织源性的细胞，无论是处于分裂期还是静止期的细胞，均能有效攻击，抗癌谱广；(2)腺病毒进入细胞后，不整合到宿主染色体，无突变和致癌的危险性，临床应用安全，使用后仅产生类似感冒样症状，无造血功能和免疫功能抑制；(3)临床应用方便，有腔隙(如腹腔、胸腔和颅腔)给药、局部或瘤体内直接注射(一点或多点)以及介入治疗等途径；(4)腺病毒在体内持续表达仅两到三周，尤其适合肿瘤治疗；(5)经静脉或局部应用仅产生轻微的炎症反应，副作用轻微；(6)临床级数量的腺病毒易于生产和纯化。基于以上的优势，越来越多的腺病毒载体被构建产生，并显示出良好的临床应用前景。Gene therapy refers to the introduction of exogenous genes into target cells to correct or compensate for diseases caused by gene defects or abnormal gene expression. As a gene therapy vector, oncolytic virus has a promising development prospect in the treatment of malignant tumors. Among the many vectors for oncolytic virus therapy, the recombinant adenovirus vector is the most widely used, and its clinical feasibility and safety have been recognized. The adenovirus gene therapy vector has the following biological advantages and clinical application advantages: (1) The infection spectrum is wide, and it can effectively attack cells of various tissue origins, whether they are in the division or quiescent phase, and has an anti-cancer spectrum. (2) After the adenovirus enters the cell, it does not integrate into the host chromosome, there is no risk of mutation and carcinogenesis, and it is safe in clinical application. It is easy to apply, and there are cavities (such as abdominal cavity, thoracic cavity and cranial cavity) administration, local or intratumoral direct injection (one or more points), and interventional therapy. It is especially suitable for tumor treatment; (5) intravenous or topical application produces only mild inflammatory response, and side effects are mild; (6) adenovirus in clinical-grade quantities is easy to produce and purify. Based on the above advantages, more and more adenovirus vectors have been constructed and produced, and they have shown good clinical application prospects.
虽然腺病毒相较于其它各种基因治疗载体表现出明显的优势，国内外现有的腺病毒治疗载体也逐步取得了一些突破性的进展，但仍然有一些不可规避的缺点限制其广泛应用，具体如下：(1)肝脏趋向性。腺病毒进入生物体后，无论是静脉系统性注射，或是瘤内注射，胸腔、腹腔等局部注射，生物体内凝血因子十(Fx)会迅速识别腺病毒衣壳蛋白Hexon，与其结合，并将腺病毒带至肝脏，凝血因子的另一侧则与肝细胞表面的硫酸类肝素蛋白多糖(Heparan Sulfate Proteoglycans,简称HSPGS)结合，即凝血因子在腺病毒与肝细胞间形成桥梁，被肝脏捕获的腺病毒则逐渐被kupffer细胞或者肝脏中的巨噬细胞吞噬。而未被清除的腺病毒在肝脏中大量复制，导致肝脏急性损伤，表现为转氨酶迅速升高。(2)腺病毒感染肿瘤细胞，依赖于肿瘤细胞表面柯萨奇腺病毒受体(CAR)。而在大多数肿瘤细胞中，受体处于低表达状态，不利于腺病毒进入肿瘤细胞内发挥杀伤作用。Although adenoviruses have obvious advantages compared with other gene therapy vectors, and the existing adenovirus therapy vectors at home and abroad have gradually made some breakthroughs, there are still some unavoidable shortcomings that limit their wide application. The details are as follows: (1) Liver tropism. After the adenovirus enters the organism, whether it is intravenous systemic injection, intratumoral injection, local injection such as thoracic cavity or abdominal cavity, the coagulation factor ten (Fx) in the organism will quickly recognize the adenovirus capsid protein Hexon, bind to it, and bind to it. The adenovirus is brought to the liver, and the other side of the coagulation factor binds to the heparan sulfate proteoglycans (HSPGS) on the surface of the hepatocyte, that is, the coagulation factor forms a bridge between the adenovirus and the hepatocyte, which is captured by the liver. Adenoviruses are gradually phagocytosed by kupffer cells or macrophages in the liver. However, the adenovirus that has not been cleared replicates in large quantities in the liver, resulting in acute liver damage, manifested as a rapid increase in transaminase. (2) Adenovirus infection of tumor cells depends on the Coxsackie adenovirus receptor (CAR) on the surface of tumor cells. In most tumor cells, the receptor is in a low expression state, which is not conducive to the entry of adenovirus into tumor cells to play a killing effect.
综上，目前肿瘤治疗技术和实践中均存在各种弊端，因此，有必要提供一种新的靶向与强效杀伤兼具的治疗应用途径，以解决现有技术的不足。To sum up, there are various drawbacks in the current tumor treatment technology and practice. Therefore, it is necessary to provide a new therapeutic application approach with both targeting and potent killing to solve the deficiencies of the existing technology.
发明内容SUMMARY OF THE INVENTION
本发明的目的之一，是提供一种携带TMTP1和tBid的溶瘤腺病毒重组体。One of the objectives of the present invention is to provide an oncolytic adenovirus recombinant carrying TMTP1 and tBid.
本发明解决上述问题的技术方案如下：一种携带TMTP1和tBid的溶瘤腺病毒重组体，该携带TMTP1和tBid的溶瘤腺病毒重组体为Ad5/ΔE1A/TMTP1/ΔADP-tBid，其核苷酸序列如SEQ ID NO.23所示，是在人类5型腺病毒基因的E1A保守序列2区缺失第920nt-946nt区域如SEQ ID NO.1所示的27个碱基，再在Hexon高变区5的第19641nt-19655nt区域插入如SEQ ID NO.15所示的编码肿瘤靶向肽TMTP1的基因序列，同时缺失E3区位于ADP基因第29477nt-29714nt区域，构成缺失区，再在所述缺失区插入如SEQ ID NO.22所示的线粒体凋亡肽tBid的基因序列并引入Cla1酶切位点。The technical solution of the present invention to solve the above problems is as follows: an oncolytic adenovirus recombinant carrying TMTP1 and tBid, the oncolytic adenovirus recombinant carrying TMTP1 and tBid is Ad5/ΔE1A/TMTP1/ΔADP-tBid, and its nucleoside The acid sequence is shown in SEQ ID NO.23, which is the deletion of 27 bases in the 920nt-946nt region shown in SEQ ID NO.1 in the E1A conserved
本申请的发明人，为了获得上述携带TMTP1和tBid的溶瘤腺病毒重组体，进行了如下工作：The inventor of the present application, in order to obtain the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid, carried out the following work:
人类5型腺病毒，英文名称为Human adenovirus type 5，简称Ad5。
在人类5型腺病毒基因的E1A保守序列2区(CR2)缺失第920nt-946nt如SEQ IDNO.1所示的27个碱基，在灭活E1a蛋白的Rb结合特性的同时，达到尽可能保留E1a转录激活特性的效果。The 27 bases from 920nt to 946nt as shown in SEQ ID NO.1 are deleted in the E1A conserved
肿瘤靶向肽TMTP1具有靶向高转移潜能肿瘤细胞、早期识别肿瘤亚临床微小转移灶、可选择性被肿瘤细胞内吞并和具备较强的肿瘤细胞毒效应等明显优势。本发明通过在人类5型腺病毒中插入如SEQ ID NO.15所示的编码肿瘤靶向肽TMTP1的基因序列，从而显著提高了携带TMTP1和tBid的溶瘤腺病毒重组体的靶向性。The tumor targeting peptide TMTP1 has obvious advantages such as targeting high metastatic potential tumor cells, early identification of tumor subclinical micrometastases, selective endocytosis by tumor cells, and strong tumor cytotoxicity. The present invention significantly improves the targeting ability of the oncolytic adenovirus recombinant carrying TMTP1 and tBid by inserting the gene sequence encoding tumor targeting peptide TMTP1 as shown in SEQ ID NO. 15 into
凋亡是各种抗癌药物杀伤肿瘤细胞的最主要环节，由BCL-2(B-celllymphoma-2)家族调控。而Bid是BCL-2家族中促进细胞凋亡的线粒体凋亡肽，当细胞感知到损伤后，caspase8被激活，激活后的caspase8则可将游离于细胞质中的Bid剪切为tBid，tBid重新在线粒体外膜上聚集，激活下游bak/bax，使其通过寡聚化形成二聚体和三聚体，从而在线粒体上打孔，引起线粒体外膜去极化(MOMP)，最终导致细胞凋亡。本发明通过在人类5型腺病毒(Ad5)插入如SEQ ID NO.22所示的编码线粒体凋亡肽活性形式tBid的基因序列，使线粒体凋亡肽tBid在肿瘤中特异性表达，促进肿瘤细胞凋亡，从而显著提高了携带TMTP1和tBid的溶瘤腺病毒重组体促凋亡作用和溶瘤病毒溶解细胞的杀伤效应。Apoptosis is the most important link in the killing of tumor cells by various anticancer drugs, which is regulated by the BCL-2 (B-celllymphoma-2) family. Bid is a mitochondrial apoptotic peptide in the BCL-2 family that promotes apoptosis. When the cell senses damage, caspase8 is activated, and the activated caspase8 can splicing Bid free in the cytoplasm into tBid, and tBid is re-initiated in the cytoplasm. Aggregates on the outer mitochondrial membrane and activates downstream bak/bax to form dimers and trimers through oligomerization, thereby punching holes in the mitochondria, causing depolarization of the mitochondrial outer membrane (MOMP), and ultimately leading to apoptosis . In the present invention, by inserting the gene sequence encoding mitochondrial apoptosis peptide active form tBid as shown in SEQ ID NO. 22 into human adenovirus type 5 (Ad5), the mitochondrial apoptosis peptide tBid is specifically expressed in tumors and promotes tumor cells Apoptosis, thereby significantly improving the pro-apoptotic effect of the oncolytic adenovirus recombinant carrying TMTP1 and tBid and the killing effect of the oncolytic virus lysing cells.
进一步的，本发明实施例的编码肿瘤靶向肽TMTP1的基因序列插入于人类5型腺病毒基因的Hexon高变区2、5和7区域，通过体外实验筛选出能够在肿瘤细胞中高拷贝数复制的最佳腺病毒插入区为高变区5。在Hexon高变区5插入编码肿瘤靶向肽TMTP1基因序列，从而破坏Hexon蛋白的正常表达，抑制Fx与Hexon的结合而阻止腺病毒被肝脏吞噬。插入肿瘤靶向肽TMTP1能降低腺病毒对肿瘤细胞表面柯萨奇腺病毒受体(CAR)的依赖性，增加腺病毒对肿瘤细胞的亲和性；破坏Hexon高变区完整性，减少腺病毒在肝脏中聚集，降低腺病毒对肝脏损害。Further, the gene sequence encoding the tumor targeting peptide TMTP1 in the embodiment of the present invention is inserted into the Hexon
综上，本发明的携带TMTP1和tBid的溶瘤腺病毒重组体能特异性识别肿瘤及其转移灶，具有更高的肿瘤靶向复制能力，利用tBid的促凋亡作用和溶瘤病毒溶解细胞的双重杀伤效应，体内外实验验证其对顺铂耐药的细胞系有强效杀伤效应，兼具理想的靶向作用和强效杀伤作用。To sum up, the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention can specifically recognize tumors and their metastases, and has higher tumor-targeted replication ability. Double killing effect, in vitro and in vivo experiments verified that it has a strong killing effect on cisplatin-resistant cell lines, and has both an ideal targeting effect and a strong killing effect.
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的有益效果是：The beneficial effects of the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention are:
1、本发明的携带TMTP1和tBid的溶瘤腺病毒重组体具有肿瘤选择性复制及治疗基因表达的强选择性；1. The oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention has strong selectivity for tumor selective replication and therapeutic gene expression;
2、肿瘤细胞内子代溶瘤腺病毒产量高，细胞裂解后在肿瘤局部可产生高浓度的病毒治疗圈，加之与治疗靶点的优势联合，可形成强效的旁观者效应；2. The progeny oncolytic adenovirus in tumor cells has a high yield, and after cell lysis, a high concentration of virus therapeutic circle can be generated in the tumor local area. Combined with the advantages of therapeutic targets, it can form a strong bystander effect;
3、肿瘤细胞内溶瘤腺病毒高效、大量扩增，同时转录出大量治疗靶基因，让肿瘤细胞成为治疗蛋白合成的“加工厂”；3. Oncolytic adenoviruses in tumor cells are efficiently and massively amplified, and at the same time, a large number of therapeutic target genes are transcribed, making tumor cells a "processing factory" for the synthesis of therapeutic proteins;
4、本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的免疫调节蛋白功能完整，一定程度上避免了机体对重组腺病毒的清除；4. The immunomodulatory protein of the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention has complete functions, and to a certain extent avoids the elimination of the recombinant adenovirus by the body;
5、本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的抗癌谱广。肿瘤动物模型的体内模型研究证实，通过肿瘤局部注射或者腹腔注射的用药途径，本发明的携带TMTP1和tBid的溶瘤腺病毒重组体对受试的所有肿瘤模型均具有显著的治疗作用，可有效抑制肿瘤转移，且对治疗动物无明显的治疗相关毒性。5. The oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention has a broad anticancer spectrum. The in vivo model study of tumor animal models confirmed that the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention has a significant therapeutic effect on all tumor models tested by local injection or intraperitoneal injection. Inhibits tumor metastasis without significant treatment-related toxicity in treated animals.
本发明的目的之二，是提供上述携带TMTP1和tBid的溶瘤腺病毒重组体的构建方法。The second objective of the present invention is to provide a method for constructing the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid.
本发明解决上述问题的技术方案如下：上述携带TMTP1和tBid的溶瘤腺病毒重组体的构建方法，包括如下步骤：The technical solution of the present invention to solve the above problem is as follows: the above-mentioned construction method of the oncolytic adenovirus recombinant carrying TMTP1 and tBid comprises the following steps:
步骤1：人类5型腺病毒基因的定向缺失Step 1: Targeted deletion of the
利用基因合成和同源重组的方式定向缺失人类5型腺病毒基因的E1A保守序列2区第920nt-946nt如SEQ ID NO.1所示的27个碱基，获得定向缺失后的人类5型腺病毒基因Ad5/ΔE1A；Using gene synthesis and homologous recombination to directionally delete 27 bases from 920nt to 946nt in the second region of the E1A conserved sequence of the
步骤2：制备Ad5/ΔE1A/TMTP1Step 2: Preparation of Ad5/ΔE1A/TMTP1
在步骤1获得的定向缺失后的人类5型腺病毒基因的Hexon高变区5的第19641nt-19655nt区域，插入如SEQ ID NO.15所示的编码肿瘤靶向肽TMTP1的基因序列，获得Ad5/ΔE1A/TMTP1；In the 19641nt-19655nt region of the Hexon hypervariable
步骤3：制备携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBidStep 3: Preparation of oncolytic adenovirus recombinant Ad5/ΔE1A/TMTP1/ΔADP-tBid carrying TMTP1 and tBid
在步骤2获得的Ad5/ΔE1A/TMTP1的E3区位于ADP基因第29477nt-29714nt区域，构成缺失区，再在所述缺失区插入如SEQ ID NO.22所示的线粒体凋亡肽tBid的基因序列并引入Cla1酶切位点，即获得如SEQ ID NO.23所示的携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBid。The E3 region of Ad5/ΔE1A/TMTP1 obtained in
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的构建方法的有益效果是：The beneficial effects of the method for constructing the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention are:
1、本发明构建的携带TMTP1和tBid的溶瘤腺病毒重组体中插入有编码肿瘤靶向肽TMTP1的基因序列和编码线粒体凋亡肽tBid的基因序列，具有肿瘤特异性复制、高度肿瘤及其转移瘤靶向性、特异性大量表达外源性治疗基因、强大的肿瘤特异性旁观者效应等优势，并具备高度的治疗指数，可以解决目前肿瘤治疗技术和实践中关键的不足之处，为肿瘤治疗提供了理想的靶向与强效杀伤兼具的治疗应用途径。1. The gene sequence encoding tumor targeting peptide TMTP1 and the gene sequence encoding mitochondrial apoptosis peptide tBid are inserted into the oncolytic adenovirus recombinant carrying TMTP1 and tBid constructed by the present invention, which has tumor-specific replication, high tumor and The advantages of metastatic tumor targeting, specific expression of a large number of exogenous therapeutic genes, strong tumor-specific bystander effect, etc., and a high therapeutic index, can solve the key deficiencies in current tumor treatment technology and practice, provide Tumor therapy provides an ideal therapeutic application approach with both targeting and potent killing.
2、本发明的构建方法简单，操作容易，成本低廉，应用前景广泛。2. The construction method of the present invention is simple, the operation is easy, the cost is low, and the application prospect is wide.
本发明的目的之三，是提供上述携带TMTP1和tBid的溶瘤腺病毒重组体的应用。The third object of the present invention is to provide the application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid.
本发明解决上述问题的技术方案如下：上述携带TMTP1和tBid的溶瘤腺病毒重组体在制备治疗肿瘤的药物中的应用。The technical solution of the present invention to solve the above problem is as follows: the application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid in the preparation of a medicine for treating tumors.
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的应用的有益效果：Beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention:
上述携带TMTP1和tBid的溶瘤腺病毒重组体可以用于制备治疗肿瘤的药物，既开拓了新的治疗肿瘤的药物，又开拓了携带TMTP1和tBid的溶瘤腺病毒重组体的用途，具有积极的药学价值和广泛的社会意义。The above-mentioned oncolytic adenovirus recombinants carrying TMTP1 and tBid can be used to prepare medicines for the treatment of tumors, which not only develops new medicines for the treatment of tumors, but also opens up the use of the oncolytic adenovirus recombinants carrying TMTP1 and tBid, and has positive effects. of pharmaceutical value and broad social significance.
本发明的目的之四，是提供上述携带TMTP1和tBid的溶瘤腺病毒重组体的另一个应用。The fourth object of the present invention is to provide another application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid.
本发明解决上述问题的技术方案如下：上述携带TMTP1和tBid的溶瘤腺病毒重组体在制备基因治疗载体中的应用。The technical solution of the present invention to solve the above problem is as follows: the application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid in the preparation of gene therapy vector.
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的应用的有益效果：Beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention:
上述携带TMTP1和tBid的溶瘤腺病毒重组体可以用于制备基因治疗载体，既开拓了新的基因治疗载体，又开拓了携带TMTP1和tBid的溶瘤腺病毒重组体的用途，具有积极的药学价值和广泛的社会意义。The above-mentioned oncolytic adenovirus recombinants carrying TMTP1 and tBid can be used to prepare gene therapy vectors, which not only develops new gene therapy vectors, but also opens up the use of oncolytic adenovirus recombinants carrying TMTP1 and tBid, and has positive pharmaceutical properties. value and wider social significance.
本发明的目的之五，是提供上述携带TMTP1和tBid的溶瘤腺病毒重组体的另一个应用。The fifth object of the present invention is to provide another application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid.
本发明解决上述问题的技术方案如下：上述携带TMTP1和tBid的溶瘤腺病毒重组体在制备改善备抗肿瘤化疗药物耐药性的药物中的应用。The technical solution of the present invention to solve the above problem is as follows: the application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid in the preparation of a drug for improving the drug resistance of anti-tumor chemotherapeutic drugs.
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的应用的有益效果：Beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention:
上述携带TMTP1和tBid的溶瘤腺病毒重组体可以用于制备改善备抗肿瘤化疗药物耐药性的药物，既开拓了新的改善备抗肿瘤化疗药物耐药性的药物，又开拓了携带TMTP1和tBid的溶瘤腺病毒重组体的用途，具有积极的药学价值和广泛的社会意义。The above-mentioned oncolytic adenovirus recombinants carrying TMTP1 and tBid can be used to prepare drugs for improving the drug resistance of anti-tumor chemotherapy drugs, which not only develops new drugs for improving the drug resistance of anti-tumor chemotherapy drugs, but also develops drugs carrying TMTP1 The use of oncolytic adenovirus recombinants of tBid and tBid has positive pharmaceutical value and broad social significance.
本发明的目的之六，是提供上述携带TMTP1和tBid的溶瘤腺病毒重组体的另一个应用。The sixth objective of the present invention is to provide another application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid.
本发明解决上述问题的技术方案如下：上述携带TMTP1和tBid的溶瘤腺病毒重组体在制备抗肿瘤化疗药物增敏剂中的应用。The technical solution of the present invention to solve the above problem is as follows: the application of the above-mentioned oncolytic adenovirus recombinant carrying TMTP1 and tBid in the preparation of an anti-tumor chemotherapeutic drug sensitizer.
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体的应用的有益效果：Beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention:
上述携带TMTP1和tBid的溶瘤腺病毒重组体可以用于制备抗肿瘤化疗药物增敏剂，既开拓了新的抗肿瘤化疗药物增敏剂，又开拓了携带TMTP1和tBid的溶瘤腺病毒重组体的用途，具有积极的药学价值和广泛的社会意义。The above-mentioned oncolytic adenovirus recombinants carrying TMTP1 and tBid can be used to prepare antitumor chemotherapeutic drug sensitizers, which not only open up new antitumor chemotherapeutic drug sensitizers, but also develop oncolytic adenovirus recombinants carrying TMTP1 and tBid. It has positive pharmaceutical value and broad social significance.
附图说明Description of drawings
图1为本发明的携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBid的结构示意图。Figure 1 is a schematic structural diagram of the oncolytic adenovirus recombinant Ad5/ΔE1A/TMTP1/ΔADP-tBid carrying TMTP1 and tBid of the present invention.
图2为本发明实施例5中Hexon高变区插入区不同的腺病毒在肿瘤细胞中复制柱状图。Figure 2 is a bar graph showing the replication of adenoviruses with different insertion regions of the Hexon hypervariable region in tumor cells in Example 5 of the present invention.
图3为本发明实施例7中对照组及实验组腺病毒在组织、肿瘤内含量的柱状图。FIG. 3 is a bar graph of the adenovirus content in tissues and tumors in the control group and the experimental group in Example 7 of the present invention.
图4为本发明实施例7中对照组及实验组腺病毒在各个组织中相对含量的柱状图。4 is a bar graph showing the relative content of adenovirus in each tissue in the control group and the experimental group in Example 7 of the present invention.
图5为本发明实施例8中皮下瘤动物模型的肿瘤体积变化的对照曲线。FIG. 5 is a control curve of the tumor volume change of the subcutaneous tumor animal model in Example 8 of the present invention.
图6为本发明实施例8中腹腔原位转移瘤模型中实验组及对照组的肿瘤体积照片。6 is a photograph of the tumor volume of the experimental group and the control group in the abdominal cavity in situ metastasis model in Example 8 of the present invention.
图7为本发明实施例8中腹腔原位转移瘤模型的实验动物的谷草转氨酶(AST)实验结果。FIG. 7 is the aspartate aminotransferase (AST) test result of the experimental animal of the peritoneal in situ metastasis model in Example 8 of the present invention.
图8为本发明实施例8中腹腔原位转移瘤模型的实验动物的谷丙转氨酶(ALT)实验结果。FIG. 8 is the result of the alanine aminotransferase (ALT) test of the experimental animal of the peritoneal in situ metastasis model in Example 8 of the present invention.
图9为本发明实施例8中腹腔原位转移瘤模型的实验动物的尿素氮(BUN)实验结果。FIG. 9 is the experimental result of blood urea nitrogen (BUN) of the experimental animal of the peritoneal orthotopic metastasis model in Example 8 of the present invention.
图10为本发明实施例8中腹腔原位转移瘤模型的实验动物的肌酐(Creatinine)实验结果。FIG. 10 is the Creatinine test result of the experimental animal of the abdominal cavity orthotopic metastasis model in Example 8 of the present invention.
具体实施方式Detailed ways
以下结合具体附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。The principles and features of the present invention will be described below with reference to the specific drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.
如图1所示，本发明采用腺病毒重组系统AdEasyTM，通过三次同源重组的方法，构建出目标腺病毒。除特别注明外，本发明中所用的酶均购自美国gibco公司，PCR引物均由北京擎科生物科技有限公司合成，细胞系均购自美国ATCC细胞库,且使用对应的培养基进行培养，培养基均购自美国gibco公司。As shown in Figure 1, the present invention adopts the adenovirus recombination system AdEasyTM to construct the target adenovirus through three homologous recombination methods. Unless otherwise specified, the enzymes used in the present invention were purchased from the American gibco company, the PCR primers were all synthesized by Beijing Qingke Biotechnology Co., Ltd., and the cell lines were all purchased from the American ATCC cell bank, and the corresponding medium was used for culturing , culture medium were purchased from the United States gibco company.
实施例1：构建pAd5/ΔE1A腺病毒包装质粒Example 1: Construction of pAd5/ΔE1A adenovirus packaging plasmid
步骤1.1：构建穿梭质粒Blunt-Zero-E1A/ΔE1AStep 1.1: Construction of Shuttle Plasmid Blunt-Zero-E1A/ΔE1A
pXC1-ΔE1A质粒，是在人类5型腺病毒基因的E1A保守序列2区缺失第920nt-946nt区域如SEQ ID NO.1所示的27个碱基。其构建方法如下：The pXC1-ΔE1A plasmid is a 27-base deletion in the 920nt-946nt region of the E1A conserved
pXC1质粒购于Microbix Biosystem Inc.(Toronato，Ontario，Canada，目录号：PD-01-03)，该质粒包含人类5型腺病毒(Ad5)22nt-5790nt序列。采用3次PCR法缺失第920nt-946nt区域。The pXC1 plasmid was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-03), which contains the human adenovirus type 5 (Ad5) 22nt-5790nt sequence. The 920nt-946nt region was deleted by three PCRs.
片段1的获取：引物1：5′-cgggatccgggcccccatttcc-3′(SEQ ID NO 2)，相当于9883-9902nt，下划线部分为BamHI酶切位点；引物2：5′-gtcactgggtggatcgatcacctccggtac-3′(SEQ ID NO 3)，相当于922nt-905nt，下划线部分为与引物3互补部分；以pXC1为模板，进行PCR反应，反应体系总体积为100μl，包括：含MgCl2的10×PCR缓冲液，10μl；2mM dNTP，10μl；10μM引物1，1μl；10μM引物2，1μl；pXC110ng/μl，1μl；pfu高保真Taq酶，2.5μl；加水至100μl；反应条件为：95℃，30s；95℃，45s；60℃，1min；72℃，2min；共28循环；72℃，延伸10min。PCR产物长940bp，形成片段1，常规电泳分离纯化后，检测浓度用于后继PCR反应。Obtainment of fragment 1: Primer 1: 5′-cg ggatcc gggcccccatttcc-3′ (SEQ ID NO 2), equivalent to 9883-9902nt, the underlined part is the BamHI restriction site; primer 2: 5′- gtcactgggtggatcgatcacctc cggtac-3′ (SEQ ID NO 3), equivalent to 922nt-905nt, the underlined part is the complementary part to
片段2的获取：引物3：5′-gaggtgatcgatccacccagtgacgacgag-3′(SEQ ID NO 4)，相当于911-947nt，下划线部分为与引物2互补部分；引物4：5′-tgctctagacacaggtgatgtcg-3′(SEQ ID NO 5)，相当于1344nt-1325nt，下划线部分为XbaI酶切位点；以pXC1为模板，进行PCR反应，反应条件同上，产物长400bp，形成片段2，常规电泳分离纯化后，检测浓度用于后继PCR反应。Acquisition of fragment 2: primer 3: 5′- gaggtgatcgatccacccagtgac gacgag-3′ (SEQ ID NO 4), equivalent to 911-947nt, the underlined part is the complementary part to
片段3的获取：将50ng/2μl片段1与25ng/1μl片段2混合，作为模板行PCR反应，上游引物为引物1，下游引物为引物4，反应条件同上，产物约为1400bp，形成片段3。Acquisition of fragment 3: Mix 50ng/
用QIAquick 8PCR产物纯化试剂盒(QIAGEN，German，Cat：28142)纯化后，用BamHI，XbaI双酶切过夜，酶切产物在1％琼脂糖凝胶电泳分离后回收酶切片段用于克隆。将pXC1用BamHI和XbaI双酶切过夜，酶切产物在1％琼脂糖凝胶电泳分离后产生2条带，约为1400bp和8500bp，回收8500bp酶切片段用于克隆。取40ng的8500bp pXC1酶切片段及90ng片段3，用DNA T4连接酶做连接反应，取1.5μl转化100μl DH5α感受态细菌，铺皿过夜培养，第二天挑取单个菌落克隆，提取其内扩增的质粒，DNA测序鉴定筛选得到缺失121-129AA 920nt-946nt的pXC1质粒突变体pXC1-ΔE1A。After purification with QIAquick 8 PCR product purification kit (QIAGEN, German, Cat: 28142), the digestion products were double digested with BamHI and XbaI overnight, and the digested products were separated by 1% agarose gel electrophoresis to recover the digested fragments for cloning. The pXC1 was double digested with BamHI and XbaI overnight, and the digested products were separated by 1% agarose gel electrophoresis to produce two bands, about 1400bp and 8500bp, and the 8500bp digested fragment was recovered for cloning. Take 40ng of 8500bp pXC1 digested fragment and
以pXC1-ΔE1A质粒为模板，进行PCR扩增反应，获得缺失27bp的E1A序列。其中，上游引物：5'-ttaattaacatcatcaataatataccttatt-3'(SEQ ID NO.6)，下游引物：5'-gatccacataatctaacacaaactc-3'(SEQ ID NO.7)。PCR扩增反应的体系为：TransStartFastPfu Fly DNA Polymerase，1μl；5×TransStartFastPfu Fly Buffer，10μl；10μM上游引物，1μl；10μM下游引物，1μl；2.5mM dNTPs，4μl；无核酸酶水，补足体系至50μl；模板，10-30ng；共计50μl。PCR扩增反应的程序为95℃，2min；95℃，20s，-5℃，20s，72℃，3mi n，35个循环；72℃，5min；4℃，保存。Using the pXC1-ΔE1A plasmid as a template, PCR amplification was performed to obtain an E1A sequence with a deletion of 27 bp. Wherein, the upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQ ID NO.6), the downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO.7). The PCR amplification reaction system is: TransStartFastPfu Fly DNA Polymerase, 1 μl; 5×TransStartFastPfu Fly Buffer, 10 μl; 10 μM upstream primer, 1 μl; 10 μM downstream primer, 1 μl; 2.5 mM dNTPs, 4 μl; nuclease-free water, make up the system to 50 μl ; Template, 10-30 ng; 50 μl total. The PCR amplification reaction program was 95°C, 2 min; 95°C, 20s, -5°C, 20s, 72°C, 3 min, 35 cycles; 72°C, 5min; 4°C, storage.
在T4连接酶(购自赛默飞世尔科技公司，货号为el0011)的作用下，将E1A序列连接至pEASY-Blunt-Zero平端连接质粒(购自北京全式金生物技术有限公司，货号为CB501)中，获得穿梭质粒Blunt-Zero-E1A/ΔE1A。Under the action of T4 ligase (purchased from Thermo Fisher Scientific Co., Ltd., item number el0011), the E1A sequence was ligated to the pEASY-Blunt-Zero blunt-end ligation plasmid (purchased from Beijing Quanshijin Biotechnology Co., Ltd., item number: CB501), the shuttle plasmid Blunt-Zero-E1A/ΔE1A was obtained.
以穿梭质粒Blunt-Zero-E1A/ΔE1A为模板，采用上述pEASY-Blunt-Zero平端连接质粒试剂盒中自带的引物进行PCR扩增反应，获得缺失27bp的E1A穿梭片段，纯化后以备同源重组。PCR反应体系及扩增反应程序同上。Using the shuttle plasmid Blunt-Zero-E1A/ΔE1A as the template, PCR amplification was performed using the primers included in the pEASY-Blunt-Zero blunt-end ligation plasmid kit to obtain the E1A shuttle fragment with a missing 27bp, which was purified for homology reorganization. The PCR reaction system and the amplification reaction procedure are the same as above.
步骤1.2：构建骨架质粒pAd-Easy-1Step 1.2: Construction of backbone plasmid pAd-Easy-1
pAd-Easy-1载体(购自安捷伦科技有限公司，目录号为240005)，用限制性内切酶pme1切断后，苯酚氯仿抽提DNA酶切产物，以其作为第一次同源重组的骨架片段，测出穿梭片段及骨架片段的DNA浓度。The pAd-Easy-1 vector (purchased from Agilent Technologies Co., Ltd., catalog number 240005) was cut with restriction endonuclease pme1, and the DNA digested product was extracted with phenol chloroform and used as the backbone of the first homologous recombination Fragments, the DNA concentrations of shuttle fragments and backbone fragments were measured.
步骤1.3：制备大肠杆菌BJ5183电转感受态Step 1.3: Preparation of E. coli BJ5183 electrocompetent
取出于-80℃保存的大肠杆菌BJ5183(购自北京科瑞思搏生物科技有限公司，目录号为st10779)，融化后，挑取1mL菌液加入到1000mL链霉素浓度为30μg/mL的LB培养基中。以250rpm/min的速度37℃摇菌7h-8h后,每10min观察一次，液体稍变浑浊即可。将菌液分装入预冷的离心管中，4℃，3000rpm/min离心10min，弃上清。适量预冷无菌双蒸水洗菌渣，两次。适量预冷无菌10％甘油洗菌渣，两次。弃上清，获得大肠杆菌BJ5183电转感受态，分装于1.5mL离心管中，-80℃保存。Take out Escherichia coli BJ5183 (purchased from Beijing Keruisibo Biotechnology Co., Ltd., catalog number st10779) stored at -80°C, and after thawing, pick 1 mL of bacterial solution and add it to 1000 mL of LB with streptomycin concentration of 30 μg/mL. in the culture medium. Shake the bacteria at 37°C for 7h-8h at a speed of 250rpm/min and observe every 10min until the liquid becomes slightly turbid. The bacterial liquid was divided into pre-cooled centrifuge tubes, centrifuged at 4°C, 3000 rpm/min for 10 min, and the supernatant was discarded. An appropriate amount of pre-cooled sterile double-distilled water was used to wash the bacterial residue twice. An appropriate amount of pre-cooled sterile 10% glycerol was used to wash the bacterial residue twice. Discard the supernatant to obtain Escherichia coli BJ5183 competent for electroporation, aliquot into 1.5 mL centrifuge tubes, and store at -80°C.
步骤1.4：同源重组构建pAd5-E1A/ΔE1A腺病毒包装质粒Step 1.4: Construction of pAd5-E1A/ΔE1A adenovirus packaging plasmid by homologous recombination
采用GenePulser Xcel lTM电转仪(购自Bio-Rad公司)，将步骤1.1获得的E1A穿梭片段以及步骤1.2获得的骨架片段，电转入步骤1.3获得的大肠杆菌BJ5183电转感受态中，电转条件为2.5Kv、25μF。采用卡那霉素抗性筛选阳性菌。挑选菌板中较小的菌斑，LB培养基中培养后，质粒小量提取，使用上游引物：5′-ttaattaacatcatcaataatataccttatt-3′(SEQID NO.8)和下游引物：5′-gatccacataatctaacacaaactc-3′(SEQ ID NO.9)进行PCR扩增反应，PCR扩增反应的体系及反应程序同步骤1.1，测序鉴定E1A区域是否缺失920bp-946bp区域。将提取的质粒转入高拷贝菌DH10B(购自赛默飞世尔科技公司,货号为18290015)中，获得pAd5-E1A/ΔE1A腺病毒包装质粒，简写为pAd5/ΔE1A。Using a GenePulser Xcel 1TM electroporator (purchased from Bio-Rad), the E1A shuttle fragment obtained in step 1.1 and the backbone fragment obtained in step 1.2 were electroporated into E. coli BJ5183 electroporation competent cells obtained in step 1.3, and the electroporation conditions were 2.5 Kv, 25μF. Positive bacteria were screened by kanamycin resistance. Smaller plaques in the bacterial plate were selected, and after culturing in LB medium, the plasmid was extracted in small quantities, using the upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQID NO. 8) and the downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO. 9) PCR amplification reaction was carried out. The system and reaction procedure of PCR amplification reaction were the same as in step 1.1. Sequencing identified whether the 920bp-946bp region was missing in the E1A region. The extracted plasmid was transferred into high-copy bacteria DH10B (purchased from Thermo Fisher Scientific, product number 18290015) to obtain pAd5-E1A/ΔE1A adenovirus packaging plasmid, abbreviated as pAd5/ΔE1A.
实施例2：构建pAd5/ΔE1A/Hexon(HVR/TMTP1)质粒载体Example 2: Construction of pAd5/ΔE1A/Hexon (HVR/TMTP1) plasmid vector
步骤2.1：构建穿梭载体pEASY-Blunt-Zero-Hexon(HVR/TMTP1)Step 2.1: Construction of shuttle vector pEASY-Blunt-Zero-Hexon (HVR/TMTP1)
pBHGE3购于Microbix Biosystem Inc.(Toronato，Ontario，Canada，目录号：PD-01-12)，该质粒包含除Ad5包装信号(194-358nt)外的全部基因组序列。pBHGE3从MicrobixBiosystem Inc.获取时，总量为10μg，先电转入感受态细菌，挑取阳性克隆，提取质粒，得到的质粒用CsCl2-EB超速离心法纯化。同源重组法获取Δ920-946Ad5重组腺病毒构建体，方法如下：pBHGE3 was purchased from Microbix Biosystem Inc. (Toronato, Ontario, Canada, catalog number: PD-01-12), and this plasmid contained the entire genomic sequence except for the Ad5 packaging signal (194-358nt). When pBHGE3 was obtained from MicrobixBiosystem Inc., the total amount was 10 μg. It was first electroporated into competent bacteria, positive clones were picked, and plasmids were extracted. The obtained plasmids were purified by CsCl2-EB ultracentrifugation. The homologous recombination method was used to obtain the Δ920-946Ad5 recombinant adenovirus construct as follows:
在15cm培养皿中种入7.5×105293细胞，培养液为10％FBS DMEM，到第二天，细胞应为1-1.5×106，大约70％的细胞融合；转染前3-4h，换成新鲜培养液。配制共转染DNA-磷酸钙溶液：将1600μl灭菌的2×HBS(280mM NaCl，43mM HEPES，10mM KCl，10mM Na2HPO4·7H2O，2％dextrose，pH值7.05-7.15)；pBHGE3和Δ920-946pXC1各42μg；加灭菌的双蒸水至2840μl混匀，缓慢加入50μl 2.5M的CaCl2，颠倒混匀，在室温下让DNA/CaCl2沉淀45-60min，形成微混浊的沉淀。加500μl上述混合液至含5ml 60mm培养皿的293细胞中，在37℃、5％CO2中孵育4-6h，吸去上述液体，用PBS洗一次。用含15％的甘油/DMEM处理1-2min以促进转染效率，用PBS洗一次，换为完全培养液。制备1.8％低溶点琼脂糖，高压灭菌，分装成5ml，用前在沸水中融化，保温在45℃，用时加入等量4％FBS DMEM，即刻铺入培养皿中。吸去培养液，加入5ml上述液体。每4-5d，加入3ml上述液体。14-21d，噬斑出现，选定6-12个噬斑。将噬斑转移到含无血清DMEM培养基，1.5ml的EP管中，在37℃孵育24h。在24孔板培养皿中种入1×105293细胞，培养液为10％FBS DMEM，到第二天，细胞应为2×105，大约70％的细胞融合，吸去液体，从上述孵育液取100μl(大约为103病毒)加入，轻轻晃动液体3次，在37℃、5％CO2中孵育90min。加入完全培养基至1ml，将细胞放置于37℃，5％CO2中孵育5-10d，直至完全的CPE出现，所谓CPE，即细胞毒效应，细胞表现为变圆、漂浮、细胞以核仁为主。假如10d后，完全的CPE未出现，则提示病毒的效价太低，需进行第二轮的扩增。将培养板进行三轮的冻/解冻的循环，释放出病毒，将裂解液收集于15ml试管，最大速度离心10min，收集上清液，冻于-80℃，该液体称为第二代病毒，大约为5×107/ml病毒。对以上病毒进行再次扩增，在75cm2培养皿中种入5×106293细胞，培养液为10ml 10％FBS DMEM，到第二天，细胞应为1×107，大约70％细胞融合；转染前3-4h，换成新鲜培养液；取1ml第二代病毒储存液加入完全培养基至1ml，用于转染；该MOI约为5；除去75cm2培养皿的液体，加入以上液体，轻轻摇动三次；在37℃、5％CO2中孵育90min；加入9ml 2％FBS DMEM，在37℃、5％CO2中孵育4-7d，用于提取病毒DNA用于阳性病毒的筛选。因为293细胞基因组包含完整的E1A基因，提取阳性病毒DNA时容易污染293细胞DNA，造成鉴定失败，为此将Δ920-946Ad5在肿瘤细胞Hela中再扩增一次，用于鉴定，步骤如下：在6孔板培养皿中种入1×105Hela细胞，培养液为10％FBS DMEM，到第二天，细胞应为2×105，大约70％的细胞融合，吸去液体，从上述滤过液取100μl(大约为103病毒)加入，轻轻晃动液体3次，在37℃、5％CO2中孵育90min。加入完全培养基至1ml，将细胞放置于37℃、5％CO2中孵育5-10d，直至完全的CPE出现，将细胞刮下，收集于1.5ml EP管，离心弃上清，加入300μl PBS溶液，进行三轮的冻/解冻的循环，释放出病毒，将裂解液最大速度离心10min，收集上清液，冻于-80℃，用Qiagen公司试剂盒mini DNA isolation kit，参照试剂盒说明提取DNA。以病毒DNA为模板，进行PCR反应，上游引物：5′-cgggatccgggcccccatttcc-3′(SEQ ID NO 10)，下游引物：5′-tgctctagacacaggtgatgtcg-3′(SEQ ID NO 11)，反应体系总体积为100μl包括：含MgCl2的10×PCR缓冲液，10μl；2mMdNTP，10μl；10μM上游引物，1μl；10μM下游引物，1μl；病毒DNA，10ng；pfu高保真Taq酶，2.5u；加水至100μl。反应条件为：95℃，30s；95℃，45s；60℃，1min；72℃，2min；共28循环；72℃延伸10min。PCR产物为1400bp，常规电泳分离纯化后，检测浓度用于DNA测序，测序引物：5′-agccggagcagagagccttg-3′(SEQ ID NO 12)，挑出测序正确克隆，即为Δ920-946Ad5。以腺病毒Δ920-946Ad5为模板，采用primer5.0软件设计引物，使用上游引物：5′-ccagagtaggtgtaataagg-3′(SEQ ID NO.13)和下游引物：5′-tagaaagtcaagtggaaatg-3′(SEQ ID NO.14)，将腺病毒的18380bp-20388bp(即Hexon区域)，采用PCR扩增反应，获得Hexon片段，并将该片段平端连接入pEASY-Blunt-Zero载体(购自北京全式金生物技术有限公司，目录号为CB501)中，获得pEASY-Blunt-Zero-Hexon质粒。Seed 7.5×10 5 293 cells in a 15cm dish in 10% FBS DMEM, by the next day, the cells should be 1-1.5×10 6 , about 70% confluent; 3-4h before transfection , replaced with fresh medium. Prepare co-transfection DNA-calcium phosphate solution: 1600 μl of sterile 2×HBS (280 mM NaCl, 43 mM HEPES, 10 mM KCl, 10 mM Na 2 HPO 4 ·7H 2 O, 2% dextrose, pH 7.05-7.15); pBHGE3 and Δ920-946pXC1 each 42μg; add sterilized double-distilled water to 2840μl and mix, slowly add 50μl 2.5M CaCl 2 , invert and mix, let DNA/CaCl 2 settle for 45-60min at room temperature to form a slightly turbid precipitate . Add 500 μl of the above mixture to 293 cells containing 5 ml of a 60 mm petri dish, incubate at 37° C., 5% CO 2 for 4-6 h, aspirate the above liquid, and wash once with PBS. Treated with 15% glycerol/DMEM for 1-2 min to promote transfection efficiency, washed once with PBS, and changed to complete medium. Prepare 1.8% low melting point agarose, autoclave, divide into 5ml, melt in boiling water before use, incubate at 45°C, add an equal amount of 4% FBS DMEM during use, and immediately spread it into a petri dish. Aspirate the culture medium and add 5 ml of the above liquid. Every 4-5d, add 3ml of the above liquid. 14-21d, plaques appeared, 6-12 plaques were selected. The plaques were transferred to a 1.5ml EP tube containing serum-free DMEM medium, and incubated at 37°C for 24h. Seed 1 x 10 5 293 cells in a 24-well plate in 10% FBS DMEM, by the next day, the cells should be 2 x 10 5 , about 70% confluent, aspirate the liquid from the
采用primer5.0软件设计引物，使用双重PCR扩增反应，将如SEQ ID O.15所示的编码肿瘤靶向肽TMTP1的基因序列分别插入Hexon高变区2、5和7区域中，获得pEASY-Hexon(HVR/TMTP1)PCR扩增产物，然后利用Hexon区域的特异性酶切位点DraⅢ和SacI，分别双酶切上述PCR产物pEASY-Hexon(HVR/TMTP1)和pEASY-Blunt-Zero-Hexon质粒，切胶回收酶切产物后，用T4连接酶连接上述产物，将连接物转化入DH10B感受态后，筛选阳性菌斑，分别获得pEASY-Blunt-Zero-Hexon(HVR2/TMTP1)穿梭质粒、pEASY-Blunt-Zero-Hexon(HVR5/TMTP1)穿梭质粒和pEASY-Blunt-Zero-Hexon(HVR7/TMTP1)穿梭质粒。Primer 5.0 software was used to design primers, and double PCR amplification reaction was used to insert the gene sequence encoding tumor-targeting peptide TMTP1 as shown in SEQ ID 0.15 into Hexon hypervariable
步骤2.2：将步骤1.4中构建的骨架质粒pAd5-E1A/ΔE1A大量提取(OMEGA，货号D6692-01)，利用特异性酶切位点AsisI，酶切后，苯酚氯仿抽提，获得线性骨架质粒。Step 2.2: Extract the backbone plasmid pAd5-E1A/ΔE1A constructed in step 1.4 (OMEGA, product number D6692-01) in large quantities, use the specific restriction site AsisI, and then extract with phenol and chloroform to obtain a linear backbone plasmid.
步骤2.3：采用步骤2.1获得的pEASY-Hexon(HVR/TMTP1)PCR产物作为同源重组片段，与步骤2.2获得的线性骨架质粒同时电转入步骤1.3获得的大肠杆菌BJ5183电转感受态中，电转条件为2.5Kv、25μF。采用卡那霉素抗性筛选阳性菌，PCR扩增反应，鉴定获得pAd5-E1A/ΔE1A-Hexon(HVR2/TMTP1)质粒、pAd5-E1A/ΔE1A-Hexon(HVR5/TMTP1)质粒和pAd5-E1A/ΔE1A-Hexon(HVR7/TMTP1)质粒。Step 2.3: The pEASY-Hexon (HVR/TMTP1) PCR product obtained in step 2.1 was used as a homologous recombination fragment, and the linear backbone plasmid obtained in step 2.2 was electroporated into the E. coli BJ5183 electrotransformation competent obtained in step 1.3. The electroporation conditions It is 2.5Kv, 25μF. The positive bacteria were screened for kanamycin resistance, and PCR amplification was used to identify and obtain pAd5-E1A/ΔE1A-Hexon(HVR2/TMTP1) plasmid, pAd5-E1A/ΔE1A-Hexon(HVR5/TMTP1) plasmid and pAd5-E1A/ ΔE1A-Hexon (HVR7/TMTP1) plasmid.
实施例3：构建Ad5/ΔE1A/TMTP1/ΔADP-tBid腺病毒载体Example 3: Construction of Ad5/ΔE1A/TMTP1/ΔADP-tBid adenovirus vector
步骤3.1构建腺病毒E3区穿梭载体Step 3.1 Construction of the shuttle vector of adenovirus E3 region
以腺病毒Δ920-946Ad5为模板(上文所述，包含腺病毒E3区完整序列)，进行PCR反应，上游引物：5′-tgtcaccactaactgctttactcg-3′(SEQ ID NO 16)，下游引物：5′-gctgccctgcgtctttcta-3′(SEQ ID NO 17)，获得26342-31140片段(即E3区完整片段)，将其连接到pEASY-Blunt-Zero载体中，获得pEASY-Blunt-Zero-E3质粒。Using adenovirus Δ920-946Ad5 as a template (described above, including the complete sequence of adenovirus E3 region), carry out PCR reaction, upstream primer: 5'-tgtcaccactaactgctttactcg-3' (SEQ ID NO 16), downstream primer: 5'- gctgccctgcgtctttcta-3' (SEQ ID NO 17) to obtain the 26342-31140 fragment (ie, the complete fragment of the E3 region), which was ligated into the pEASY-Blunt-Zero vector to obtain the pEASY-Blunt-Zero-E3 plasmid.
步骤3.2：质粒pcDNA3.1-E3/ΔADP为骨架质粒，其内插入完整腺病毒E3区但缺失29477bp-29714bp(腺病毒两个EcoRI酶切位点之间的片段，即ADP区)片段，且两端有EcoRI酶切位点的片段。其构建方法如下：Step 3.2: Plasmid pcDNA3.1-E3/ΔADP is a backbone plasmid into which the complete adenovirus E3 region is inserted but the 29477bp-29714bp (the fragment between the two EcoRI restriction sites of the adenovirus, namely the ADP region) fragment is deleted, and Fragments with EcoRI restriction sites at both ends. Its construction method is as follows:
采用3次PCR法缺失Ad5 E3区29477-29714nt。片段1的获取：引物1：5′-atacgcgcccaccgaaac-3′(SEQ ID NO 18)，相当于27306nt-27323nt；引物2：5′-aatctatgg atatcgatagggtgggtcgctgtagtt-3′(SEQ ID NO 19)，相当于29477-29495nt，下划线部分为与引物3互补部分，atcgat为Cla I酶切位点；以Ad5 DNA为模板，进行PCR反应，反应体系总体积为100μl包括：含MgCl2的10×PCR缓冲液，10μl；2mM dNTP，10μl；10μM引物1，1μl；10μM引物2，1μl；Ad5 DNA 200ng/μl，1μl；pfu高保真Taq酶，2.5u；加水至100μl。反应条件为：94℃，30s；94℃，30s；46℃，1min；72℃，1min；共30循环；72℃延伸10min。PCR产物为2207bp(片段1)，常规电泳分离纯化后，检测浓度用于后继PCR反应。The Ad5 E3 region 29477-29714nt was deleted by 3 PCRs. Obtainment of fragment 1: primer 1: 5'-atacgcgcccaccgaaac-3' (SEQ ID NO 18), equivalent to 27306nt-27323nt; primer 2: 5'- aatctatgg at atcgatagggtgggtcgctgtagtt-3' (SEQ ID NO 19), equivalent to 29477 -29495nt, the underlined part is the complementary part of
片段2的获取：引物3：5′-cgacccaccctatcgatatccatagattggacggactg-3′(SEQ IDNO 20)，相当于29714nt-29734n，下划线部分为与引物2互补部分，atcgat为Cla I酶切位点；引物4：5′-atgtctttgaggcttggagg-3′(SEQ ID NO21)，相当于30137nt-30118nt；PCR反应条件同上，产物为422bp(片段2)，常规电泳分离纯化后，检测浓度用于后继PCR反应。Obtaining fragment 2: primer 3: 5′- cgacccaccctat cgatatccatagattggacggactg-3′ (SEQ ID NO 20), equivalent to 29714nt-29734n, the underlined part is the complementary part to
片段3的获取：将片段1与片段2等量混合，作为模板行PCR反应，上游引物为引物1，下游引物为引物4，反应条件同上，产物约为2612bp(片段3)，用QIAquick 8PCR产物纯化试剂盒(QIAGEN，German，Cat：28142)纯化后，用EcoRI酶切过夜，酶切产物在1％琼脂糖凝胶电泳分离后回收，酶切片段用于后继连接反应。将pcDNA3.1(Invitrogen，U.S.A.，Cat：V79020)用EcoRI酶切过夜，酶切产物在1％琼脂糖凝胶电泳分离后回收酶切片段，去磷酸化后用于后继连接反应。将PCR反应产物片段3与酶切、去磷酸化后pcDNA3.1连接，取1.5μl转入100μlDH5α感受态细菌，挑取阳性克隆，质粒小提，DNA测序鉴定筛选得到缺失29477nt-29714nt的pcDNA3.1质粒突变体-pcDNA3.1-E3/ΔADP。Acquisition of fragment 3: Mix
将如SEQ ID NO.22所示的线粒体凋亡肽tBid的基因序列引入Cla1酶切位点。ClaI酶切连接入pcDNA3.1-E3/ΔADP质粒中。The gene sequence of mitochondrial apoptotic peptide tBid as shown in SEQ ID NO. 22 was introduced into the Cla1 restriction site. ClaI digested and ligated into pcDNA3.1-E3/ΔADP plasmid.
步骤3.3：将步骤3.2构建的质粒通过EcoRI酶切，连接入步骤3.1构建的pEASY-Blunt-Zero-E3质粒中，采用上述pEASY-Blunt-Zero-E3质粒试剂盒中自带的引物进行PCR扩增反应，获得E3/tBid片段。Step 3.3: The plasmid constructed in Step 3.2 was digested with EcoRI and ligated into the pEASY-Blunt-Zero-E3 plasmid constructed in Step 3.1. PCR amplification was carried out using the primers included in the pEASY-Blunt-Zero-E3 plasmid kit. The reaction was amplified to obtain the E3/tBid fragment.
步骤3.4：SpeI酶切步骤2.3构建的pAd5-E1A/ΔE1A-Hexon(HVR2/TMTP1)质粒、pAd5-E1A/ΔE1A-Hexon(HVR5/TMTP1)质粒和pAd5-E1A/ΔE1A-Hexon(HVR7/TMTP1)质粒，苯酚氯仿抽提，回收线性骨架质粒。Step 3.4: SpeI digestion of the pAd5-E1A/ΔE1A-Hexon(HVR2/TMTP1) plasmid, pAd5-E1A/ΔE1A-Hexon(HVR5/TMTP1) plasmid and pAd5-E1A/ΔE1A-Hexon(HVR7/TMTP1) plasmid constructed in step 2.3 The plasmid was extracted with phenol and chloroform, and the linear backbone plasmid was recovered.
将线性骨架质粒和步骤3.3获得的E3/tBid片段，电转入步骤1.3获得的大肠杆菌BJ5183电转感受肽中，电转条件为2.5Kv、25μF，同源重组，分别获得pAd5/ΔE1A/HVR2/TMTP1/ΔADP-tBid质粒、pAd5/ΔE1A/HVR5/TMTP1/ΔADP-tBid质粒和pAd5/ΔE1A/HVR7/TMTP1/ΔADP-tBid质粒。The linear backbone plasmid and the E3/tBid fragment obtained in step 3.3 were electroporated into E. coli BJ5183 electrocompetent peptide obtained in step 1.3, and the electroporation conditions were 2.5Kv, 25μF, and homologous recombination to obtain pAd5/ΔE1A/HVR2/TMTP1 respectively. /ΔADP-tBid plasmid, pAd5/ΔE1A/HVR5/TMTP1/ΔADP-tBid plasmid and pAd5/ΔE1A/HVR7/TMTP1/ΔADP-tBid plasmid.
实施例4：携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBid的获得、扩增和纯化Example 4: Acquisition, Amplification and Purification of Oncolytic Adenovirus Recombinant Ad5/ΔE1A/TMTP1/ΔADP-tBid Carrying TMTP1 and tBid
步骤4.1：携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBid的获得Step 4.1: Acquisition of oncolytic adenovirus recombinant Ad5/ΔE1A/TMTP1/ΔADP-tBid carrying TMTP1 and tBid
步骤4.1.1：将步骤3.4获得的pAd5/ΔE1A/HVR2/TMTP1/ΔADP-tBid质粒、pAd5/ΔE1A/HVR5/TMTP1/ΔADP-tBid质粒和pAd5/ΔE1A/HVR7/TMTP1/ΔADP-tBid质粒分别大量提取，使用PacI酶切质粒，苯酚氯仿抽提，回收酶切产物。Step 4.1.1: The pAd5/ΔE1A/HVR2/TMTP1/ΔADP-tBid plasmid, pAd5/ΔE1A/HVR5/TMTP1/ΔADP-tBid plasmid and pAd5/ΔE1A/HVR7/TMTP1/ΔADP-tBid plasmid obtained in step 3.4 Extract, digest the plasmid with PacI, extract with phenol and chloroform, and recover the digested product.
步骤4.1.2：利用lipo3000脂质体(购自赛默飞世尔科技公司，货号为L3000001)转染方法，将步骤4.1.1回收的产物转入到融合度为30％的293细胞24孔板中。每隔2天加入新鲜的DMEM培养基，待293细胞的融合度达到100％以上，收集细胞，并反复冻融使病毒裂解释放到DMEM培养基中,离心收集上清病毒液。Step 4.1.2: Using the transfection method of lipo3000 liposome (purchased from Thermo Fisher Scientific, the product number is L3000001), the product recovered in step 4.1.1 was transferred into 24 wells of 293 cells with a confluence of 30% in the board. Fresh DMEM medium was added every 2 days. When the confluence of 293 cells reached more than 100%, the cells were collected, and the virus was lysed and released into DMEM medium by repeated freezing and thawing. The supernatant virus liquid was collected by centrifugation.
步骤4.1.3：采用含有腺病毒的DMEM培养基，再次转染293细胞，重复大约2周，直至293细胞出现CPE效应，收集细胞并反复冻融裂解，获得上清病毒液。Step 4.1.3: Use DMEM medium containing adenovirus to transfect 293 cells again, and repeat for about 2 weeks until the CPE effect occurs in 293 cells, collect the cells and repeat freeze-thaw lysis to obtain the supernatant virus solution.
步骤4.1.4：鉴定步骤4.1.3获得的三种Ad5/ΔE1A/TMTP1/ΔADP-tBid，分别用相应引物PCR后测序E1A、Hexon和E3/ADP区。Step 4.1.4: Identify the three Ad5/ΔE1A/TMTP1/ΔADP-tBids obtained in step 4.1.3, and sequence the E1A, Hexon and E3/ADP regions after PCR with corresponding primers, respectively.
步骤4.2：携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBid的扩增、纯化和滴度测定Step 4.2: Amplification, purification and titer determination of oncolytic adenovirus recombinant Ad5/ΔE1A/TMTP1/ΔADP-tBid carrying TMTP1 and tBid
步骤4.2.1：扩增步骤4.1.4测序正确的携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/TMTP1/ΔADP-tBid，检测腺病毒种子的滴度(MOI)，根据MOI值确定感染293细胞的病毒量，在48h-72h内出现CPE效应时收集细胞。如此反复扩增直至病毒量达到实验需求(109pfu/ml-1011pfu/ml)。Step 4.2.1: Amplify Step 4.1.4 Sequencing the correct oncolytic adenovirus recombinant Ad5/ΔE1A/TMTP1/ΔADP-tBid carrying TMTP1 and tBid, and detect the adenovirus seed titer (MOI), which is determined according to the MOI value The amount of virus infecting 293 cells was collected when the CPE effect appeared within 48h-72h. The amplification was repeated until the amount of virus reached the experimental requirement (10 9 pfu/ml-10 11 pfu/ml).
步骤4.2.2：采用传统CsCl梯度离心法纯化腺病毒，病毒透析液透析获得纯化后的腺病毒。使用紫外吸收法检测病毒颗粒数，使用Adeno-XTM快速滴度试剂盒(Clonetech,货号632250)检测MOI。Step 4.2.2: The adenovirus was purified by the traditional CsCl gradient centrifugation method, and the purified adenovirus was obtained by dialysis of the virus dialysate. The number of viral particles was determined by UV absorption and the MOI was determined using the Adeno-XTM Rapid Titer Kit (Clonetech, Cat. No. 632250).
实施例5：携带TMTP1和tBid的溶瘤腺病毒重组体最佳插入区域筛选Example 5: Screening of the optimal insertion region of oncolytic adenovirus recombinants carrying TMTP1 and tBid
本实施例的目的在于确定插入Hexon高变区2、5、7三种不同区域的携带TMTP1和tBid的溶瘤腺病毒重组体对肿瘤细胞感染效率，以挑选出对肿瘤细胞感染效应最好的靶向肽插入区域。The purpose of this example is to determine the infection efficiency of oncolytic adenovirus recombinants carrying TMTP1 and tBid inserted into three different regions of Hexon
在6孔培养板中种入约2×105个skov3肿瘤细胞，培养液为10％FBS DMEM培养基，到第二天大约70％细胞融合，吸取液体，加入2mL新鲜的10％FBS DMEM培养基，将MOI＝1对应的溶瘤腺病毒数量用培养基稀释至100μL后加入培养孔中，轻轻晃动液体三次，在37℃、5％CO2的培养箱中孵育12h、24h和36h后，收细胞渣，提取DNA，用实时定量PCR鉴定不同处理组腺病毒DNA的相对拷贝数。结果如图2显示，携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid在肿瘤细胞中复制最为活跃，其核苷酸序列如SEQ ID NO.23所示。通过体外实验,筛选出能够在肿瘤细胞中高拷贝数复制的最佳腺病毒插入区——高变区5，即HVR5为最终区域。Seed about 2 × 10 5 skov3 tumor cells in a 6-well culture plate, the culture medium is 10% FBS DMEM medium, about 70% of the cells are confluent on the next day, aspirate the liquid, add 2 mL of fresh 10% FBS DMEM to culture The number of oncolytic adenoviruses corresponding to MOI= 1 was diluted with medium to 100 μL and added to the culture wells, and the liquid was gently shaken three times. , the cell residue was collected, DNA was extracted, and the relative copy number of adenovirus DNA in different treatment groups was identified by real-time quantitative PCR. The results are shown in Figure 2. The oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid carrying TMTP1 and tBid is the most active in tumor cell replication, and its nucleotide sequence is shown in SEQ ID NO.23. Through in vitro experiments, the optimal adenovirus insertion region that can replicate in high copy number in tumor cells,
实施例6：携带TMTP1和tBid的溶瘤腺病毒重组体的治疗效应的鉴定Example 6: Identification of Therapeutic Effects of Oncolytic Adenovirus Recombinants Carrying TMTP1 and tBid
步骤6.1：携带TMTP1和tBid的溶瘤腺病毒重组体体外肿瘤细胞杀伤效应的鉴定Step 6.1: Identification of in vitro tumor cell killing effect of oncolytic adenovirus recombinants carrying TMTP1 and tBid
本实施例的目的在于鉴定Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid溶瘤腺病毒对一系列肿瘤细胞及正常细胞的杀伤效应。选用的细胞系如表1所示，具有不同的P53,Rb基因表型；具有不同的组织源性，因此实验结果可代表不同类型肿瘤。The purpose of this example is to identify the killing effect of Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid oncolytic adenovirus on a series of tumor cells and normal cells. The selected cell lines are shown in Table 1, with different P53, Rb gene phenotypes; with different tissue origins, so the experimental results can represent different types of tumors.
在96孔培养板中种入5×103个肿瘤细胞，培养液为10％FBS DMEM或RIPM1640，到第二天大约70％细胞融合，吸取液体，将MOI＝10对应的溶瘤腺病毒数量用培养基稀释至100μl后加入培养孔中，轻轻晃动液体三次，在37℃、5％CO2的培养箱中孵育72h后，加入cck-8测量病毒存活率。
实验结果表明，当病毒MOI＝10时，44.5％的肿瘤细胞表现为极度敏感，存活率小于25％；38.8％的肿瘤细胞对腺病毒有良好的反应，存活率介于25％-75％；但仍有16.7％的肿瘤细胞对腺病毒反应轻微，具体实验结果见表1。The experimental results show that when the virus MOI=10, 44.5% of tumor cells are extremely sensitive, and the survival rate is less than 25%; 38.8% of tumor cells have a good response to adenovirus, and the survival rate is between 25%-75%; However, 16.7% of tumor cells still had mild reaction to adenovirus. The specific experimental results are shown in Table 1.
步骤6.2：携带TMTP1和tBid的溶瘤腺病毒重组体对化疗药顺铂增敏效应的鉴定Step 6.2: Identification of the sensitizing effect of oncolytic adenovirus recombinants carrying TMTP1 and tBid on the chemotherapeutic drug cisplatin
鉴于携带TMTP1和tBid的溶瘤腺病毒重组体并非对所有肿瘤细胞均表现出强效杀伤效应，设想将携带TMTP1和tBid的溶瘤腺病毒重组体与顺铂(DDP)联合杀伤不敏感的肿瘤细胞。Given that the oncolytic adenovirus recombinants carrying TMTP1 and tBid did not show potent killing effect on all tumor cells, it is envisaged that the oncolytic adenovirus recombinants carrying TMTP1 and tBid combined with cisplatin (DDP) to kill insensitive tumors cell.
在96孔细胞培养板中种入5×103个肿瘤细胞，同时混入一定浓度的携带TMTP1和tBid的溶瘤腺病毒重组体及其对照病毒Ad5/ΔE1A。该对照病毒Ad5/ΔE1A仅E1A缺失27bp的溶瘤腺病毒，由本实验室构建并保存。第二天用不同浓度的DDP培养基孵育48h后，测量细胞存活率，计算在腺病毒孵育后，每个细胞系对DDP的IC50(即引起肿瘤细胞凋亡50％时DDP的浓度)，并计算出携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid与DDP的协同指数CI。CI值小于1说明两药有协同效应，且CI值越小表明协同效应越强[1]。具体实验结果见表1。5×10 3 tumor cells were seeded in a 96-well cell culture plate, and a certain concentration of oncolytic adenovirus recombinant carrying TMTP1 and tBid and its control virus Ad5/ΔE1A were mixed. The control virus Ad5/ΔE1A is an oncolytic adenovirus with only E1A deletion of 27bp, which was constructed and preserved by our laboratory. On the second day, after 48h incubation with different concentrations of DDP medium, the cell viability was measured, and the IC50 of each cell line for DDP after adenovirus incubation (ie the concentration of DDP that caused 50% of tumor cell apoptosis) was calculated, And the synergy index CI of the oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid carrying TMTP1 and tBid with DDP was calculated. The CI value less than 1 indicates that the two drugs have a synergistic effect, and the smaller the CI value, the stronger the synergistic effect [1] . The specific experimental results are shown in Table 1.
表1Table 1
实验结果表明，对于大部分肿瘤细胞，Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid与DDP联用有增敏效应，对于耐药细胞系如ES2、C13K、MDA-MB-231尤为明显，CI值分别为0.2、0.37和0.04。以上结果充分说明，本发明的携带TMTP1和tBid的溶瘤腺病毒重组体对化疗药顺铂增敏，不论是对DDP原发敏感或者是原发耐药的细胞系。The experimental results show that for most tumor cells, Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid combined with DDP has a sensitizing effect, especially for drug-resistant cell lines such as ES2, C13K, MDA-MB-231, and the CI value were 0.2, 0.37 and 0.04, respectively. The above results fully demonstrate that the oncolytic adenovirus recombinant carrying TMTP1 and tBid of the present invention is sensitized to the chemotherapeutic drug cisplatin, whether it is a primary sensitive or primary resistant cell line to DDP.
实施例7：体内实验验证携带TMTP1和tBid的溶瘤腺病毒重组体可逃避肝脏摄取并可特异性靶向肿瘤细胞Example 7: In vivo experiments verify that oncolytic adenovirus recombinants carrying TMTP1 and tBid can escape liver uptake and can specifically target tumor cells
4-6周大小BALB/c nu裸鼠，腹腔种植2×106个skov3卵巢癌细胞，4周后，将裸鼠分为两组(n＝4)，分别腹腔注射1×108PFU携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid及其对照病毒Ad5/ΔE1A。48h后，取出每只小鼠的肝脏、脾脏、肺及肿瘤组织，提取各个组织DNA，检测400ng组织DNA中，腺病毒的DNA含量，结果如图3所示：对照病毒Ad5/ΔE1A在肝脏、脾脏、肺中均有聚集，在肿瘤组织中分布较少，Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid主要分布在肿瘤组织中，几乎不被肝脏摄取；图4显示裸鼠体内各个组织中，腺病毒的相对含量，注射Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid的小鼠中，腺病毒主要集中在肿瘤组织中，在肿瘤组织中的含量是对照病毒的近100倍，而对照病毒Ad5/ΔE1A则大量被肝脏摄取，在肿瘤组织中几乎检测不到腺病毒基因。4-6 weeks old BALB/c nu nude mice were implanted intraperitoneally with 2×10 6 skov3 ovarian cancer cells. After 4 weeks, the nude mice were divided into two groups (n=4) and injected intraperitoneally with 1×10 8 PFU cells Oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid of TMTP1 and tBid and its control virus Ad5/ΔE1A. After 48 hours, the liver, spleen, lung and tumor tissues of each mouse were taken out, DNA of each tissue was extracted, and the DNA content of adenovirus in 400 ng of tissue DNA was detected. Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid was mainly distributed in tumor tissue and was hardly taken up by liver; Figure 4 shows that in various tissues in nude mice, The relative content of adenovirus, in mice injected with Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid, adenovirus was mainly concentrated in tumor tissue, and the content in tumor tissue was nearly 100 times that of control virus, while the control virus Ad5 /ΔE1A was taken up in large amounts by the liver, and almost no adenovirus genes were detected in tumor tissues.
以上实验结果充分表明，本发明的携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid克服了肝嗜性，几乎不再被肝脏摄取，且极大增强了对肿瘤的靶向性。The above experimental results fully show that the oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid carrying TMTP1 and tBid of the present invention overcomes liver tropism, is almost no longer taken up by the liver, and greatly enhances the response to the liver. tumor targeting.
实施例8：体内实验验证携带TMTP1和tBid的溶瘤腺病毒重组体对肿瘤的杀伤效应及其安全性Example 8: In vivo experiments to verify the killing effect and safety of oncolytic adenovirus recombinants carrying TMTP1 and tBid on tumors
步骤8.1：皮下瘤动物模型Step 8.1: Subcutaneous Tumor Animal Model
4-6周大小BALB/c nu裸鼠，单侧腋窝脂肪垫下种植2×106个skov3卵巢癌细胞，四周后，待肿瘤黄豆大小，将小鼠分为六组(n＝6)，六组分别为：a-空白组，b-单加DDP，c-单注射Ad5/ΔE1A，d-注射Ad5/ΔE1A加DDP，e-单注射Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid，f-注射Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid加DDP。4-6 weeks old BALB/c nu nude mice were implanted with 2×10 6 skov3 ovarian cancer cells under the unilateral axillary fat pad. The six groups are: a-blank group, b-single injection of DDP, c-single injection of Ad5/ΔE1A, d- injection of Ad5/ΔE1A plus DDP, e- single injection of Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid, f - Injection of Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid plus DDP.
其中，c组、d组、e组和f组连续肿瘤内注射腺病毒1×108PFU，三天后b组、d组和f组腹腔内注射DDP，含量为2.5mg/kg，隔一天一次，共注射四次。从肿瘤种植后开始，每三天测量肿瘤体积，肿瘤体积变化如图5所示。Among them, groups c, d, e and f were continuously injected with
实验组f组中6只小鼠，肿瘤全部消失，而实验组e组中，单注射Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid并不能很好控制肿瘤生长。In the 6 mice in the experimental group f, all the tumors disappeared, while in the experimental group e, the single injection of Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid could not control the tumor growth very well.
步骤8.2：腹腔原位转移瘤模型Step 8.2: Abdominal Orthotopic Metastasis Model
4-6周大小BALB/c nu裸鼠，腹腔种植2×106skov3卵巢癌细胞，四周后，将小鼠分为六组(n＝16-21)，分组同上，且给药方式时间同上，病毒给药方式改为腹腔注射，其余同皮下瘤模型，15d后，每组随机选出三只老鼠，取出腹腔中最大肿瘤，如图6所示：实验组f组中肿瘤大小，明显小于各实验组及对照组(p<0.0001)。4-6 weeks old BALB/c nu nude mice were implanted with 2×10 6 skov3 ovarian cancer cells in the abdominal cavity. After four weeks, the mice were divided into six groups (n=16-21), the groups were the same as above, and the administration time was the same as above , the virus administration method was changed to intraperitoneal injection, and the rest were the same as the subcutaneous tumor model. After 15 days, three mice were randomly selected from each group, and the largest tumor in the abdominal cavity was taken out, as shown in Figure 6: the size of the tumor in the experimental group f group was significantly smaller than Each experimental group and control group (p<0.0001).
步骤8.3：携带TMTP1和tBid的溶瘤腺病毒重组体安全性检测Step 8.3: Safety detection of oncolytic adenovirus recombinants carrying TMTP1 and tBid
在上述腹腔原位转移瘤模型中，将小鼠麻醉后，眼眶取血，测量各组中实验小鼠的肝肾功能，包括：谷丙转氨酶(ALT)、谷草转氨酶(AST)、肌酐(Creatinine)、尿素氮(BUN)，结果如图7-图10所示，对照组与实验组中，肾功能无明显损伤，但是注射对照病毒Ad5/ΔE1A的小鼠，谷丙转氨酶(ALT)、谷草转氨酶(AST)明显升高，而注射Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid的小鼠，肝功能正常。In the above-mentioned intraperitoneal orthotopic metastasis model, the mice were anesthetized, and the orbital blood was collected to measure the liver and kidney functions of the experimental mice in each group, including: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Creatinine) ), urea nitrogen (BUN), the results are shown in Figure 7-Figure 10, in the control group and the experimental group, there was no obvious damage to renal function, but the mice injected with the control virus Ad5/ΔE1A, alanine aminotransferase (ALT), aspartate Transaminases (AST) were significantly elevated, and mice injected with Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid had normal liver function.
以上结果表明，携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid可以逃避肝脏摄取，对肝脏无损伤，且可特异性靶向肿瘤组织。在体内及体外均对肿瘤细胞有杀伤性，且对化疗药物顺铂的增敏效应尤为明显，皮下瘤模型及腹腔种植模型中二者联用均可控制肿瘤进展，且作为基因治疗载体安全可靠，对实验动物无毒副作用。The above results indicated that the oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid carrying TMTP1 and tBid could escape hepatic uptake, did not damage the liver, and could specifically target tumor tissues. Both in vivo and in vitro are cytotoxic to tumor cells, and the sensitizing effect to the chemotherapeutic drug cisplatin is particularly obvious. The combination of the two can control tumor progression in subcutaneous tumor models and intraperitoneal implantation models, and it is safe and reliable as a gene therapy carrier. , has no toxic and side effects on experimental animals.
因此，本发明的携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid，可以用于制备治疗肿瘤的药物，既开拓了新的治疗肿瘤的药物，又开拓了携带TMTP1和tBid的溶瘤腺病毒重组体的用途，具有积极的药学价值和广泛的社会意义。Therefore, the oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid carrying TMTP1 and tBid of the present invention can be used to prepare a drug for the treatment of tumors, which not only opens up new drugs for the treatment of tumors, but also develops new drugs for the treatment of tumors. The use of oncolytic adenovirus recombinants carrying TMTP1 and tBid has positive pharmaceutical value and broad social significance.
本发明的携带TMTP1和tBid的溶瘤腺病毒重组体Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid，可以用于制备基因治疗载体，既开拓了新的基因治疗载体，又开拓了携带TMTP1和tBid的溶瘤腺病毒重组体的用途，具有积极的药学价值和广泛的社会意义。The oncolytic adenovirus recombinant Ad5/ΔE1A/HVR5/TMTP1/ΔADP-tBid carrying TMTP1 and tBid of the present invention can be used to prepare a gene therapy vector, which not only develops a new gene therapy vector, but also develops a new gene therapy vector carrying TMTP1 and tBid. The use of the oncolytic adenovirus recombinant has positive pharmaceutical value and broad social significance.
参考文献：references:
[1].Chou TC,Motzer RJ,Tong Y,Bosl GJ(1994)Computerized quantitationof synergism and antagonism of taxol,topotecan,and cisplatin against humanteratocarcinoma cell growth:A rational approach to clinical protocol design.JNatl Cancer Inst 86:1517–1524.[1]. Chou TC, Motzer RJ, Tong Y, Bosl GJ (1994) Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatin against humanteratocarcinoma cell growth: A rational approach to clinical protocol design. JNatl Cancer Inst 86:1517– 1524.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.
序列表sequence listing
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<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 4<400> 4
gaggtgatcg atccacccag tgacgacgag 30gaggtgatcg atccacccag tgacgacgag 30
<210> 5<210> 5
<211> 23<211> 23
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 5<400> 5
tgctctagac acaggtgatg tcg 23tgctctagac acaggtgatg tcg 23
<210> 6<210> 6
<211> 31<211> 31
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 6<400> 6
ttaattaaca tcatcaataa tataccttat t 31ttaattaaca tcatcaataa tataccttat t 31
<210> 7<210> 7
<211> 25<211> 25
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 7<400> 7
gatccacata atctaacaca aactc 25gatccacata atctaacaca aactc 25
<210> 8<210> 8
<211> 31<211> 31
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 8<400> 8
ttaattaaca tcatcaataa tataccttat t 31ttaattaaca tcatcaataa tataccttat t 31
<210> 9<210> 9
<211> 25<211> 25
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 9<400> 9
gatccacata atctaacaca aactc 25gatccacata atctaacaca aactc 25
<210> 10<210> 10
<211> 22<211> 22
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 10<400> 10
cgggatccgg gcccccattt cc 22cgggatccgg gcccccattt cc 22
<210> 11<210> 11
<211> 23<211> 23
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 11<400> 11
tgctctagac acaggtgatg tcg 23tgctctagac acaggtgatg tcg 23
<210> 12<210> 12
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 12<400> 12
agccggagca gagagccttg 20
<210> 13<210> 13
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 13<400> 13
ccagagtagg tgtaataagg 20
<210> 14<210> 14
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 14<400> 14
tagaaagtca agtggaaatg 20
<210> 15<210> 15
<211> 15<211> 15
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 15<400> 15
aacgtggtgc gtcaa 15aacgtggtgc gtcaa 15
<210> 16<210> 16
<211> 24<211> 24
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 16<400> 16
tgtcaccact aactgcttta ctcg 24tgtcaccact aactgcttta ctcg 24
<210> 17<210> 17
<211> 19<211> 19
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 17<400> 17
gctgccctgc gtctttcta 19gctgccctgc gtctttcta 19
<210> 18<210> 18
<211> 18<211> 18
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 18<400> 18
atacgcgccc accgaaac 18atacgcgccc accgaaac 18
<210> 19<210> 19
<211> 36<211> 36
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 19<400> 19
aatctatgga tatcgatagg gtgggtcgct gtagtt 36aatctatgga tatcgatagg gtgggtcgct gtagtt 36
<210> 20<210> 20
<211> 38<211> 38
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 20<400> 20
cgacccaccc tatcgatatc catagattgg acggactg 38cgacccaccc tatcgatatc catagattgg acggactg 38
<210> 21<210> 21
<211> 20<211> 20
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 21<400> 21
atgtctttga ggcttggagg 20
<210> 22<210> 22
<211> 408<211> 408
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 22<400> 22
ggcaaccgca gcagccactc ccgcttggga agaatagagg cagattctga aagtcaagaa 60ggcaaccgca gcagccactc ccgcttggga agaatagagg cagattctga aagtcaagaa 60
gacatcatcc ggaatattgc caggcacctc gcccaggtcg gggacagcat ggaccgtagc 120gacatcatcc ggaatattgc caggcacctc gcccaggtcg gggacagcat ggaccgtagc 120
atccctccgg gcctggtgaa cggcctggcc ctgcagctca ggaacaccag ccggtcggag 180atccctccgg gcctggtgaa cggcctggcc ctgcagctca ggaacaccag ccggtcggag 180
gaggaccgga acagggacct ggccactgcc ctggagcagc tgctgcaggc ctaccctaga 240gaggaccgga acagggacct ggccactgcc ctggagcagc tgctgcaggc ctaccctaga 240
gacatggaga aggagaagac catgctggtg ctggccctgc tgctggccaa gaaggtggcc 300gacatggaga aggagaagac catgctggtg ctggccctgc tgctggccaa gaaggtggcc 300
agtcacacgc cgtccttgct ccgtgatgtc tttcacacaa cagtgaattt tattaaccag 360agtcacacgc cgtccttgct ccgtgatgtc tttcacacaa cagtgaattt tattaaccag 360
aacctacgca cctacgtgag gagcttagcc agaaatggga tggatgaa 408aacctacgca cctacgtgag gagcttagcc agaaatggga tggatgaa 408
<210> 23<210> 23
<211> 36210<211> 36210
<212> DNA<212> DNA
<213> 人工序列(Artificial Sequence)<213> Artificial Sequence
<400> 23<400> 23
catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60
ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120
gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180
gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240
taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300
agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360
gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420
cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 480cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 480
tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc 540tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc 540
tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga 600tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga 600
aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc 660aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc 660
tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc 720tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc 720
cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt 780cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt 780
gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca 840gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca 840
cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa 900cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa 900
ccttgtaccg gaggtgatcc cacccagtga cgacgaggat gaagagggtg aggagtttgt 960ccttgtaccg gaggtgatcc cacccagtga cgacgaggat gaagagggtg aggagtttgt 960
gttagattat gtggagcacc ccgggcacgg ttgcaggtct tgtcattatc accggaggaa 1020gttagattat gtggagcacc ccgggcacgg ttgcaggtct tgtcattatc accggaggaa 1020
tacgggggac ccagatatta tgtgttcgct ttgctatatg aggacctgtg gcatgtttgt 1080tacgggggac ccagatatta tgtgttcgct ttgctatatg aggacctgtg gcatgtttgt 1080
ctacagtaag tgaaaattat gggcagtggg tgatagagtg gtgggtttgg tgtggtaatt 1140ctacagtaag tgaaaattat gggcagtggg tgatagagtg gtgggtttgg tgtggtaatt 1140
ttttttttaa tttttacagt tttgtggttt aaagaatttt gtattgtgat ttttttaaaa 1200ttttttttaa ttttttacagt tttgtggttt aaagaatttt gtattgtgat ttttttaaaa 1200
ggtcctgtgt ctgaacctga gcctgagccc gagccagaac cggagcctgc aagacctacc 1260ggtcctgtgt ctgaacctga gcctgagccc gagccagaac cggagcctgc aagacctacc 1260
cgccgtccta aaatggcgcc tgctatcctg agacgcccga catcacctgt gtctagagaa 1320cgccgtccta aaatggcgcc tgctatcctg agacgcccga catcacctgt gtctagagaa 1320
tgcaatagta gtacggatag ctgtgactcc ggtccttcta acacacctcc tgagatacac 1380tgcaatagta gtacggatag ctgtgactcc ggtccttcta acacacctcc tgagatacac 1380
ccggtggtcc cgctgtgccc cattaaacca gttgccgtga gagttggtgg gcgtcgccag 1440ccggtggtcc cgctgtgccc cattaaacca gttgccgtga gagttggtgg gcgtcgccag 1440
gctgtggaat gtatcgagga cttgcttaac gagcctgggc aacctttgga cttgagctgt 1500gctgtggaat gtatcgagga cttgcttaac gagcctgggc aacctttgga cttgagctgt 1500
aaacgcccca ggccataagg tgtaaacctg tgattgcgtg tgtggttaac gcctttgttt 1560aaacgcccca ggccataagg tgtaaacctg tgattgcgtg tgtggttaac gcctttgttt 1560
gctgaatgag ttgatgtaag tttaataaag ggtgagataa tgtttaactt gcatggcgtg 1620gctgaatgag ttgatgtaag tttaataaag ggtgagataa tgtttaactt gcatggcgtg 1620
ttaaatgggg cggggcttaa agggtatata atgcgccgtg ggctaatctt ggttacatct 1680ttaaatgggg cggggcttaa agggtatata atgcgccgtg ggctaatctt ggttacatct 1680
gacctcatgg aggcttggga gtgtttggaa gatttttctg ctgtgcgtaa cttgctggaa 1740gacctcatgg aggcttggga gtgtttggaa gattttttctg ctgtgcgtaa cttgctggaa 1740
cagagctcta acagtacctc ttggttttgg aggtttctgt ggggctcatc ccaggcaaag 1800cagagctcta acagtacctc ttggttttgg aggtttctgt ggggctcatc ccaggcaaag 1800
ttagtctgca gaattaagga ggattacaag tgggaatttg aagagctttt gaaatcctgt 1860ttagtctgca gaattaagga ggattacaag tgggaatttg aagagctttt gaaatcctgt 1860
ggtgagctgt ttgattcttt gaatctgggt caccaggcgc ttttccaaga gaaggtcatc 1920ggtgagctgt ttgattcttt gaatctgggt caccaggcgc ttttccaaga gaaggtcatc 1920
aagactttgg atttttccac accggggcgc gctgcggctg ctgttgcttt tttgagtttt 1980aagactttgg atttttccac accggggcgc gctgcggctg ctgttgcttt tttgagtttt 1980
ataaaggata aatggagcga agaaacccat ctgagcgggg ggtacctgct ggattttctg 2040ataaaggata aatggagcga agaaacccat ctgagcgggg ggtacctgct ggattttctg 2040
gccatgcatc tgtggagagc ggttgtgaga cacaagaatc gcctgctact gttgtcttcc 2100gccatgcatc tgtggagagc ggttgtgaga cacaagaatc gcctgctact gttgtcttcc 2100
gtccgcccgg cgataatacc gacggaggag cagcagcagc agcaggagga agccaggcgg 2160gtccgcccgg cgataatacc gacggaggag cagcagcagc agcaggagga agccaggcgg 2160
cggcggcagg agcagagccc atggaacccg agagccggcc tggaccctcg ggaatgaatg 2220cggcggcagg agcagagccc atggaacccg agagccggcc tggaccctcg ggaatgaatg 2220
ttgtacaggt ggctgaactg tatccagaac tgagacgcat tttgacaatt acagaggatg 2280ttgtacaggt ggctgaactg tatccagaac tgagacgcat tttgacaatt acagaggatg 2280
ggcaggggct aaagggggta aagagggagc ggggggcttg tgaggctaca gaggaggcta 2340ggcaggggct aaagggggta aagagggagc ggggggcttg tgaggctaca gaggaggcta 2340
ggaatctagc ttttagctta atgaccagac accgtcctga gtgtattact tttcaacaga 2400ggaatctagc ttttagctta atgaccagac accgtcctga gtgtattact tttcaacaga 2400
tcaaggataa ttgcgctaat gagcttgatc tgctggcgca gaagtattcc atagagcagc 2460tcaaggataa ttgcgctaat gagcttgatc tgctggcgca gaagtattcc atagagcagc 2460
tgaccactta ctggctgcag ccaggggatg attttgagga ggctattagg gtatatgcaa 2520tgaccactta ctggctgcag ccaggggatg attttgagga ggctattagg gtatatgcaa 2520
aggtggcact taggccagat tgcaagtaca agatcagcaa acttgtaaat atcaggaatt 2580aggtggcact taggccagat tgcaagtaca agatcagcaa acttgtaaat atcaggaatt 2580
gttgctacat ttctgggaac ggggccgagg tggagataga tacggaggat agggtggcct 2640gttgctacat ttctgggaac ggggccgagg tggagataga tacggaggat agggtggcct 2640
ttagatgtag catgataaat atgtggccgg gggtgcttgg catggacggg gtggttatta 2700ttagatgtag catgataaat atgtggccgg gggtgcttgg catggacggg gtggttatta 2700
tgaatgtaag gtttactggc cccaatttta gcggtacggt tttcctggcc aataccaacc 2760tgaatgtaag gtttactggc cccaatttta gcggtacggt tttcctggcc aataccaacc 2760
ttatcctaca cggtgtaagc ttctatgggt ttaacaatac ctgtgtggaa gcctggaccg 2820ttatcctaca cggtgtaagc ttctatgggt ttaacaatac ctgtgtggaa gcctggaccg 2820
atgtaagggt tcggggctgt gccttttact gctgctggaa gggggtggtg tgtcgcccca 2880atgtaagggt tcggggctgt gccttttact gctgctggaa gggggtggtg tgtcgcccca 2880
aaagcagggc ttcaattaag aaatgcctct ttgaaaggtg taccttgggt atcctgtctg 2940aaagcagggc ttcaattaag aaatgcctct ttgaaaggtg taccttgggt atcctgtctg 2940
agggtaactc cagggtgcgc cacaatgtgg cctccgactg tggttgcttc atgctagtga 3000agggtaactc cagggtgcgc cacaatgtgg cctccgactg tggttgcttc atgctagtga 3000
aaagcgtggc tgtgattaag cataacatgg tatgtggcaa ctgcgaggac agggcctctc 3060aaagcgtggc tgtgattaag cataacatgg tatgtggcaa ctgcgaggac agggcctctc 3060
agatgctgac ctgctcggac ggcaactgtc acctgctgaa gaccattcac gtagccagcc 3120agatgctgac ctgctcggac ggcaactgtc acctgctgaa gaccattcac gtagccagcc 3120
actctcgcaa ggcctggcca gtgtttgagc ataacatact gacccgctgt tccttgcatt 3180actctcgcaa ggcctggcca gtgtttgagc ataacatact gacccgctgt tccttgcatt 3180
tgggtaacag gaggggggtg ttcctacctt accaatgcaa tttgagtcac actaagatat 3240tgggtaacag gaggggggtg ttcctacctt accaatgcaa tttgagtcac actaagatat 3240
tgcttgagcc cgagagcatg tccaaggtga acctgaacgg ggtgtttgac atgaccatga 3300tgcttgagcc cgagagcatg tccaaggtga acctgaacgg ggtgtttgac atgaccatga 3300
agatctggaa ggtgctgagg tacgatgaga cccgcaccag gtgcagaccc tgcgagtgtg 3360agatctggaa ggtgctgagg tacgatgaga cccgcaccag gtgcagaccc tgcgagtgtg 3360
gcggtaaaca tattaggaac cagcctgtga tgctggatgt gaccgaggag ctgaggcccg 3420gcggtaaaca tattaggaac cagcctgtga tgctggatgt gaccgaggag ctgaggcccg 3420
atcacttggt gctggcctgc acccgcgctg agtttggctc tagcgatgaa gatacagatt 3480atcacttggt gctggcctgc acccgcgctg agtttggctc tagcgatgaa gatacagatt 3480
gaggtactga aatgtgtggg cgtggcttaa gggtgggaaa gaatatataa ggtgggggtc 3540gaggtactga aatgtgtggg cgtggcttaa gggtgggaaa gaatatataa ggtgggggtc 3540
ttatgtagtt ttgtatctgt tttgcagcag ccgccgccgc catgagcacc aactcgtttg 3600ttatgtagtt ttgtatctgt tttgcagcag ccgccgccgc catgagcacc aactcgtttg 3600
atggaagcat tgtgagctca tatttgacaa cgcgcatgcc cccatgggcc ggggtgcgtc 3660atggaagcat tgtgagctca tatttgacaa cgcgcatgcc cccatgggcc ggggtgcgtc 3660
agaatgtgat gggctccagc attgatggtc gccccgtcct gcccgcaaac tctactacct 3720agaatgtgat gggctccagc attgatggtc gccccgtcct gcccgcaaac tctactacct 3720
tgacctacga gaccgtgtct ggaacgccgt tggagactgc agcctccgcc gccgcttcag 3780tgacctacga gaccgtgtct ggaacgccgt tggagactgc agcctccgcc gccgcttcag 3780
ccgctgcagc caccgcccgc gggattgtga ctgactttgc tttcctgagc ccgcttgcaa 3840ccgctgcagc caccgcccgc gggattgtga ctgactttgc tttcctgagc ccgcttgcaa 3840
gcagtgcagc ttcccgttca tccgcccgcg atgacaagtt gacggctctt ttggcacaat 3900gcagtgcagc ttcccgttca tccgcccgcg atgacaagtt gacggctctt ttggcacaat 3900
tggattcttt gacccgggaa cttaatgtcg tttctcagca gctgttggat ctgcgccagc 3960tggattcttt gacccgggaa cttaatgtcg tttctcagca gctgttggat ctgcgccagc 3960
aggtttctgc cctgaaggct tcctcccctc ccaatgcggt ttaaaacata aataaaaaac 4020aggtttctgc cctgaaggct tcctcccctc ccaatgcggt ttaaaacata aataaaaaac 4020
cagactctgt ttggatttgg atcaagcaag tgtcttgctg tctttattta ggggttttgc 4080cagactctgt ttggatttgg atcaagcaag tgtcttgctg tctttattta ggggttttgc 4080
gcgcgcggta ggcccgggac cagcggtctc ggtcgttgag ggtcctgtgt attttttcca 4140gcgcgcggta ggcccgggac cagcggtctc ggtcgttgag ggtcctgtgt attttttcca 4140
ggacgtggta aaggtgactc tggatgttca gatacatggg cataagcccg tctctggggt 4200ggacgtggta aaggtgactc tggatgttca gatacatggg cataagcccg tctctggggt 4200
ggaggtagca ccactgcaga gcttcatgct gcggggtggt gttgtagatg atccagtcgt 4260ggaggtagca ccactgcaga gcttcatgct gcggggtggt gttgtagatg atccagtcgt 4260
agcaggagcg ctgggcgtgg tgcctaaaaa tgtctttcag tagcaagctg attgccaggg 4320agcaggagcg ctgggcgtgg tgcctaaaaa tgtctttcag tagcaagctg attgccaggg 4320
gcaggccctt ggtgtaagtg tttacaaagc ggttaagctg ggatgggtgc atacgtgggg 4380gcaggccctt ggtgtaagtg tttacaaagc ggttaagctg ggatgggtgc atacgtgggg 4380
atatgagatg catcttggac tgtattttta ggttggctat gttcccagcc atatccctcc 4440atatgagatg catcttggac tgtattttta ggttggctat gttcccagcc atatccctcc 4440
ggggattcat gttgtgcaga accaccagca cagtgtatcc ggtgcacttg ggaaatttgt 4500ggggattcat gttgtgcaga accaccagca cagtgtatcc ggtgcacttg ggaaatttgt 4500
catgtagctt agaaggaaat gcgtggaaga acttggagac gcccttgtga cctccaagat 4560catgtagctt agaaggaaat gcgtggaaga acttggagac gcccttgtga cctccaagat 4560
tttccatgca ttcgtccata atgatggcaa tgggcccacg ggcggcggcc tgggcgaaga 4620tttccatgca ttcgtccata atgatggcaa tgggcccacg ggcggcggcc tgggcgaaga 4620
tatttctggg atcactaacg tcatagttgt gttccaggat gagatcgtca taggccattt 4680tatttctggg atcactaacg tcatagttgt gttccaggat gagatcgtca taggccattt 4680
ttacaaagcg cgggcggagg gtgccagact gcggtataat ggttccatcc ggcccagggg 4740ttacaaagcg cgggcggagg gtgccagact gcggtataat ggttccatcc ggcccagggg 4740
cgtagttacc ctcacagatt tgcatttccc acgctttgag ttcagatggg gggatcatgt 4800cgtagttacc ctcacagatt tgcatttccc acgctttgag ttcagatggg gggatcatgt 4800
ctacctgcgg ggcgatgaag aaaacggttt ccggggtagg ggagatcagc tgggaagaaa 4860ctacctgcgg ggcgatgaag aaaacggttt ccggggtagg ggagatcagc tgggaagaaa 4860
gcaggttcct gagcagctgc gacttaccgc agccggtggg cccgtaaatc acacctatta 4920gcaggttcct gagcagctgc gacttaccgc agccggtggg cccgtaaatc acacctatta 4920
ccgggtgcaa ctggtagtta agagagctgc agctgccgtc atccctgagc aggggggcca 4980ccgggtgcaa ctggtagtta agagagctgc agctgccgtc atccctgagc aggggggcca 4980
cttcgttaag catgtccctg actcgcatgt tttccctgac caaatccgcc agaaggcgct 5040cttcgttaag catgtccctg actcgcatgt tttccctgac caaatccgcc agaaggcgct 5040
cgccgcccag cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt ttgagaccgt 5100cgccgcccag cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt ttgagaccgt 5100
ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc cacagctcgg 5160ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc cacagctcgg 5160
tcacctgctc tacggcatct cgatccagca tatctcctcg tttcgcgggt tggggcggct 5220tcacctgctc tacggcatct cgatccagca tatctcctcg tttcgcgggt tggggcggct 5220
ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt ctttccacgg 5280ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt ctttccacgg 5280
gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc 5340gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc 5340
gctggccagg gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc 5400gctggccagg gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc 5400
ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag tccagcccct ccgcggcgtg 5460ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag tccagcccct ccgcggcgtg 5460
gcccttggcg cgcagcttgc ccttggagga ggcgccgcac gaggggcagt gcagactttt 5520gcccttggcg cgcagcttgc ccttggagga ggcgccgcac gaggggcagt gcagactttt 5520
gagggcgtag agcttgggcg cgagaaatac cgattccggg gagtaggcat ccgcgccgca 5580gagggcgtag agcttgggcg cgagaaatac cgattccggg gagtaggcat ccgcgccgca 5580
ggccccgcag acggtctcgc attccacgag ccaggtgagc tctggccgtt cggggtcaaa 5640ggccccgcag acggtctcgc attccacgag ccaggtgagc tctggccgtt cggggtcaaa 5640
aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg 5700aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg 5700
tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga gaggcctgtc 5760tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga gaggcctgtc 5760
ctcgagcggt gttccgcggt cctcctcgta tagaaactcg gaccactctg agacaaaggc 5820ctcgagcggt gttccgcggt cctcctcgta tagaaactcg gaccactctg agacaaaggc 5820
tcgcgtccag gccagcacga aggaggctaa gtgggagggg tagcggtcgt tgtccactag 5880tcgcgtccag gccagcacga aggaggctaa gtgggagggg tagcggtcgt tgtccactag 5880
ggggtccact cgctccaggg tgtgaagaca catgtcgccc tcttcggcat caaggaaggt 5940ggggtccact cgctccaggg tgtgaagaca catgtcgccc tcttcggcat caaggaaggt 5940
gattggtttg taggtgtagg ccacgtgacc gggtgttcct gaaggggggc tataaaaggg 6000gattggtttg taggtgtagg ccacgtgacc gggtgttcct gaaggggggc tataaaaggg 6000
ggtgggggcg cgttcgtcct cactctcttc cgcatcgctg tctgcgaggg ccagctgttg 6060ggtgggggcg cgttcgtcct cactctcttc cgcatcgctg tctgcgaggg ccagctgttg 6060
gggtgagtac tccctctgaa aagcgggcat gacttctgcg ctaagattgt cagtttccaa 6120gggtgagtac tccctctgaa aagcgggcat gacttctgcg ctaagattgt cagtttccaa 6120
aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg tggccgcatc 6180aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg tggccgcatc 6180
catctggtca gaaaagacaa tctttttgtt gtcaagcttg gtggcaaacg acccgtagag 6240catctggtca gaaaagacaa tctttttgtt gtcaagcttg gtggcaaacg acccgtagag 6240
ggcgttggac agcaacttgg cgatggagcg cagggtttgg tttttgtcgc gatcggcgcg 6300ggcgttggac agcaacttgg cgatggagcg cagggtttgg tttttgtcgc gatcggcgcg 6300
ctccttggcc gcgatgttta gctgcacgta ttcgcgcgca acgcaccgcc attcgggaaa 6360ctccttggcc gcgatgttta gctgcacgta ttcgcgcgca acgcaccgcc attcgggaaa 6360
gacggtggtg cgctcgtcgg gcaccaggtg cacgcgccaa ccgcggttgt gcagggtgac 6420gacggtggtg cgctcgtcgg gcaccaggtg cacgcgccaa ccgcggttgt gcagggtgac 6420
aaggtcaacg ctggtggcta cctctccgcg taggcgctcg ttggtccagc agaggcggcc 6480aaggtcaacg ctggtggcta cctctccgcg taggcgctcg ttggtccagc agaggcggcc 6480
gcccttgcgc gagcagaatg gcggtagggg gtctagctgc gtctcgtccg gggggtctgc 6540gcccttgcgc gagcagaatg gcggtagggg gtctagctgc gtctcgtccg gggggtctgc 6540
gtccacggta aagaccccgg gcagcaggcg cgcgtcgaag tagtctatct tgcatccttg 6600gtccacggta aagaccccgg gcagcaggcg cgcgtcgaag tagtctatct tgcatccttg 6600
caagtctagc gcctgctgcc atgcgcgggc ggcaagcgcg cgctcgtatg ggttgagtgg 6660caagtctagc gcctgctgcc atgcgcgggc ggcaagcgcg cgctcgtatg ggttgagtgg 6660
gggaccccat ggcatggggt gggtgagcgc ggaggcgtac atgccgcaaa tgtcgtaaac 6720gggaccccat ggcatggggt gggtgagcgc ggaggcgtac atgccgcaaa tgtcgtaaac 6720
gtagaggggc tctctgagta ttccaagata tgtagggtag catcttccac cgcggatgct 6780gtagaggggc tctctgagta ttccaagata tgtagggtag catcttccac cgcggatgct 6780
ggcgcgcacg taatcgtata gttcgtgcga gggagcgagg aggtcgggac cgaggttgct 6840ggcgcgcacg taatcgtata gttcgtgcga gggagcgagg aggtcgggac cgaggttgct 6840
acgggcgggc tgctctgctc ggaagactat ctgcctgaag atggcatgtg agttggatga 6900acgggcgggc tgctctgctc ggaagactat ctgcctgaag atggcatgtg agttggatga 6900
tatggttgga cgctggaaga cgttgaagct ggcgtctgtg agacctaccg cgtcacgcac 6960tatggttgga cgctggaaga cgttgaagct ggcgtctgtg agacctaccg cgtcacgcac 6960
gaaggaggcg taggagtcgc gcagcttgtt gaccagctcg gcggtgacct gcacgtctag 7020gaaggaggcg taggagtcgc gcagcttgtt gaccagctcg gcggtgacct gcacgtctag 7020
ggcgcagtag tccagggttt ccttgatgat gtcatactta tcctgtccct tttttttcca 7080ggcgcagtag tccagggttt ccttgatgat gtcatactta tcctgtccct tttttttcca 7080
cagctcgcgg ttgaggacaa actcttcgcg gtctttccag tactcttgga tcggaaaccc 7140cagctcgcgg ttgaggacaa actcttcgcg gtctttccag tactcttgga tcggaaaccc 7140
gtcggcctcc gaacggtaag agcctagcat gtagaactgg ttgacggcct ggtaggcgca 7200gtcggcctcc gaacggtaag agcctagcat gtagaactgg ttgacggcct ggtaggcgca 7200
gcatcccttt tctacgggta gcgcgtatgc ctgcgcggcc ttccggagcg aggtgtgggt 7260gcatcccttt tctacgggta gcgcgtatgc ctgcgcggcc ttccggagcg aggtgtgggt 7260
gagcgcaaag gtgtccctga ccatgacttt gaggtactgg tatttgaagt cagtgtcgtc 7320gagcgcaaag gtgtccctga ccatgacttt gaggtactgg tatttgaagt cagtgtcgtc 7320
gcatccgccc tgctcccaga gcaaaaagtc cgtgcgcttt ttggaacgcg gatttggcag 7380gcatccgccc tgctcccaga gcaaaaagtc cgtgcgcttt ttggaacgcg gatttggcag 7380
ggcgaaggtg acatcgttga agagtatctt tcccgcgcga ggcataaagt tgcgtgtgat 7440ggcgaaggtg acatcgttga agagtatctt tcccgcgcga ggcataaagt tgcgtgtgat 7440
gcggaagggt cccggcacct cggaacggtt gttaattacc tgggcggcga gcacgatctc 7500gcggaagggt cccggcacct cggaacggtt gttaattacc tgggcggcga gcacgatctc 7500
gtcaaagccg ttgatgttgt ggcccacaat gtaaagttcc aagaagcgcg ggatgccctt 7560gtcaaagccg ttgatgttgt ggcccacaat gtaaagttcc aagaagcgcg ggatgccctt 7560
gatggaaggc aattttttaa gttcctcgta ggtgagctct tcaggggagc tgagcccgtg 7620gatggaaggc aattttttaa gttcctcgta ggtgagctct tcaggggagc tgagcccgtg 7620
ctctgaaagg gcccagtctg caagatgagg gttggaagcg acgaatgagc tccacaggtc 7680ctctgaaagg gcccagtctg caagatgagg gttggaagcg acgaatgagc tccacaggtc 7680
acgggccatt agcatttgca ggtggtcgcg aaaggtccta aactggcgac ctatggccat 7740acgggccatt agcatttgca ggtggtcgcg aaaggtccta aactggcgac ctatggccat 7740
tttttctggg gtgatgcagt agaaggtaag cgggtcttgt tcccagcggt cccatccaag 7800ttttttctggg gtgatgcagt agaaggtaag cgggtcttgt tcccagcggt cccatccaag 7800
gttcgcggct aggtctcgcg cggcagtcac tagaggctca tctccgccga acttcatgac 7860gttcgcggct aggtctcgcg cggcagtcac tagaggctca tctccgccga acttcatgac 7860
cagcatgaag ggcacgagct gcttcccaaa ggcccccatc caagtatagg tctctacatc 7920cagcatgaag ggcacgagct gcttcccaaa ggcccccatc caagtatagg tctctacatc 7920
gtaggtgaca aagagacgct cggtgcgagg atgcgagccg atcgggaaga actggatctc 7980gtaggtgaca aagagacgct cggtgcgagg atgcgagccg atcgggaaga actggatctc 7980
ccgccaccaa ttggaggagt ggctattgat gtggtgaaag tagaagtccc tgcgacgggc 8040ccgccaccaa ttggaggagt ggctattgat gtggtgaaag tagaagtccc tgcgacgggc 8040
cgaacactcg tgctggcttt tgtaaaaacg tgcgcagtac tggcagcggt gcacgggctg 8100cgaacactcg tgctggcttt tgtaaaaacg tgcgcagtac tggcagcggt gcacgggctg 8100
tacatcctgc acgaggttga cctgacgacc gcgcacaagg aagcagagtg ggaatttgag 8160tacatcctgc acgaggttga cctgacgacc gcgcacaagg aagcagagtg ggaatttgag 8160
cccctcgcct ggcgggtttg gctggtggtc ttctacttcg gctgcttgtc cttgaccgtc 8220cccctcgcct ggcgggtttg gctggtggtc ttctacttcg gctgcttgtc cttgaccgtc 8220
tggctgctcg aggggagtta cggtggatcg gaccaccacg ccgcgcgagc ccaaagtcca 8280tggctgctcg aggggagtta cggtggatcg gaccaccacg ccgcgcgagc ccaaagtcca 8280
gatgtccgcg cgcggcggtc ggagcttgat gacaacatcg cgcagatggg agctgtccat 8340gatgtccgcg cgcggcggtc ggagcttgat gacaacatcg cgcagatggg agctgtccat 8340
ggtctggagc tcccgcggcg tcaggtcagg cgggagctcc tgcaggttta cctcgcatag 8400ggtctggagc tcccgcggcg tcaggtcagg cgggagctcc tgcaggttta cctcgcatag 8400
acgggtcagg gcgcgggcta gatccaggtg atacctaatt tccaggggct ggttggtggc 8460acgggtcagg gcgcgggcta gatccaggtg atacctaatt tccaggggct ggttggtggc 8460
ggcgtcgatg gcttgcaaga ggccgcatcc ccgcggcgcg actacggtac cgcgcggcgg 8520ggcgtcgatg gcttgcaaga ggccgcatcc ccgcggcgcg actacggtac cgcgcggcgg 8520
gcggtgggcc gcgggggtgt ccttggatga tgcatctaaa agcggtgacg cgggcgagcc 8580gcggtgggcc gcgggggtgt ccttggatga tgcatctaaa agcggtgacg cgggcgagcc 8580
cccggaggta gggggggctc cggacccgcc gggagagggg gcaggggcac gtcggcgccg 8640cccggaggta gggggggctc cggacccgcc gggagagggg gcaggggcac gtcggcgccg 8640
cgcgcgggca ggagctggtg ctgcgcgcgt aggttgctgg cgaacgcgac gacgcggcgg 8700cgcgcgggca ggagctggtg ctgcgcgcgt aggttgctgg cgaacgcgac gacgcggcgg 8700
ttgatctcct gaatctggcg cctctgcgtg aagacgacgg gcccggtgag cttgagcctg 8760ttgatctcct gaatctggcg cctctgcgtg aagacgacgg gcccggtgag cttgagcctg 8760
aaagagagtt cgacagaatc aatttcggtg tcgttgacgg cggcctggcg caaaatctcc 8820aaagagagtt cgacagaatc aatttcggtg tcgttgacgg cggcctggcg caaaatctcc 8820
tgcacgtctc ctgagttgtc ttgataggcg atctcggcca tgaactgctc gatctcttcc 8880tgcacgtctc ctgagttgtc ttgataggcg atctcggcca tgaactgctc gatctcttcc 8880
tcctggagat ctccgcgtcc ggctcgctcc acggtggcgg cgaggtcgtt ggaaatgcgg 8940tcctggagat ctccgcgtcc ggctcgctcc acggtggcgg cgaggtcgtt ggaaatgcgg 8940
gccatgagct gcgagaaggc gttgaggcct ccctcgttcc agacgcggct gtagaccacg 9000gccatgagct gcgagaaggc gttgaggcct ccctcgttcc agacgcggct gtagaccacg 9000
cccccttcgg catcgcgggc gcgcatgacc acctgcgcga gattgagctc cacgtgccgg 9060cccccttcgg catcgcgggc gcgcatgacc acctgcgcga gattgagctc cacgtgccgg 9060
gcgaagacgg cgtagtttcg caggcgctga aagaggtagt tgagggtggt ggcggtgtgt 9120gcgaagacgg cgtagtttcg caggcgctga aagaggtagt tgagggtggt ggcggtgtgt 9120
tctgccacga agaagtacat aacccagcgt cgcaacgtgg attcgttgat atcccccaag 9180tctgccacga agaagtacat aacccagcgt cgcaacgtgg attcgttgat atcccccaag 9180
gcctcaaggc gctccatggc ctcgtagaag tccacggcga agttgaaaaa ctgggagttg 9240gcctcaaggc gctccatggc ctcgtagaag tccacggcga agttgaaaaa ctgggagttg 9240
cgcgccgaca cggttaactc ctcctccaga agacggatga gctcggcgac agtgtcgcgc 9300cgcgccgaca cggttaactc ctcctccaga agacggatga gctcggcgac agtgtcgcgc 9300
acctcgcgct caaaggctac aggggcctct tcttcttctt caatctcctc ttccataagg 9360acctcgcgct caaaggctac aggggcctct tcttcttctt caatctcctc ttccataagg 9360
gcctcccctt cttcttcttc tggcggcggt gggggagggg ggacacggcg gcgacgacgg 9420gcctcccctt cttcttcttc tggcggcggt gggggagggg ggacacggcg gcgacgacgg 9420
cgcaccggga ggcggtcgac aaagcgctcg atcatctccc cgcggcgacg gcgcatggtc 9480cgcaccggga ggcggtcgac aaagcgctcg atcatctccc cgcggcgacg gcgcatggtc 9480
tcggtgacgg cgcggccgtt ctcgcggggg cgcagttgga agacgccgcc cgtcatgtcc 9540tcggtgacgg cgcggccgtt ctcgcggggg cgcagttgga agacgccgcc cgtcatgtcc 9540
cggttatggg ttggcggggg gctgccatgc ggcagggata cggcgctaac gatgcatctc 9600cggttatggg ttggcggggg gctgccatgc ggcagggata cggcgctaac gatgcatctc 9600
aacaattgtt gtgtaggtac tccgccgccg agggacctga gcgagtccgc atcgaccgga 9660aacaattgtt gtgtaggtac tccgccgccg agggacctga gcgagtccgc atcgaccgga 9660
tcggaaaacc tctcgagaaa ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc 9720tcggaaaacc tctcgagaaa ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc 9720
gtggcgggcg gcagcgggcg gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg 9780gtggcgggcg gcagcgggcg gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg 9780
taattaaagt aggcggtctt gagacggcgg atggtcgaca gaagcaccat gtccttgggt 9840taattaaagt aggcggtctt gagacggcgg atggtcgaca gaagcaccat gtccttgggt 9840
ccggcctgct gaatgcgcag gcggtcggcc atgccccagg cttcgttttg acatcggcgc 9900ccggcctgct gaatgcgcag gcggtcggcc atgccccagg cttcgttttg acatcggcgc 9900
aggtctttgt agtagtcttg catgagcctt tctaccggca cttcttcttc tccttcctct 9960aggtctttgt agtagtcttg catgagcctt tctaccggca cttcttcttc tccttcctct 9960
tgtcctgcat ctcttgcatc tatcgctgcg gcggcggcgg agtttggccg taggtggcgc 10020tgtcctgcat ctcttgcatc tatcgctgcg gcggcggcgg agtttggccg taggtggcgc 10020
cctcttcctc ccatgcgtgt gaccccgaag cccctcatcg gctgaagcag ggctaggtcg 10080cctcttcctc ccatgcgtgt gaccccgaag cccctcatcg gctgaagcag ggctaggtcg 10080
gcgacaacgc gctcggctaa tatggcctgc tgcacctgcg tgagggtaga ctggaagtca 10140gcgacaacgc gctcggctaa tatggcctgc tgcacctgcg tgagggtaga ctggaagtca 10140
tccatgtcca caaagcggtg gtatgcgccc gtgttgatgg tgtaagtgca gttggccata 10200tccatgtcca caaagcggtg gtatgcgccc gtgttgatgg tgtaagtgca gttggccata 10200
acggaccagt taacggtctg gtgacccggc tgcgagagct cggtgtacct gagacgcgag 10260acggaccagt taacggtctg gtgacccggc tgcgagagct cggtgtacct gagacgcgag 10260
taagccctcg agtcaaatac gtagtcgttg caagtccgca ccaggtactg gtatcccacc 10320taagccctcg agtcaaatac gtagtcgttg caagtccgca ccaggtactg gtatcccacc 10320
aaaaagtgcg gcggcggctg gcggtagagg ggccagcgta gggtggccgg ggctccgggg 10380aaaaagtgcg gcggcggctg gcggtagagg ggccagcgta gggtggccgg ggctccgggg 10380
gcgagatctt ccaacataag gcgatgatat ccgtagatgt acctggacat ccaggtgatg 10440gcgagatctt ccaacataag gcgatgatat ccgtagatgt acctggacat ccaggtgatg 10440
ccggcggcgg tggtggaggc gcgcggaaag tcgcggacgc ggttccagat gttgcgcagc 10500ccggcggcgg tggtggaggc gcgcggaaag tcgcggacgc ggttccagat gttgcgcagc 10500
ggcaaaaagt gctccatggt cgggacgctc tggccggtca ggcgcgcgca atcgttgacg 10560ggcaaaaagt gctccatggt cgggacgctc tggccggtca ggcgcgcgca atcgttgacg 10560
ctctagaccg tgcaaaagga gagcctgtaa gcgggcactc ttccgtggtc tggtggataa 10620ctctagaccg tgcaaaagga gagcctgtaa gcgggcactc ttccgtggtc tggtggataa 10620
attcgcaagg gtatcatggc ggacgaccgg ggttcgagcc ccgtatccgg ccgtccgccg 10680attcgcaagg gtatcatggc ggacgaccgg ggttcgagcc ccgtatccgg ccgtccgccg 10680
tgatccatgc ggttaccgcc cgcgtgtcga acccaggtgt gcgacgtcag acaacggggg 10740tgatccatgc ggttaccgcc cgcgtgtcga acccaggtgt gcgacgtcag acaacggggg 10740
agtgctcctt ttggcttcct tccaggcgcg gcggctgctg cgctagcttt tttggccact 10800agtgctcctt ttggcttcct tccaggcgcg gcggctgctg cgctagcttt tttggccact 10800
ggccgcgcgc agcgtaagcg gttaggctgg aaagcgaaag cattaagtgg ctcgctccct 10860ggccgcgcgc agcgtaagcg gttaggctgg aaagcgaaag cattaagtgg ctcgctccct 10860
gtagccggag ggttattttc caagggttga gtcgcgggac ccccggttcg agtctcggac 10920gtagccggag ggttattttc caagggttga gtcgcgggac ccccggttcg agtctcggac 10920
cggccggact gcggcgaacg ggggtttgcc tccccgtcat gcaagacccc gcttgcaaat 10980cggccggact gcggcgaacg ggggtttgcc tccccgtcat gcaagacccc gcttgcaaat 10980
tcctccggaa acagggacga gccccttttt tgcttttccc agatgcatcc ggtgctgcgg 11040tcctccggaa acagggacga gccccttttt tgcttttccc agatgcatcc ggtgctgcgg 11040
cagatgcgcc cccctcctca gcagcggcaa gagcaagagc agcggcagac atgcagggca 11100cagatgcgcc cccctcctca gcagcggcaa gagcaagagc agcggcagac atgcagggca 11100
ccctcccctc ctcctaccgc gtcaggaggg gcgacatccg cggttgacgc ggcagcagat 11160ccctcccctc ctcctaccgc gtcaggaggg gcgacatccg cggttgacgc ggcagcagat 11160
ggtgattacg aacccccgcg gcgccgggcc cggcactacc tggacttgga ggagggcgag 11220ggtgattacg aacccccgcg gcgccgggcc cggcactacc tggacttgga ggagggcgag 11220
ggcctggcgc ggctaggagc gccctctcct gagcggtacc caagggtgca gctgaagcgt 11280ggcctggcgc ggctaggagc gccctctcct gagcggtacc caagggtgca gctgaagcgt 11280
gatacgcgtg aggcgtacgt gccgcggcag aacctgtttc gcgaccgcga gggagaggag 11340gatacgcgtg aggcgtacgt gccgcggcag aacctgtttc gcgaccgcga gggagaggag 11340
cccgaggaga tgcgggatcg aaagttccac gcagggcgcg agctgcggca tggcctgaat 11400cccgaggaga tgcgggatcg aaagttccac gcagggcgcg agctgcggca tggcctgaat 11400
cgcgagcggt tgctgcgcga ggaggacttt gagcccgacg cgcgaaccgg gattagtccc 11460cgcgagcggt tgctgcgcga ggaggacttt gagcccgacg cgcgaaccgg gattagtccc 11460
gcgcgcgcac acgtggcggc cgccgacctg gtaaccgcat acgagcagac ggtgaaccag 11520gcgcgcgcac acgtggcggc cgccgacctg gtaaccgcat acgagcagac ggtgaaccag 11520
gagattaact ttcaaaaaag ctttaacaac cacgtgcgta cgcttgtggc gcgcgaggag 11580gagattaact ttcaaaaaag ctttaacaac cacgtgcgta cgcttgtggc gcgcgaggag 11580
gtggctatag gactgatgca tctgtgggac tttgtaagcg cgctggagca aaacccaaat 11640gtggctatag gactgatgca tctgtgggac tttgtaagcg cgctggagca aaacccaaat 11640
agcaagccgc tcatggcgca gctgttcctt atagtgcagc acagcaggga caacgaggca 11700agcaagccgc tcatggcgca gctgttcctt atagtgcagc acagcaggga caacgaggca 11700
ttcagggatg cgctgctaaa catagtagag cccgagggcc gctggctgct cgatttgata 11760ttcagggatg cgctgctaaa catagtagag cccgagggcc gctggctgct cgatttgata 11760
aacatcctgc agagcatagt ggtgcaggag cgcagcttga gcctggctga caaggtggcc 11820aacatcctgc agagcatagt ggtgcaggag cgcagcttga gcctggctga caaggtggcc 11820
gccatcaact attccatgct tagcctgggc aagttttacg cccgcaagat ataccatacc 11880gccatcaact attccatgct tagcctgggc aagttttacg cccgcaagat ataccatacc 11880
ccttacgttc ccatagacaa ggaggtaaag atcgaggggt tctacatgcg catggcgctg 11940ccttacgttc ccatagacaa ggaggtaaag atcgaggggt tctacatgcg catggcgctg 11940
aaggtgctta ccttgagcga cgacctgggc gtttatcgca acgagcgcat ccacaaggcc 12000aaggtgctta ccttgagcga cgacctgggc gtttatcgca acgagcgcat ccacaaggcc 12000
gtgagcgtga gccggcggcg cgagctcagc gaccgcgagc tgatgcacag cctgcaaagg 12060gtgagcgtga gccggcggcg cgagctcagc gaccgcgagc tgatgcacag cctgcaaagg 12060
gccctggctg gcacgggcag cggcgataga gaggccgagt cctactttga cgcgggcgct 12120gccctggctg gcacgggcag cggcgataga gaggccgagt cctactttga cgcgggcgct 12120
gacctgcgct gggccccaag ccgacgcgcc ctggaggcag ctggggccgg acctgggctg 12180gacctgcgct gggccccaag ccgacgcgcc ctggaggcag ctggggccgg acctgggctg 12180
gcggtggcac ccgcgcgcgc tggcaacgtc ggcggcgtgg aggaatatga cgaggacgat 12240gcggtggcac ccgcgcgcgc tggcaacgtc ggcggcgtgg aggaatatga cgaggacgat 12240
gagtacgagc cagaggacgg cgagtactaa gcggtgatgt ttctgatcag atgatgcaag 12300gagtacgagc cagaggacgg cgagtactaa gcggtgatgt ttctgatcag atgatgcaag 12300
acgcaacgga cccggcggtg cgggcggcgc tgcagagcca gccgtccggc cttaactcca 12360acgcaacgga cccggcggtg cgggcggcgc tgcagagcca gccgtccggc cttaactcca 12360
cggacgactg gcgccaggtc atggaccgca tcatgtcgct gactgcgcgc aatcctgacg 12420cggacgactg gcgccaggtc atggaccgca tcatgtcgct gactgcgcgc aatcctgacg 12420
cgttccggca gcagccgcag gccaaccggc tctccgcaat tctggaagcg gtggtcccgg 12480cgttccggca gcagccgcag gccaaccggc tctccgcaat tctggaagcg gtggtcccgg 12480
cgcgcgcaaa ccccacgcac gagaaggtgc tggcgatcgt aaacgcgctg gccgaaaaca 12540cgcgcgcaaa ccccacgcac gagaaggtgc tggcgatcgt aaacgcgctg gccgaaaaca 12540
gggccatccg gcccgacgag gccggcctgg tctacgacgc gctgcttcag cgcgtggctc 12600gggccatccg gcccgacgag gccggcctgg tctacgacgc gctgcttcag cgcgtggctc 12600
gttacaacag cggcaacgtg cagaccaacc tggaccggct ggtgggggat gtgcgcgagg 12660gttacaacag cggcaacgtg cagaccaacc tggaccggct ggtgggggat gtgcgcgagg 12660
ccgtggcgca gcgtgagcgc gcgcagcagc agggcaacct gggctccatg gttgcactaa 12720ccgtggcgca gcgtgagcgc gcgcagcagc agggcaacct gggctccatg gttgcactaa 12720
acgccttcct gagtacacag cccgccaacg tgccgcgggg acaggaggac tacaccaact 12780acgccttcct gagtacacag cccgccaacg tgccgcgggg acaggaggac tacaccaact 12780
ttgtgagcgc actgcggcta atggtgactg agacaccgca aagtgaggtg taccagtctg 12840ttgtgagcgc actgcggcta atggtgactg agacaccgca aagtgaggtg taccagtctg 12840
ggccagacta ttttttccag accagtagac aaggcctgca gaccgtaaac ctgagccagg 12900ggccagacta ttttttccag accagtagac aaggcctgca gaccgtaaac ctgagccagg 12900
ctttcaaaaa cttgcagggg ctgtgggggg tgcgggctcc cacaggcgac cgcgcgaccg 12960ctttcaaaaa cttgcagggg ctgtgggggg tgcgggctcc cacaggcgac cgcgcgaccg 12960
tgtctagctt gctgacgccc aactcgcgcc tgttgctgct gctaatagcg cccttcacgg 13020tgtctagctt gctgacgccc aactcgcgcc tgttgctgct gctaatagcg cccttcacgg 13020
acagtggcag cgtgtcccgg gacacatacc taggtcactt gctgacactg taccgcgagg 13080acagtggcag cgtgtcccgg gacacatacc taggtcactt gctgacactg taccgcgagg 13080
ccataggtca ggcgcatgtg gacgagcata ctttccagga gattacaagt gtcagccgcg 13140ccataggtca ggcgcatgtg gacgagcata ctttccagga gattacaagt gtcagccgcg 13140
cgctggggca ggaggacacg ggcagcctgg aggcaaccct aaactacctg ctgaccaacc 13200cgctggggca ggaggacacg ggcagcctgg aggcaaccct aaactacctg ctgaccaacc 13200
ggcggcagaa gatcccctcg ttgcacagtt taaacagcga ggaggagcgc attttgcgct 13260ggcggcagaa gatcccctcg ttgcacagtt taaacagcga ggaggagcgc attttgcgct 13260
acgtgcagca gagcgtgagc cttaacctga tgcgcgacgg ggtaacgccc agcgtggcgc 13320acgtgcagca gagcgtgagc cttaacctga tgcgcgacgg ggtaacgccc agcgtggcgc 13320
tggacatgac cgcgcgcaac atggaaccgg gcatgtatgc ctcaaaccgg ccgtttatca 13380tggacatgac cgcgcgcaac atggaaccgg gcatgtatgc ctcaaaccgg ccgtttatca 13380
accgcctaat ggactacttg catcgcgcgg ccgccgtgaa ccccgagtat ttcaccaatg 13440accgcctaat ggactacttg catcgcgcgg ccgccgtgaa ccccgagtat ttcaccaatg 13440
ccatcttgaa cccgcactgg ctaccgcccc ctggtttcta caccggggga ttcgaggtgc 13500ccatcttgaa cccgcactgg ctaccgcccc ctggtttcta caccggggga ttcgaggtgc 13500
ccgagggtaa cgatggattc ctctgggacg acatagacga cagcgtgttt tccccgcaac 13560ccgagggtaa cgatggattc ctctgggacg acatagacga cagcgtgttt tccccgcaac 13560
cgcagaccct gctagagttg caacagcgcg agcaggcaga ggcggcgctg cgaaaggaaa 13620cgcagaccct gctagagttg caacagcgcg agcaggcaga ggcggcgctg cgaaaggaaa 13620
gcttccgcag gccaagcagc ttgtccgatc taggcgctgc ggccccgcgg tcagatgcta 13680gcttccgcag gccaagcagc ttgtccgatc taggcgctgc ggccccgcgg tcagatgcta 13680
gtagcccatt tccaagcttg atagggtctc ttaccagcac tcgcaccacc cgcccgcgcc 13740gtagcccatt tccaagcttg atagggtctc ttaccagcac tcgcaccacc cgcccgcgcc 13740
tgctgggcga ggaggagtac ctaaacaact cgctgctgca gccgcagcgc gaaaaaaacc 13800tgctgggcga ggaggagtac ctaaacaact cgctgctgca gccgcagcgc gaaaaaaacc 13800
tgcctccggc atttcccaac aacgggatag agagcctagt ggacaagatg agtagatgga 13860tgcctccggc atttcccaac aacgggatag agagcctagt ggacaagatg agtagatgga 13860
agacgtacgc gcaggagcac agggacgtgc caggcccgcg cccgcccacc cgtcgtcaaa 13920agacgtacgc gcaggagcac agggacgtgc caggcccgcg cccgcccacc cgtcgtcaaa 13920
ggcacgaccg tcagcggggt ctggtgtggg aggacgatga ctcggcagac gacagcagcg 13980ggcacgaccg tcagcggggt ctggtgtggg aggacgatga ctcggcagac gacagcagcg 13980
tcctggattt gggagggagt ggcaacccgt ttgcgcacct tcgccccagg ctggggagaa 14040tcctggattt gggagggagt ggcaacccgt ttgcgcacct tcgccccagg ctggggagaa 14040
tgttttaaaa aaaaaaaagc atgatgcaaa ataaaaaact caccaaggcc atggcaccga 14100tgttttaaaa aaaaaaaagc atgatgcaaa ataaaaaact caccaaggcc atggcaccga 14100
gcgttggttt tcttgtattc cccttagtat gcggcgcgcg gcgatgtatg aggaaggtcc 14160gcgttggttt tcttgtattc cccttagtat gcggcgcgcg gcgatgtatg aggaaggtcc 14160
tcctccctcc tacgagagtg tggtgagcgc ggcgccagtg gcggcggcgc tgggttctcc 14220tcctccctcc tacgagagtg tggtgagcgc ggcgccagtg gcggcggcgc tgggttctcc 14220
cttcgatgct cccctggacc cgccgtttgt gcctccgcgg tacctgcggc ctaccggggg 14280cttcgatgct cccctggacc cgccgtttgt gcctccgcgg tacctgcggc ctaccggggg 14280
gagaaacagc atccgttact ctgagttggc acccctattc gacaccaccc gtgtgtacct 14340gagaaacagc atccgttact ctgagttggc acccctattc gacaccaccc gtgtgtacct 14340
ggtggacaac aagtcaacgg atgtggcatc cctgaactac cagaacgacc acagcaactt 14400ggtggacaac aagtcaacgg atgtggcatc cctgaactac cagaacgacc acagcaactt 14400
tctgaccacg gtcattcaaa acaatgacta cagcccgggg gaggcaagca cacagaccat 14460tctgaccacg gtcattcaaa acaatgacta cagcccgggg gaggcaagca cacagaccat 14460
caatcttgac gaccggtcgc actggggcgg cgacctgaaa accatcctgc ataccaacat 14520caatcttgac gaccggtcgc actggggcgg cgacctgaaa accatcctgc ataccaacat 14520
gccaaatgtg aacgagttca tgtttaccaa taagtttaag gcgcgggtga tggtgtcgcg 14580gccaaatgtg aacgagttca tgtttaccaa taagtttaag gcgcgggtga tggtgtcgcg 14580
cttgcctact aaggacaatc aggtggagct gaaatacgag tgggtggagt tcacgctgcc 14640cttgcctact aaggacaatc aggtggagct gaaatacgag tgggtggagt tcacgctgcc 14640
cgagggcaac tactccgaga ccatgaccat agaccttatg aacaacgcga tcgtggagca 14700cgagggcaac tactccgaga ccatgaccat agaccttatg aacaacgcga tcgtggagca 14700
ctacttgaaa gtgggcagac agaacggggt tctggaaagc gacatcgggg taaagtttga 14760ctacttgaaa gtgggcagac agaacggggt tctggaaagc gacatcgggg taaagtttga 14760
cacccgcaac ttcagactgg ggtttgaccc cgtcactggt cttgtcatgc ctggggtata 14820cacccgcaac ttcagactgg ggtttgaccc cgtcactggt cttgtcatgc ctggggtata 14820
tacaaacgaa gccttccatc cagacatcat tttgctgcca ggatgcgggg tggacttcac 14880tacaaacgaa gccttccatc cagacatcat tttgctgcca ggatgcgggg tggacttcac 14880
ccacagccgc ctgagcaact tgttgggcat ccgcaagcgg caacccttcc aggagggctt 14940ccacagccgc ctgagcaact tgttgggcat ccgcaagcgg caacccttcc aggagggctt 14940
taggatcacc tacgatgatc tggagggtgg taacattccc gcactgttgg atgtggacgc 15000taggatcacc tacgatgatc tggagggtgg taacattccc gcactgttgg atgtggacgc 15000
ctaccaggcg agcttgaaag atgacaccga acagggcggg ggtggcgcag gcggcagcaa 15060ctaccaggcg agcttgaaag atgacaccga acagggcggg ggtggcgcag gcggcagcaa 15060
cagcagtggc agcggcgcgg aagagaactc caacgcggca gccgcggcaa tgcagccggt 15120cagcagtggc agcggcgcgg aagagaactc caacgcggca gccgcggcaa tgcagccggt 15120
ggaggacatg aacgatcatg ccattcgcgg cgacaccttt gccacacggg ctgaggagaa 15180ggaggacatg aacgatcatg ccattcgcgg cgacaccttt gccacacggg ctgaggagaa 15180
gcgcgctgag gccgaagcag cggccgaagc tgccgccccc gctgcgcaac ccgaggtcga 15240gcgcgctgag gccgaagcag cggccgaagc tgccgccccc gctgcgcaac ccgaggtcga 15240
gaagcctcag aagaaaccgg tgatcaaacc cctgacagag gacagcaaga aacgcagtta 15300gaagcctcag aagaaaccgg tgatcaaacc cctgacagag gacagcaaga aacgcagtta 15300
caacctaata agcaatgaca gcaccttcac ccagtaccgc agctggtacc ttgcatacaa 15360caacctaata agcaatgaca gcaccttcac ccagtaccgc agctggtacc ttgcatacaa 15360
ctacggcgac cctcagaccg gaatccgctc atggaccctg ctttgcactc ctgacgtaac 15420ctacggcgac cctcagaccg gaatccgctc atggaccctg ctttgcactc ctgacgtaac 15420
ctgcggctcg gagcaggtct actggtcgtt gccagacatg atgcaagacc ccgtgacctt 15480ctgcggctcg gagcaggtct actggtcgtt gccagacatg atgcaagacc ccgtgacctt 15480
ccgctccacg cgccagatca gcaactttcc ggtggtgggc gccgagctgt tgcccgtgca 15540ccgctccacg cgccagatca gcaactttcc ggtggtgggc gccgagctgt tgcccgtgca 15540
ctccaagagc ttctacaacg accaggccgt ctactcccaa ctcatccgcc agtttacctc 15600ctccaagagc ttctacaacg accaggccgt ctactcccaa ctcatccgcc agtttacctc 15600
tctgacccac gtgttcaatc gctttcccga gaaccagatt ttggcgcgcc cgccagcccc 15660tctgacccac gtgttcaatc gctttcccga gaaccagatt ttggcgcgcc cgccagcccc 15660
caccatcacc accgtcagtg aaaacgttcc tgctctcaca gatcacggga cgctaccgct 15720caccatcacc accgtcagtg aaaacgttcc tgctctcaca gatcacggga cgctaccgct 15720
gcgcaacagc atcggaggag tccagcgagt gaccattact gacgccagac gccgcacctg 15780gcgcaacagc atcggaggag tccagcgagt gaccattact gacgccagac gccgcacctg 15780
cccctacgtt tacaaggccc tgggcatagt ctcgccgcgc gtcctatcga gccgcacttt 15840cccctacgtt tacaaggccc tgggcatagt ctcgccgcgc gtcctatcga gccgcacttt 15840
ttgagcaagc atgtccatcc ttatatcgcc cagcaataac acaggctggg gcctgcgctt 15900ttgagcaagc atgtccatcc ttatatcgcc cagcaataac acaggctggg gcctgcgctt 15900
cccaagcaag atgtttggcg gggccaagaa gcgctccgac caacacccag tgcgcgtgcg 15960cccaagcaag atgtttggcg gggccaagaa gcgctccgac caacacccag tgcgcgtgcg 15960
cgggcactac cgcgcgccct ggggcgcgca caaacgcggc cgcactgggc gcaccaccgt 16020cgggcactac cgcgcgccct ggggcgcgca caaacgcggc cgcactgggc gcaccaccgt 16020
cgatgacgcc atcgacgcgg tggtggagga ggcgcgcaac tacacgccca cgccgccacc 16080cgatgacgcc atcgacgcgg tggtggagga ggcgcgcaac tacacgccca cgccgccacc 16080
agtgtccaca gtggacgcgg ccattcagac cgtggtgcgc ggagcccggc gctatgctaa 16140agtgtccaca gtggacgcgg ccattcagac cgtggtgcgc ggagcccggc gctatgctaa 16140
aatgaagaga cggcggaggc gcgtagcacg tcgccaccgc cgccgacccg gcactgccgc 16200aatgaagaga cggcggaggc gcgtagcacg tcgccaccgc cgccgacccg gcactgccgc 16200
ccaacgcgcg gcggcggccc tgcttaaccg cgcacgtcgc accggccgac gggcggccat 16260ccaacgcgcg gcggcggccc tgcttaaccg cgcacgtcgc accggccgac gggcggccat 16260
gcgggccgct cgaaggctgg ccgcgggtat tgtcactgtg ccccccaggt ccaggcgacg 16320gcgggccgct cgaaggctgg ccgcgggtat tgtcactgtg ccccccaggt ccaggcgacg 16320
agcggccgcc gcagcagccg cggccattag tgctatgact cagggtcgca ggggcaacgt 16380agcggccgcc gcagcagccg cggccattag tgctatgact cagggtcgca ggggcaacgt 16380
gtattgggtg cgcgactcgg ttagcggcct gcgcgtgccc gtgcgcaccc gccccccgcg 16440gtattgggtg cgcgactcgg ttagcggcct gcgcgtgccc gtgcgcaccc gccccccgcg 16440
caactagatt gcaagaaaaa actacttaga ctcgtactgt tgtatgtatc cagcggcggc 16500caactagatt gcaagaaaaa actacttaga ctcgtactgt tgtatgtatc cagcggcggc 16500
ggcgcgcaac gaagctatgt ccaagcgcaa aatcaaagaa gagatgctcc aggtcatcgc 16560ggcgcgcaac gaagctatgt ccaagcgcaa aatcaaagaa gagatgctcc aggtcatcgc 16560
gccggagatc tatggccccc cgaagaagga agagcaggat tacaagcccc gaaagctaaa 16620gccggagatc tatggccccc cgaagaagga agagcaggat tacaagcccc gaaagctaaa 16620
gcgggtcaaa aagaaaaaga aagatgatga tgatgaactt gacgacgagg tggaactgct 16680gcgggtcaaa aagaaaaaga aagatgatga tgatgaactt gacgacgagg tggaactgct 16680
gcacgctacc gcgcccaggc gacgggtaca gtggaaaggt cgacgcgtaa aacgtgtttt 16740gcacgctacc gcgcccaggc gacgggtaca gtggaaaggt cgacgcgtaa aacgtgtttt 16740
gcgacccggc accaccgtag tctttacgcc cggtgagcgc tccacccgca cctacaagcg 16800gcgacccggc accaccgtag tctttacgcc cggtgagcgc tccacccgca cctacaagcg 16800
cgtgtatgat gaggtgtacg gcgacgagga cctgcttgag caggccaacg agcgcctcgg 16860cgtgtatgat gaggtgtacg gcgacgagga cctgcttgag caggccaacg agcgcctcgg 16860
ggagtttgcc tacggaaagc ggcataagga catgctggcg ttgccgctgg acgagggcaa 16920ggagttttgcc tacggaaagc ggcataagga catgctggcg ttgccgctgg acgagggcaa 16920
cccaacacct agcctaaagc ccgtaacact gcagcaggtg ctgcccgcgc ttgcaccgtc 16980cccaacacct agcctaaagc ccgtaacact gcagcaggtg ctgcccgcgc ttgcaccgtc 16980
cgaagaaaag cgcggcctaa agcgcgagtc tggtgacttg gcacccaccg tgcagctgat 17040cgaagaaaag cgcggcctaa agcgcgagtc tggtgacttg gcacccaccg tgcagctgat 17040
ggtacccaag cgccagcgac tggaagatgt cttggaaaaa atgaccgtgg aacctgggct 17100ggtacccaag cgccagcgac tggaagatgt cttggaaaaa atgaccgtgg aacctgggct 17100
ggagcccgag gtccgcgtgc ggccaatcaa gcaggtggcg ccgggactgg gcgtgcagac 17160ggagcccgag gtccgcgtgc ggccaatcaa gcaggtggcg ccgggactgg gcgtgcagac 17160
cgtggacgtt cagataccca ctaccagtag caccagtatt gccaccgcca cagagggcat 17220cgtggacgtt cagataccca ctaccagtag caccagtatt gccaccgcca cagagggcat 17220
ggagacacaa acgtccccgg ttgcctcagc ggtggcggat gccgcggtgc aggcggtcgc 17280ggagacacaa acgtccccgg ttgcctcagc ggtggcggat gccgcggtgc aggcggtcgc 17280
tgcggccgcg tccaagacct ctacggaggt gcaaacggac ccgtggatgt ttcgcgtttc 17340tgcggccgcg tccaagacct ctacggaggt gcaaacggac ccgtggatgt ttcgcgtttc 17340
agccccccgg cgcccgcgcg gttcgaggaa gtacggcgcc gccagcgcgc tactgcccga 17400agccccccgg cgcccgcgcg gttcgaggaa gtacggcgcc gccagcgcgc tactgcccga 17400
atatgcccta catccttcca ttgcgcctac ccccggctat cgtggctaca cctaccgccc 17460atatgcccta catccttcca ttgcgcctac ccccggctat cgtggctaca cctaccgccc 17460
cagaagacga gcaactaccc gacgccgaac caccactgga acccgccgcc gccgtcgccg 17520cagaagacga gcaactaccc gacgccgaac caccactgga acccgccgcc gccgtcgccg 17520
tcgccagccc gtgctggccc cgatttccgt gcgcagggtg gctcgcgaag gaggcaggac 17580tcgccagccc gtgctggccc cgatttccgt gcgcagggtg gctcgcgaag gaggcaggac 17580
cctggtgctg ccaacagcgc gctaccaccc cagcatcgtt taaaagccgg tctttgtggt 17640cctggtgctg ccaacagcgc gctaccaccc cagcatcgtt taaaagccgg tctttgtggt 17640
tcttgcagat atggccctca cctgccgcct ccgtttcccg gtgccgggat tccgaggaag 17700tcttgcagat atggccctca cctgccgcct ccgtttcccg gtgccgggat tccgaggaag 17700
aatgcaccgt aggaggggca tggccggcca cggcctgacg ggcggcatgc gtcgtgcgca 17760aatgcaccgt aggaggggca tggccggcca cggcctgacg ggcggcatgc gtcgtgcgca 17760
ccaccggcgg cggcgcgcgt cgcaccgtcg catgcgcggc ggtatcctgc ccctccttat 17820ccaccggcgg cggcgcgcgt cgcaccgtcg catgcgcggc ggtatcctgc ccctccttat 17820
tccactgatc gccgcggcga ttggcgccgt gcccggaatt gcatccgtgg ccttgcaggc 17880tccactgatc gccgcggcga ttggcgccgt gcccggaatt gcatccgtgg ccttgcaggc 17880
gcagagacac tgattaaaaa caagttgcat gtggaaaaat caaaataaaa agtctggact 17940gcagagacac tgattaaaaa caagttgcat gtggaaaaat caaaataaaa agtctggact 17940
ctcacgctcg cttggtcctg taactatttt gtagaatgga agacatcaac tttgcgtctc 18000ctcacgctcg cttggtcctg taactatttt gtagaatgga agacatcaac tttgcgtctc 18000
tggccccgcg acacggctcg cgcccgttca tgggaaactg gcaagatatc ggcaccagca 18060tggccccgcg acacggctcg cgcccgttca tgggaaactg gcaagatatc ggcaccagca 18060
atatgagcgg tggcgccttc agctggggct cgctgtggag cggcattaaa aatttcggtt 18120atatgagcgg tggcgccttc agctggggct cgctgtggag cggcattaaa aatttcggtt 18120
ccaccgttaa gaactatggc agcaaggcct ggaacagcag cacaggccag atgctgaggg 18180ccaccgttaa gaactatggc agcaaggcct ggaacagcag cacaggccag atgctgaggg 18180
ataagttgaa agagcaaaat ttccaacaaa aggtggtaga tggcctggcc tctggcatta 18240ataagttgaa agagcaaaat ttccaacaaa aggtggtaga tggcctggcc tctggcatta 18240
gcggggtggt ggacctggcc aaccaggcag tgcaaaataa gattaacagt aagcttgatc 18300gcggggtggt ggacctggcc aaccaggcag tgcaaaataa gattaacagt aagcttgatc 18300
cccgccctcc cgtagaggag cctccaccgg ccgtggagac agtgtctcca gaggggcgtg 18360cccgccctcc cgtagaggag cctccaccgg ccgtggagac agtgtctcca gaggggcgtg 18360
gcgaaaagcg tccgcgcccc gacagggaag aaactctggt gacgcaaata gacgagcctc 18420gcgaaaagcg tccgcgcccc gacagggaag aaactctggt gacgcaaata gacgagcctc 18420
cctcgtacga ggaggcacta aagcaaggcc tgcccaccac ccgtcccatc gcgcccatgg 18480cctcgtacga ggaggcacta aagcaaggcc tgcccaccac ccgtcccatc gcgcccatgg 18480
ctaccggagt gctgggccag cacacacccg taacgctgga cctgcctccc cccgccgaca 18540ctaccggagt gctgggccag cacacacccg taacgctgga cctgcctccc cccgccgaca 18540
cccagcagaa acctgtgctg ccaggcccga ccgccgttgt tgtaacccgt cctagccgcg 18600cccagcagaa acctgtgctg ccaggcccga ccgccgttgt tgtaacccgt cctagccgcg 18600
cgtccctgcg ccgcgccgcc agcggtccgc gatcgttgcg gcccgtagcc agtggcaact 18660cgtccctgcg ccgcgccgcc agcggtccgc gatcgttgcg gcccgtagcc agtggcaact 18660
ggcaaagcac actgaacagc atcgtgggtc tgggggtgca atccctgaag cgccgacgat 18720ggcaaagcac actgaacagc atcgtgggtc tgggggtgca atccctgaag cgccgacgat 18720
gcttctgaat agctaacgtg tcgtatgtgt gtcatgtatg cgtccatgtc gccgccagag 18780gcttctgaat agctaacgtg tcgtatgtgt gtcatgtatg cgtccatgtc gccgccagag 18780
gagctgctga gccgccgcgc gcccgctttc caagatggct accccttcga tgatgccgca 18840gagctgctga gccgccgcgc gcccgctttc caagatggct accccttcga tgatgccgca 18840
gtggtcttac atgcacatct cgggccagga cgcctcggag tacctgagcc ccgggctggt 18900gtggtcttac atgcacatct cgggccagga cgcctcggag tacctgagcc ccgggctggt 18900
gcagtttgcc cgcgccaccg agacgtactt cagcctgaat aacaagttta gaaaccccac 18960gcagtttgcc cgcgccaccg agacgtactt cagcctgaat aacaagttta gaaaccccac 18960
ggtggcgcct acgcacgacg tgaccacaga ccggtcccag cgtttgacgc tgcggttcat 19020ggtggcgcct acgcacgacg tgaccacaga ccggtcccag cgtttgacgc tgcggttcat 19020
ccctgtggac cgtgaggata ctgcgtactc gtacaaggcg cggttcaccc tagctgtggg 19080ccctgtggac cgtgaggata ctgcgtactc gtacaaggcg cggttcaccc tagctgtggg 19080
tgataaccgt gtgctggaca tggcttccac gtactttgac atccgcggcg tgctggacag 19140tgataaccgt gtgctggaca tggcttccac gtactttgac atccgcggcg tgctggacag 19140
gggccctact tttaagccct actctggcac tgcctacaac gccctggctc ccaagggtgc 19200gggccctact tttaagccct actctggcac tgcctacaac gccctggctc ccaagggtgc 19200
cccaaatcct tgcgaatggg atgaagctgc tactgctctt gaaataaacc tagaagaaga 19260cccaaatcct tgcgaatggg atgaagctgc tactgctctt gaaataaacc tagaagaaga 19260
ggacgatgac aacgaagacg aagtagacga gcaagctgag cagcaaaaaa ctcacgtatt 19320ggacgatgac aacgaagacg aagtagacga gcaagctgag cagcaaaaaa ctcacgtatt 19320
tgggcaggcg ccttattctg gtataaatat tacaaaggag ggtattcaaa taggtgtcgg 19380tgggcaggcg ccttattctg gtataaatat tacaaaggag ggtattcaaa taggtgtcgg 19380
aggaggagga tcaaacgtgg tgcgtcaagg tggtggtggt tctcctaaat atgccgataa 19440aggaggagga tcaaacgtgg tgcgtcaagg tggtggtggt tctcctaaat atgccgataa 19440
aacatttcaa cctgaacctc aaataggaga atctcagtgg tacgaaactg aaattaatca 19500aacatttcaa cctgaacctc aaataggaga atctcagtgg tacgaaactg aaattaatca 19500
tgcagctggg agagtcctta aaaagactac cccaatgaaa ccatgttacg gttcatatgc 19560tgcagctggg agagtcctta aaaagactac cccaatgaaa ccatgttacg gttcatatgc 19560
aaaacccaca aatgaaaatg gagggcaagg cattcttgta aagcaacaaa atggaaagct 19620aaaacccaca aatgaaaatg gagggcaagg cattcttgta aagcaacaaa atggaaagct 19620
agaaagtcaa gtggaaatgc aatttttctc aactactgag gcgaccgcag gcaatggtga 19680agaaagtcaa gtggaaatgc aatttttctc aactactgag gcgaccgcag gcaatggtga 19680
taacttgact cctaaagtgg tattgtacag tgaagatgta gatatagaaa ccccagacac 19740taacttgact cctaaagtgg tattgtacag tgaagatgta gatatagaaa ccccagacac 19740
tcatatttct tacatgccca ctattaagga aggtaactca cgagaactaa tgggccaaca 19800tcatatttct tacatgccca ctattaagga aggtaactca cgagaactaa tgggccaaca 19800
atctatgccc aacaggccta attacattgc ttttagggac aattttattg gtctaatgta 19860atctatgccc aacaggccta attacattgc ttttagggac aattttattg gtctaatgta 19860
ttacaacagc acgggtaata tgggtgttct ggcgggccaa gcatcgcagt tgaatgctgt 19920ttacaacagc acgggtaata tgggtgttct ggcgggccaa gcatcgcagt tgaatgctgt 19920
tgtagatttg caagacagaa acacagagct ttcataccag cttttgcttg attccattgg 19980tgtagatttg caagacagaa acacagagct ttcataccag cttttgcttg attccattgg 19980
tgatagaacc aggtactttt ctatgtggaa tcaggctgtt gacagctatg atccagatgt 20040tgatagaacc aggtactttt ctatgtggaa tcaggctgtt gacagctatg atccagatgt 20040
tagaattatt gaaaatcatg gaactgaaga tgaacttcca aattactgct ttccactggg 20100tagaattatt gaaaatcatg gaactgaaga tgaacttcca aattactgct ttccactggg 20100
aggtgtgatt aatacagaga ctcttaccaa ggtaaaacct aaaacaggtc aggaaaatgg 20160aggtgtgatt aatacagaga ctcttaccaa ggtaaaacct aaaacaggtc aggaaaatgg 20160
atgggaaaaa gatgctacag aattttcaga taaaaatgaa ataagagttg gaaataattt 20220atgggaaaaa gatgctacag aattttcaga taaaaatgaa ataagagttg gaaataattt 20220
tgccatggaa atcaatctaa atgccaacct gtggagaaat ttcctgtact ccaacatagc 20280tgccatggaa atcaatctaa atgccaacct gtggagaaat ttcctgtact ccaacatagc 20280
gctgtatttg cccgacaagc taaagtacag tccttccaac gtaaaaattt ctgataaccc 20340gctgtatttg cccgacaagc taaagtacag tccttccaac gtaaaaattt ctgataaccc 20340
aaacacctac gactacatga acaagcgagt ggtggctccc gggttagtgg actgctacat 20400aaacacctac gactacatga acaagcgagt ggtggctccc gggttagtgg actgctacat 20400
taaccttgga gcacgctggt cccttgacta tatggacaac gtcaacccat ttaaccacca 20460taaccttgga gcacgctggt cccttgacta tatggacaac gtcaacccat ttaaccacca 20460
ccgcaatgct ggcctgcgct accgctcaat gttgctgggc aatggtcgct atgtgccctt 20520ccgcaatgct ggcctgcgct accgctcaat gttgctgggc aatggtcgct atgtgccctt 20520
ccacatccag gtgcctcaga agttctttgc cattaaaaac ctccttctcc tgccgggctc 20580ccacatccag gtgcctcaga agttctttgc cattaaaaac ctccttctcc tgccgggctc 20580
atacacctac gagtggaact tcaggaagga tgttaacatg gttctgcaga gctccctagg 20640atacacctac gagtggaact tcaggaagga tgttaacatg gttctgcaga gctccctagg 20640
aaatgaccta agggttgacg gagccagcat taagtttgat agcatttgcc tttacgccac 20700aaatgaccta agggttgacg gagccagcat taagtttgat agcatttgcc tttacgccac 20700
cttcttcccc atggcccaca acaccgcctc cacgcttgag gccatgctta gaaacgacac 20760cttcttcccc atggcccaca acaccgcctc cacgcttgag gccatgctta gaaacgacac 20760
caacgaccag tcctttaacg actatctctc cgccgccaac atgctctacc ctatacccgc 20820caacgaccag tcctttaacg actatctctc cgccgccaac atgctctacc ctatacccgc 20820
caacgctacc aacgtgccca tatccatccc ctcccgcaac tgggcggctt tccgcggctg 20880caacgctacc aacgtgccca tatccatccc ctcccgcaac tgggcggctt tccgcggctg 20880
ggccttcacg cgccttaaga ctaaggaaac cccatcactg ggctcgggct acgaccctta 20940ggccttcacg cgccttaaga ctaaggaaac cccatcactg ggctcgggct acgaccctta 20940
ttacacctac tctggctcta taccctacct agatggaacc ttttacctca accacacctt 21000ttacacctac tctggctcta taccctacct agatggaacc ttttacctca accacacctt 21000
taagaaggtg gccattacct ttgactcttc tgtcagctgg cctggcaatg accgcctgct 21060taagaaggtg gccattacct ttgactcttc tgtcagctgg cctggcaatg accgcctgct 21060
tacccccaac gagtttgaaa ttaagcgctc agttgacggg gagggttaca acgttgccca 21120tacccccaac gagtttgaaa ttaagcgctc agttgacggg gagggttaca acgttgccca 21120
gtgtaacatg accaaagact ggttcctggt acaaatgcta gctaactaca acattggcta 21180gtgtaacatg accaaagact ggttcctggt acaaatgcta gctaactaca acattggcta 21180
ccagggcttc tatatcccag agagctacaa ggaccgcatg tactccttct ttagaaactt 21240ccagggcttc tatatcccag agagctacaa ggaccgcatg tactccttct ttagaaactt 21240
ccagcccatg agccgtcagg tggtggatga tactaaatac aaggactacc aacaggtggg 21300ccagcccatg agccgtcagg tggtggatga tactaaatac aaggactacc aacaggtggg 21300
catcctacac caacacaaca actctggatt tgttggctac cttgccccca ccatgcgcga 21360catcctacac caacacaaca actctggatt tgttggctac cttgccccca ccatgcgcga 21360
aggacaggcc taccctgcta acttccccta tccgcttata ggcaagaccg cagttgacag 21420aggacaggcc taccctgcta acttccccta tccgcttata ggcaagaccg cagttgacag 21420
cattacccag aaaaagtttc tttgcgatcg caccctttgg cgcatcccat tctccagtaa 21480cattacccag aaaaagtttc tttgcgatcg caccctttgg cgcatcccat tctccagtaa 21480
ctttatgtcc atgggcgcac tcacagacct gggccaaaac cttctctacg ccaactccgc 21540ctttatgtcc atgggcgcac tcacagacct gggccaaaac cttctctacg ccaactccgc 21540
ccacgcgcta gacatgactt ttgaggtgga tcccatggac gagcccaccc ttctttatgt 21600ccacgcgcta gacatgactt ttgaggtgga tcccatggac gagcccaccc ttctttatgt 21600
tttgtttgaa gtctttgacg tggtccgtgt gcaccggccg caccgcggcg tcatcgaaac 21660tttgtttgaa gtctttgacg tggtccgtgt gcaccggccg caccgcggcg tcatcgaaac 21660
cgtgtacctg cgcacgccct tctcggccgg caacgccaca acataaagaa gcaagcaaca 21720cgtgtacctg cgcacgccct tctcggccgg caacgccaca acataaagaa gcaagcaaca 21720
tcaacaacag ctgccgccat gggctccagt gagcaggaac tgaaagccat tgtcaaagat 21780tcaacaacag ctgccgccat gggctccagt gagcaggaac tgaaagccat tgtcaaagat 21780
cttggttgtg ggccatattt tttgggcacc tatgacaagc gctttccagg ctttgtttct 21840cttggttgtg ggccatattt tttgggcacc tatgacaagc gctttccagg ctttgtttct 21840
ccacacaagc tcgcctgcgc catagtcaat acggccggtc gcgagactgg gggcgtacac 21900ccacacaagc tcgcctgcgc catagtcaat acggccggtc gcgagactgg gggcgtacac 21900
tggatggcct ttgcctggaa cccgcactca aaaacatgct acctctttga gccctttggc 21960tggatggcct ttgcctggaa cccgcactca aaaacatgct acctctttga gccctttggc 21960
ttttctgacc agcgactcaa gcaggtttac cagtttgagt acgagtcact cctgcgccgt 22020ttttctgacc agcgactcaa gcaggtttac cagtttgagt acgagtcact cctgcgccgt 22020
agcgccattg cttcttcccc cgaccgctgt ataacgctgg aaaagtccac ccaaagcgta 22080agcgccattg cttcttcccc cgaccgctgt ataacgctgg aaaagtccac ccaaagcgta 22080
caggggccca actcggccgc ctgtggacta ttctgctgca tgtttctcca cgcctttgcc 22140caggggccca actcggccgc ctgtggacta ttctgctgca tgtttctcca cgcctttgcc 22140
aactggcccc aaactcccat ggatcacaac cccaccatga accttattac cggggtaccc 22200aactggcccc aaactcccat ggatcacaac cccaccatga accttattac cggggtaccc 22200
aactccatgc tcaacagtcc ccaggtacag cccaccctgc gtcgcaacca ggaacagctc 22260aactccatgc tcaacagtcc ccaggtacag cccaccctgc gtcgcaacca ggaacagctc 22260
tacagcttcc tggagcgcca ctcgccctac ttccgcagcc acagtgcgca gattaggagc 22320tacagcttcc tggagcgcca ctcgccctac ttccgcagcc acagtgcgca gattaggagc 22320
gccacttctt tttgtcactt gaaaaacatg taaaaataat gtactagaga cactttcaat 22380gccacttctt tttgtcactt gaaaaacatg taaaaataat gtactagaga cactttcaat 22380
aaaggcaaat gcttttattt gtacactctc gggtgattat ttacccccac ccttgccgtc 22440aaaggcaaat gctttttattt gtacactctc gggtgattat ttacccccac ccttgccgtc 22440
tgcgccgttt aaaaatcaaa ggggttctgc cgcgcatcgc tatgcgccac tggcagggac 22500tgcgccgttt aaaaatcaaa ggggttctgc cgcgcatcgc tatgcgccac tggcagggac 22500
acgttgcgat actggtgttt agtgctccac ttaaactcag gcacaaccat ccgcggcagc 22560acgttgcgat actggtgttt agtgctccac ttaaactcag gcacaaccat ccgcggcagc 22560
tcggtgaagt tttcactcca caggctgcgc accatcacca acgcgtttag caggtcgggc 22620tcggtgaagt tttcactcca caggctgcgc accatcacca acgcgtttag caggtcgggc 22620
gccgatatct tgaagtcgca gttggggcct ccgccctgcg cgcgcgagtt gcgatacaca 22680gccgatatct tgaagtcgca gttggggcct ccgccctgcg cgcgcgagtt gcgatacaca 22680
gggttgcagc actggaacac tatcagcgcc gggtggtgca cgctggccag cacgctcttg 22740gggttgcagc actggaacac tatcagcgcc gggtggtgca cgctggccag cacgctcttg 22740
tcggagatca gatccgcgtc caggtcctcc gcgttgctca gggcgaacgg agtcaacttt 22800tcggagatca gatccgcgtc caggtcctcc gcgttgctca gggcgaacgg agtcaacttt 22800
ggtagctgcc ttcccaaaaa gggcgcgtgc ccaggctttg agttgcactc gcaccgtagt 22860ggtagctgcc ttcccaaaaa gggcgcgtgc ccaggctttg agttgcactc gcaccgtagt 22860
ggcatcaaaa ggtgaccgtg cccggtctgg gcgttaggat acagcgcctg cataaaagcc 22920ggcatcaaaa ggtgaccgtg cccggtctgg gcgttaggat acagcgcctg cataaaagcc 22920
ttgatctgct taaaagccac ctgagccttt gcgccttcag agaagaacat gccgcaagac 22980ttgatctgct taaaagccac ctgagccttt gcgccttcag agaagaacat gccgcaagac 22980
ttgccggaaa actgattggc cggacaggcc gcgtcgtgca cgcagcacct tgcgtcggtg 23040ttgccggaaa actgattggc cggacaggcc gcgtcgtgca cgcagcacct tgcgtcggtg 23040
ttggagatct gcaccacatt tcggccccac cggttcttca cgatcttggc cttgctagac 23100ttggagatct gcaccacatt tcggccccac cggttcttca cgatcttggc cttgctagac 23100
tgctccttca gcgcgcgctg cccgttttcg ctcgtcacat ccatttcaat cacgtgctcc 23160tgctccttca gcgcgcgctg cccgttttcg ctcgtcacat ccatttcaat cacgtgctcc 23160
ttatttatca taatgcttcc gtgtagacac ttaagctcgc cttcgatctc agcgcagcgg 23220ttatttatca taatgcttcc gtgtagacac ttaagctcgc cttcgatctc agcgcagcgg 23220
tgcagccaca acgcgcagcc cgtgggctcg tgatgcttgt aggtcacctc tgcaaacgac 23280tgcagccaca acgcgcagcc cgtgggctcg tgatgcttgt aggtcacctc tgcaaacgac 23280
tgcaggtacg cctgcaggaa tcgccccatc atcgtcacaa aggtcttgtt gctggtgaag 23340tgcaggtacg cctgcaggaa tcgccccatc atcgtcacaa aggtcttgtt gctggtgaag 23340
gtcagctgca acccgcggtg ctcctcgttc agccaggtct tgcatacggc cgccagagct 23400gtcagctgca acccgcggtg ctcctcgttc agccaggtct tgcatacggc cgccagagct 23400
tccacttggt caggcagtag tttgaagttc gcctttagat cgttatccac gtggtacttg 23460tccacttggt caggcagtag tttgaagttc gcctttagat cgttatccac gtggtacttg 23460
tccatcagcg cgcgcgcagc ctccatgccc ttctcccacg cagacacgat cggcacactc 23520tccatcagcg cgcgcgcagc ctccatgccc ttctcccacg cagacacgat cggcacactc 23520
agcgggttca tcaccgtaat ttcactttcc gcttcgctgg gctcttcctc ttcctcttgc 23580agcgggttca tcaccgtaat ttcactttcc gcttcgctgg gctcttcctc ttcctcttgc 23580
gtccgcatac cacgcgccac tgggtcgtct tcattcagcc gccgcactgt gcgcttacct 23640gtccgcatac cacgcgccac tgggtcgtct tcattcagcc gccgcactgt gcgcttacct 23640
cctttgccat gcttgattag caccggtggg ttgctgaaac ccaccatttg tagcgccaca 23700cctttgccat gcttgattag caccggtggg ttgctgaaac ccaccatttg tagcgccaca 23700
tcttctcttt cttcctcgct gtccacgatt acctctggtg atggcgggcg ctcgggcttg 23760tcttctcttt cttcctcgct gtccacgatt acctctggtg atggcgggcg ctcgggcttg 23760
ggagaagggc gcttcttttt cttcttgggc gcaatggcca aatccgccgc cgaggtcgat 23820ggagaagggc gcttcttttt cttcttgggc gcaatggcca aatccgccgc cgaggtcgat 23820
ggccgcgggc tgggtgtgcg cggcaccagc gcgtcttgtg atgagtcttc ctcgtcctcg 23880ggccgcgggc tgggtgtgcg cggcaccagc gcgtcttgtg atgagtcttc ctcgtcctcg 23880
gactcgatac gccgcctcat ccgctttttt gggggcgccc ggggaggcgg cggcgacggg 23940gactcgatac gccgcctcat ccgctttttt gggggcgccc ggggaggcgg cggcgacggg 23940
gacggggacg acacgtcctc catggttggg ggacgtcgcg ccgcaccgcg tccgcgctcg 24000gacggggacg acacgtcctc catggttggg ggacgtcgcg ccgcaccgcg tccgcgctcg 24000
ggggtggttt cgcgctgctc ctcttcccga ctggccattt ccttctccta taggcagaaa 24060ggggtggttt cgcgctgctc ctcttcccga ctggccattt ccttctccta taggcagaaa 24060
aagatcatgg agtcagtcgg aagaaggaca gcctaaccgc cccctctgag ttcgccacca 24120aagatcatgg agtcagtcgg aagaaggaca gcctaaccgc cccctctgag ttcgccacca 24120
ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg 24180ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg 24180
aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggacc 24240aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggacc 24240
gctcagtacc aacagaggat aaaaagcaag accaggacaa cgcagaggca aacgaggaac 24300gctcagtacc aacagaggat aaaaagcaag accaggacaa cgcagaggca aacgaggaac 24300
aagtcgggcg gggggacgaa aggcatggcg actacctaga tgtgggagac gacgtgctgt 24360aagtcgggcg gggggacgaa aggcatggcg actacctaga tgtggggac gacgtgctgt 24360
tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg 24420tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg 24420
tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctattc tcaccgcgcg 24480tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctattc tcaccgcgcg 24480
taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc 24540taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc 24540
ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga 24600ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga 24600
tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg 24660tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg 24660
gcgctgtcat acctgatatc gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg 24720gcgctgtcat acctgatatc gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg 24720
gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc 24780gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc 24780
actctggagt gttggtggaa ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca 24840actctggagt gttggtggaa ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca 24840
gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gtcatgagca 24900gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gtcatgagca 24900
cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc 24960cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc 24960
aagaacaaac agaggagggc ctacccgcag ttggcgacga gcagctagcg cgctggcttc 25020aagaacaaac agaggagggc ctacccgcag ttggcgacga gcagctagcg cgctggcttc 25020
aaacgcgcga gcctgccgac ttggaggagc gacgcaaact aatgatggcc gcagtgctcg 25080aaacgcgcga gcctgccgac ttggaggagc gacgcaaact aatgatggcc gcagtgctcg 25080
ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc 25140ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc 25140
tagaggaaac attgcactac acctttcgac agggctacgt acgccaggcc tgcaagatct 25200tagaggaaac attgcactac acctttcgac agggctacgt acgccaggcc tgcaagatct 25200
ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgccttg 25260ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgccttg 25260
ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact 25320ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact 25320
gcgtttactt atttctatgc tacacctggc agacggccat gggcgtttgg cagcagtgct 25380gcgtttactt atttctatgc tacacctggc agacggccat gggcgtttgg cagcagtgct 25380
tggaggagtg caacctcaag gagctgcaga aactgctaaa gcaaaacttg aaggacctat 25440tggaggagtg caacctcaag gagctgcaga aactgctaaa gcaaaacttg aaggacctat 25440
ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacatcatt ttccccgaac 25500ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacatcatt ttccccgaac 25500
gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaga 25560gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaga 25560
actttaggaa ctttatccta gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc 25620actttaggaa ctttatccta gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc 25620
ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc gccgctttgg ggccactgct 25680ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc gccgctttgg ggccactgct 25680
accttctgca gctagccaac taccttgcct accactctga cataatggaa gacgtgagcg 25740accttctgca gctagccaac taccttgcct accactctga cataatggaa gacgtgagcg 25740
gtgacggtct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg 25800gtgacggtct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg 25800
tttgcaattc gcagctgctt aacgaaagtc aaattatcgg tacctttgag ctgcagggtc 25860tttgcaattc gcagctgctt aacgaaagtc aaattatcgg tacctttgag ctgcagggtc 25860
cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt 25920cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt 25920
cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg 25980cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg 25980
aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca 26040aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca 26040
ttcttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac 26100ttcttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac 26100
ggggggttta cttggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc 26160ggggggttta cttggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc 26160
agccctatca gcagcagccg cgggcccttg cttcccagga tggcacccaa aaagaagctg 26220agccctatca gcagcagccg cgggcccttg cttcccagga tggcacccaa aaagaagctg 26220
cagctgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt 26280cagctgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt 26280
ttggacgagg aggaggagga catgatggaa gactgggaga gcctagacga ggaagcttcc 26340ttggacgagg aggaggagga catgatggaa gactgggaga gcctagacga ggaagcttcc 26340
gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg 26400gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg 26400
ccccagaaat cggcaaccgg ttccagcatg gctacaacct ccgctcctca ggcgccgccg 26460ccccagaaat cggcaaccgg ttccagcatg gctacaacct ccgctcctca ggcgccgccg 26460
gcactgcccg ttcgccgacc caaccgtaga tgggacacca ctggaaccag ggccggtaag 26520gcactgcccg ttcgccgacc caaccgtaga tgggacacca ctggaaccag ggccggtaag 26520
tccaagcagc cgccgccgtt agcccaagag caacaacagc gccaaggcta ccgctcatgg 26580tccaagcagc cgccgccgtt agcccaagag caacaacagc gccaaggcta ccgctcatgg 26580
cgcgggcaca agaacgccat agttgcttgc ttgcaagact gtgggggcaa catctccttc 26640cgcgggcaca agaacgccat agttgcttgc ttgcaagact gtgggggcaa catctccttc 26640
gcccgccgct ttcttctcta ccatcacggc gtggccttcc cccgtaacat cctgcattac 26700gcccgccgct ttcttctcta ccatcacggc gtggccttcc cccgtaacat cctgcattac 26700
taccgtcatc tctacagccc atactgcacc ggcggcagcg gcagcggcag caacagcagc 26760taccgtcatc tctacagccc atactgcacc ggcggcagcg gcagcggcag caacagcagc 26760
ggccacacag aagcaaaggc gaccggatag caagactctg acaaagccca agaaatccac 26820ggccacacag aagcaaaggc gaccggatag caagactctg acaaagccca agaaatccac 26820
agcggcggca gcagcaggag gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac 26880agcggcggca gcagcaggag gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac 26880
ccgcgagctt agaaacagga tttttcccac tctgtatgct atatttcaac agagcagggg 26940ccgcgagctt agaaacagga tttttcccac tctgtatgct atatttcaac agagcagggg 26940
ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga tccctcaccc gcagctgcct 27000ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga tccctcaccc gcagctgcct 27000
gtatcacaaa agcgaagatc agcttcggcg cacgctggaa gacgcggagg ctctcttcag 27060gtatcacaaa agcgaagatc agcttcggcg cacgctggaa gacgcggagg ctctcttcag 27060
taaatactgc gcgctgactc ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg 27120taaatactgc gcgctgactc ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg 27120
aaaactacgt catctccagc ggccacaccc ggcgccagca cctgtcgtca gcgccattat 27180aaaactacgt catctccagc ggccacaccc ggcgccagca cctgtcgtca gcgccattat 27180
gagcaaggaa attcccacgc cctacatgtg gagttaccag ccacaaatgg gacttgcggc 27240gagcaaggaa attcccacgc cctacatgtg gagttaccag ccacaaatgg gacttgcggc 27240
tggagctgcc caagactact caacccgaat aaactacatg agcgcgggac cccacatgat 27300tggagctgcc caagactact caacccgaat aaactacatg agcgcgggac cccacatgat 27300
atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ctcttggaac aggcggctat 27360atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ctcttggaac aggcggctat 27360
taccaccaca cctcgtaata accttaatcc ccgtagttgg cccgctgccc tggtgtacca 27420taccaccaca cctcgtaata accttaatcc ccgtagttgg cccgctgccc tggtgtacca 27420
ggaaagtccc gctcccacca ctgtggtact tcccagagac gcccaggccg aagttcagat 27480ggaaagtccc gctcccacca ctgtggtact tcccagagac gcccaggccg aagttcagat 27480
gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca 27540gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca 27540
gggtataact cacctgacaa tcagagggcg aggtattcag ctcaacgacg agtcggtgag 27600gggtataact cacctgacaa tcagagggcg aggtattcag ctcaacgacg agtcggtgag 27600
ctcctcgctt ggtctccgtc cggacgggac atttcagatc ggcggcgccg gccgtccttc 27660ctcctcgctt ggtctccgtc cggacgggac atttcagatc ggcggcgccg gccgtccttc 27660
attcacgcct cgtcaggcaa tcctaactct gcagacctcg tcctctgagc cgcgctctgg 27720attcacgcct cgtcaggcaa tcctaactct gcagacctcg tcctctgagc cgcgctctgg 27720
aggcattgga actctgcaat ttattgagga gtttgtgcca tcggtctact ttaacccctt 27780aggcattgga actctgcaat ttattgagga gtttgtgcca tcggtctact ttaacccctt 27780
ctcgggacct cccggccact atccggatca atttattcct aactttgacg cggtaaagga 27840ctcgggacct cccggccact atccggatca atttattcct aactttgacg cggtaaagga 27840
ctcggcggac ggctacgact gaatgttaag tggagaggca gagcaactgc gcctgaaaca 27900ctcggcggac ggctacgact gaatgttaag tggagaggca gagcaactgc gcctgaaaca 27900
cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac tccggtgagt tttgctactt 27960cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac tccggtgagt tttgctactt 27960
tgaattgccc gaggatcata tcgagggccc ggcgcacggc gtccggctta ccgcccaggg 28020tgaattgccc gaggatcata tcgagggccc ggcgcacggc gtccggctta ccgcccaggg 28020
agagcttgcc cgtagcctga ttcgggagtt tacccagcgc cccctgctag ttgagcggga 28080agagcttgcc cgtagcctga ttcgggagtt tacccagcgc cccctgctag ttgagcggga 28080
caggggaccc tgtgttctca ctgtgatttg caactgtcct aaccttggat tacatcaaga 28140caggggaccc tgtgttctca ctgtgatttg caactgtcct aaccttggat tacatcaaga 28140
tctttgttgc catctctgtg ctgagtataa taaatacaga aattaaaata tactggggct 28200tctttgttgc catctctgtg ctgagtataa taaatacaga aattaaaata tactggggct 28200
cctatcgcca tcctgtaaac gccaccgtct tcacccgccc aagcaaacca aggcgaacct 28260cctatcgcca tcctgtaaac gccaccgtct tcacccgccc aagcaaacca aggcgaacct 28260
tacctggtac ttttaacatc tctccctctg tgatttacaa cagtttcaac ccagacggag 28320tacctggtac ttttaacatc tctccctctg tgatttacaa cagtttcaac ccagacggag 28320
tgagtctacg agagaacctc tccgagctca gctactccat cagaaaaaac accaccctcc 28380tgagtctacg agagaacctc tccgagctca gctactccat cagaaaaaac accaccctcc 28380
ttacctgccg ggaacgtacg agtgcgtcac cggccgctgc accacaccta ccgcctgacc 28440ttacctgccg ggaacgtacg agtgcgtcac cggccgctgc accacaccta ccgcctgacc 28440
gtaaaccaga ctttttccgg acagacctca ataactctgt ttaccagaac aggaggtgag 28500gtaaaccaga ctttttccgg acagacctca ataactctgt ttaccagaac aggaggtgag 28500
cttagaaaac ccttagggta ttaggccaaa ggcgcagcta ctgtggggtt tatgaacaat 28560cttagaaaac ccttagggta ttaggccaaa ggcgcagcta ctgtggggtt tatgaacaat 28560
tcaagcaact ctacgggcta ttctaattca ggtttctcta gaatcggggt tggggttatt 28620tcaagcaact ctacgggcta ttctaattca ggtttctcta gaatcggggt tggggttatt 28620
ctctgtcttg tgattctctt tattcttata ctaacgcttc tctgcctaag gctcgccgcc 28680ctctgtcttg tgattctctt tattcttata ctaacgcttc tctgcctaag gctcgccgcc 28680
tgctgtgtgc acatttgcat ttattgtcag ctttttaaac gctggggtcg ccacccaaga 28740tgctgtgtgc acatttgcat ttattgtcag ctttttaaac gctggggtcg ccacccaaga 28740
tgattaggta cataatccta ggtttactca cccttgcgtc agcccacggt accacccaaa 28800tgattaggta cataatccta ggtttactca cccttgcgtc agcccacggt accacccaaa 28800
aggtggattt taaggagcca gcctgtaatg ttacattcgc agctgaagct aatgagtgca 28860aggtggattt taaggagcca gcctgtaatg ttacattcgc agctgaagct aatgagtgca 28860
ccactcttat aaaatgcacc acagaacatg aaaagctgct tattcgccac aaaaacaaaa 28920ccactcttat aaaatgcacc acagaacatg aaaagctgct tattcgccac aaaaacaaaa 28920
ttggcaagta tgctgtttat gctatttggc agccaggtga cactacagag tataatgtta 28980ttggcaagta tgctgtttat gctatttggc agccaggtga cactacagag tataatgtta 28980
cagttttcca gggtaaaagt cataaaactt ttatgtatac ttttccattt tatgaaatgt 29040cagttttcca gggtaaaagt cataaaactt ttatgtatac ttttccattt tatgaaatgt 29040
gcgacattac catgtacatg agcaaacagt ataagttgtg gcccccacaa aattgtgtgg 29100gcgacattac catgtacatg agcaaacagt ataagttgtg gcccccacaa aattgtgtgg 29100
aaaacactgg cactttctgc tgcactgcta tgctaattac agtgctcgct ttggtctgta 29160aaaacactgg cactttctgc tgcactgcta tgctaattac agtgctcgct ttggtctgta 29160
ccctactcta tattaaatac aaaagcagac gcagctttat tgaggaaaag aaaatgcctt 29220ccctactcta tattaaatac aaaagcagac gcagctttat tgaggaaaag aaaatgcctt 29220
aatttactaa gttacaaagc taatgtcacc actaactgct ttactcgctg cttgcaaaac 29280aatttactaa gttacaaagc taatgtcacc actaactgct ttactcgctg cttgcaaaac 29280
aaattcaaaa agttagcatt ataattagaa taggatttaa accccccggt catttcctgc 29340aaattcaaaa agttagcatt ataattagaa taggatttaa accccccggt catttcctgc 29340
tcaataccat tcccctgaac aattgactct atgtgggata tgctccagcg ctacaacctt 29400tcaataccat tcccctgaac aattgactct atgtgggata tgctccagcg ctacaacctt 29400
gaagtcaggc ttcctggatg tcagcatctg actttggcca gcacctgtcc cgcggatttg 29460gaagtcaggc ttcctggatg tcagcatctg actttggcca gcacctgtcc cgcggatttg 29460
ttccagtcca actacagcga cccaccctaa cagagatgac caacacaacc aacgcggccg 29520ttccagtcca actacagcga cccaccctaa cagagatgac caacacaacc aacgcggccg 29520
ccgctaccgg acttacatct accacaaata caccccaagt ttctgccttt gtcaataact 29580ccgctaccgg acttacatct accacaaata caccccaagt ttctgccttt gtcaataact 29580
gggatgcaac cgcagcagcc actcccgctt gggaagaata gaggcagatt ctgaaagtca 29640gggatgcaac cgcagcagcc actcccgctt gggaagaata gaggcagatt ctgaaagtca 29640
agaagacatc atccggaata ttgccaggca cctcgcccag gtcggggaca gcatggaccg 29700agaagacatc atccggaata ttgccaggca cctcgcccag gtcggggaca gcatggaccg 29700
tagcatccct ccgggcctgg tgaacggcct ggccctgcag ctcaggaaca ccagccggtc 29760tagcatccct ccgggcctgg tgaacggcct ggccctgcag ctcaggaaca ccagccggtc 29760
ggaggaggac cggaacaggg acctggccac tgccctggag cagctgctgc aggcctaccc 29820ggaggaggac cggaacaggg acctggccac tgccctggag cagctgctgc aggcctaccc 29820
tagagacatg gagaaggaga agaccatgct ggtgctggcc ctgctgctgg ccaagaaggt 29880tagagacatg gagaaggaga agaccatgct ggtgctggcc ctgctgctgg ccaagaaggt 29880
ggccagtcac acgccgtcct tgctccgtga tgtctttcac acaacagtga attttattaa 29940ggccagtcac acgccgtcct tgctccgtga tgtctttcac acaacagtga attttattaa 29940
ccagaaccta cgcacctacg tgaggagctt agccagaaat gggatggatg caaatccata 30000ccagaaccta cgcacctacg tgaggagctt agccagaaat gggatggatg caaatccata 30000
gattggacgg actgaaacac atgttctttt ctcttacagt atgattaaat gagacatgat 30060gattggacgg actgaaacac atgttctttt ctcttacagt atgattaaat gagacatgat 30060
tcctcgagtt tttatattac tgacccttgt tgcgcttttt tgtgcgtgct ccacattggc 30120tcctcgagtt tttatattac tgacccttgt tgcgcttttt tgtgcgtgct ccacattggc 30120
tgcggtttct cacatcgaag tagactgcat tccagccttc acagtctatt tgctttacgg 30180tgcggtttct cacatcgaag tagactgcat tccagccttc acagtctatt tgctttacgg 30180
atttgtcacc ctcacgctca tctgcagcct catcactgtg gtcatcgcct ttatccagtg 30240atttgtcacc ctcacgctca tctgcagcct catcactgtg gtcatcgcct ttatccagtg 30240
cattgactgg gtctgtgtgc gctttgcata tctcagacac catccccagt acagggacag 30300cattgactgg gtctgtgtgc gctttgcata tctcagacac catccccagt acagggacag 30300
gactatagct gagcttctta gaattcttta attatgaaat ttactgtgac ttttctgctg 30360gactatagct gagcttctta gaattcttta attatgaaat ttactgtgac ttttctgctg 30360
attatttgca ccctatctgc gttttgttcc ccgacctcca agcctcaaag acatatatca 30420attatttgca ccctatctgc gttttgttcc ccgacctcca agcctcaaag acatatatca 30420
tgcagattca ctcgtatatg gaatattcca agttgctaca atgaaaaaag cgatctttcc 30480tgcagattca ctcgtatatg gaatattcca agttgctaca atgaaaaaag cgatctttcc 30480
gaagcctggt tatatgcaat catctctgtt atggtgttct gcagtaccat cttagcccta 30540gaagcctggt tatatgcaat catctctgtt atggtgttct gcagtaccat cttagcccta 30540
gctatatatc cctaccttga cattggctgg aaacgaatag atgccatgaa ccacccaact 30600gctatatatc cctaccttga cattggctgg aaacgaatag atgccatgaa ccacccaact 30600
ttccccgcgc ccgctatgct tccactgcaa caagttgttg ccggcggctt tgtcccagcc 30660ttccccgcgc ccgctatgct tccactgcaa caagttgttg ccggcggctt tgtcccagcc 30660
aatcagcctc gccccacttc tcccaccccc actgaaatca gctactttaa tctaacagga 30720aatcagcctc gccccacttc tcccaccccc actgaaatca gctactttaa tctaacagga 30720
ggagatgact gacaccctag atctagaaat ggacggaatt attacagagc agcgcctgct 30780ggagatgact gacaccctag atctagaaat ggacggaatt attacagagc agcgcctgct 30780
agaaagacgc agggcagcgg ccgagcaaca gcgcatgaat caagagctcc aagacatggt 30840agaaagacgc agggcagcgg ccgagcaaca gcgcatgaat caagagctcc aagacatggt 30840
taacttgcac cagtgcaaaa ggggtatctt ttgtctggta aagcaggcca aagtcaccta 30900taacttgcac cagtgcaaaa ggggtatctt ttgtctggta aagcaggcca aagtcaccta 30900
cgacagtaat accaccggac accgccttag ctacaagttg ccaaccaagc gtcagaaatt 30960cgacagtaat accaccggac accgccttag ctacaagttg ccaaccaagc gtcagaaatt 30960
ggtggtcatg gtgggagaaa agcccattac cataactcag cactcggtag aaaccgaagg 31020ggtggtcatg gtgggagaaa agcccattac cataactcag cactcggtag aaaccgaagg 31020
ctgcattcac tcaccttgtc aaggacctga ggatctctgc acccttatta agaccctgtg 31080ctgcattcac tcaccttgtc aaggacctga ggatctctgc acccttatta agaccctgtg 31080
cggtctcaaa gatcttattc cctttaacta ataaaaaaaa ataataaagc atcacttact 31140cggtctcaaa gatcttattc cctttaacta ataaaaaaaa ataataaagc atcacttact 31140
taaaatcagt tagcaaattt ctgtccagtt tattcagcag cacctccttg ccctcctccc 31200taaaatcagt tagcaaattt ctgtccagtt tattcagcag cacctccttg ccctcctccc 31200
agctctggta ttgcagcttc ctcctggctg caaactttct ccacaatcta aatggaatgt 31260agctctggta ttgcagcttc ctcctggctg caaactttct ccacaatcta aatggaatgt 31260
cagtttcctc ctgttcctgt ccatccgcac ccactatctt catgttgttg cagatgaagc 31320cagtttcctc ctgttcctgt ccatccgcac ccactatctt catgttgttg cagatgaagc 31320
gcgcaagacc gtctgaagat accttcaacc ccgtgtatcc atatgacacg gaaaccggtc 31380gcgcaagacc gtctgaagat accttcaacc ccgtgtatcc atatgacacg gaaaccggtc 31380
ctccaactgt gccttttctt actcctccct ttgtatcccc caatgggttt caagagagtc 31440ctccaactgt gccttttctt actcctccct ttgtatcccc caatgggttt caagagagtc 31440
cccctggggt actctctttg cgcctatccg aacctctagt tacctccaat ggcatgcttg 31500cccctggggt actctctttg cgcctatccg aacctctagt tacctccaat ggcatgcttg 31500
cgctcaaaat gggcaacggc ctctctctgg acgaggccgg caaccttacc tcccaaaatg 31560cgctcaaaat gggcaacggc ctctctctgg acgaggccgg caaccttacc tcccaaaatg 31560
taaccactgt gagcccacct ctcaaaaaaa ccaagtcaaa cataaacctg gaaatatctg 31620taaccactgt gagcccacct ctcaaaaaaa ccaagtcaaa cataaacctg gaaatatctg 31620
cacccctcac agttacctca gaagccctaa ctgtggctgc cgccgcacct ctaatggtcg 31680cacccctcac agttacctca gaagccctaa ctgtggctgc cgccgcacct ctaatggtcg 31680
cgggcaacac actcaccatg caatcacagg ccccgctaac cgtgcacgac tccaaactta 31740cgggcaacac actcaccatg caatcacagg ccccgctaac cgtgcacgac tccaaactta 31740
gcattgccac ccaaggaccc ctcacagtgt cagaaggaaa gctagccctg caaacatcag 31800gcattgccac ccaaggaccc ctcacagtgt cagaaggaaa gctagccctg caaacatcag 31800
gccccctcac caccaccgat agcagtaccc ttactatcac tgcctcaccc cctctaacta 31860gccccctcac caccaccgat agcagtaccc ttactatcac tgcctcaccc cctctaacta 31860
ctgccactgg tagcttgggc attgacttga aagagcccat ttatacacaa aatggaaaac 31920ctgccactgg tagcttgggc attgacttga aagagcccat ttatacacaa aatggaaaac 31920
taggactaaa gtacggggct cctttgcatg taacagacga cctaaacact ttgaccgtag 31980taggactaaa gtacggggct cctttgcatg taacagacga cctaaacact ttgaccgtag 31980
caactggtcc aggtgtgact attaataata cttccttgca aactaaagtt actggagcct 32040caactggtcc aggtgtgact attaataata cttccttgca aactaaagtt actggagcct 32040
tgggttttga ttcacaaggc aatatgcaac ttaatgtagc aggaggacta aggattgatt 32100tgggttttga ttcacaaggc aatatgcaac ttaatgtagc aggaggacta aggattgatt 32100
ctcaaaacag acgccttata cttgatgtta gttatccgtt tgatgctcaa aaccaactaa 32160ctcaaaacag acgccttata cttgatgtta gttatccgtt tgatgctcaa aaccaactaa 32160
atctaagact aggacagggc cctcttttta taaactcagc ccacaacttg gatattaact 32220atctaagact aggacagggc cctcttttta taaactcagc ccacaacttg gatattaact 32220
acaacaaagg cctttacttg tttacagctt caaacaattc caaaaagctt gaggttaacc 32280acaacaaagg cctttacttg tttacagctt caaacaattc caaaaagctt gaggttaacc 32280
taagcactgc caaggggttg atgtttgacg ctacagccat agccattaat gcaggagatg 32340taagcactgc caaggggttg atgtttgacg ctacagccat agccattaat gcaggagatg 32340
ggcttgaatt tggttcacct aatgcaccaa acacaaatcc cctcaaaaca aaaattggcc 32400ggcttgaatt tggttcacct aatgcaccaa acacaaatcc cctcaaaaca aaaattggcc 32400
atggcctaga atttgattca aacaaggcta tggttcctaa actaggaact ggccttagtt 32460atggcctaga atttgattca aacaaggcta tggttcctaa actaggaact ggccttagtt 32460
ttgacagcac aggtgccatt acagtaggaa acaaaaataa tgataagcta actttgtgga 32520ttgacagcac aggtgccatt acagtaggaa acaaaaataa tgataagcta actttgtgga 32520
ccacaccagc tccatctcct aactgtagac taaatgcaga gaaagatgct aaactcactt 32580ccacaccagc tccatctcct aactgtagac taaatgcaga gaaagatgct aaactcactt 32580
tggtcttaac aaaatgtggc agtcaaatac ttgctacagt ttcagttttg gctgttaaag 32640tggtcttaac aaaatgtggc agtcaaatac ttgctacagt ttcagttttg gctgttaaag 32640
gcagtttggc tccaatatct ggaacagttc aaagtgctca tcttattata agatttgacg 32700gcagtttggc tccaatatct ggaacagttc aaagtgctca tcttattata agatttgacg 32700
aaaatggagt gctactaaac aattccttcc tggacccaga atattggaac tttagaaatg 32760aaaatggagt gctactaaac aattccttcc tggacccaga atattggaac tttagaaatg 32760
gagatcttac tgaaggcaca gcctatacaa acgctgttgg atttatgcct aacctatcag 32820gagatcttac tgaaggcaca gcctatacaa acgctgttgg atttatgcct aacctatcag 32820
cttatccaaa atctcacggt aaaactgcca aaagtaacat tgtcagtcaa gtttacttaa 32880cttatccaaa atctcacggt aaaactgcca aaagtaacat tgtcagtcaa gtttacttaa 32880
acggagacaa aactaaacct gtaacactaa ccattacact aaacggtaca caggaaacag 32940acggagacaa aactaaacct gtaacactaa ccattacact aaacggtaca caggaaacag 32940
gagacacaac tccaagtgca tactctatgt cattttcatg ggactggtct ggccacaact 33000gagacacaac tccaagtgca tactctatgt cattttcatg ggactggtct ggccacaact 33000
acattaatga aatatttgcc acatcctctt acactttttc atacattgcc caagaataaa 33060acattaatga aatatttgcc acatcctctt acactttttc atacattgcc caagaataaa 33060
gaatcgtttg tgttatgttt caacgtgttt atttttcaat tgcagaaaat ttcaagtcat 33120gaatcgtttg tgttatgttt caacgtgttt atttttcaat tgcagaaaat ttcaagtcat 33120
ttttcattca gtagtatagc cccaccacca catagcttat acagatcacc gtaccttaat 33180ttttcattca gtagtatagc cccaccacca catagcttat acagatcacc gtaccttaat 33180
caaactcaca gaaccctagt attcaacctg ccacctccct cccaacacac agagtacaca 33240caaactcaca gaaccctagt attcaacctg ccacctccct cccaacacac agagtacaca 33240
gtcctttctc cccggctggc cttaaaaagc atcatatcat gggtaacaga catattctta 33300gtcctttctc cccggctggc cttaaaaagc atcatatcat gggtaacaga catattctta 33300
ggtgttatat tccacacggt ttcctgtcga gccaaacgct catcagtgat attaataaac 33360ggtgttatat tccacacggt ttcctgtcga gccaaacgct catcagtgat attaataaac 33360
tccccgggca gctcacttaa gttcatgtcg ctgtccagct gctgagccac aggctgctgt 33420tccccgggca gctcacttaa gttcatgtcg ctgtccagct gctgagccac aggctgctgt 33420
ccaacttgcg gttgcttaac gggcggcgaa ggagaagtcc acgcctacat gggggtagag 33480ccaacttgcg gttgcttaac gggcggcgaa ggagaagtcc acgcctacat gggggtagag 33480
tcataatcgt gcatcaggat agggcggtgg tgctgcagca gcgcgcgaat aaactgctgc 33540tcataatcgt gcatcaggat agggcggtgg tgctgcagca gcgcgcgaat aaactgctgc 33540
cgccgccgct ccgtcctgca ggaatacaac atggcagtgg tctcctcagc gatgattcgc 33600cgccgccgct ccgtcctgca ggaatacaac atggcagtgg tctcctcagc gatgattcgc 33600
accgcccgca gcataaggcg ccttgtcctc cgggcacagc agcgcaccct gatctcactt 33660accgcccgca gcataaggcg ccttgtcctc cgggcacagc agcgcaccct gatctcactt 33660
aaatcagcac agtaactgca gcacagcacc acaatattgt tcaaaatccc acagtgcaag 33720aaatcagcac agtaactgca gcacagcacc acaatattgt tcaaaatccc acagtgcaag 33720
gcgctgtatc caaagctcat ggcggggacc acagaaccca cgtggccatc ataccacaag 33780gcgctgtatc caaagctcat ggcggggacc acagaaccca cgtggccatc ataccacaag 33780
cgcaggtaga ttaagtggcg acccctcata aacacgctgg acataaacat tacctctttt 33840cgcaggtaga ttaagtggcg acccctcata aacacgctgg acataaacat tacctctttt 33840
ggcatgttgt aattcaccac ctcccggtac catataaacc tctgattaaa catggcgcca 33900ggcatgttgt aattcaccac ctcccggtac catataaacc tctgattaaa catggcgcca 33900
tccaccacca tcctaaacca gctggccaaa acctgcccgc cggctataca ctgcagggaa 33960tccaccacca tcctaaacca gctggccaaa acctgcccgc cggctataca ctgcagggaa 33960
ccgggactgg aacaatgaca gtggagagcc caggactcgt aaccatggat catcatgctc 34020ccgggactgg aacaatgaca gtggagagcc caggactcgt aaccatggat catcatgctc 34020
gtcatgatat caatgttggc acaacacagg cacacgtgca tacacttcct caggattaca 34080gtcatgatat caatgttggc acaacacagg cacacgtgca tacacttcct caggattaca 34080
agctcctccc gcgttagaac catatcccag ggaacaaccc attcctgaat cagcgtaaat 34140agctcctccc gcgttagaac catatcccag ggaacaaccc attcctgaat cagcgtaaat 34140
cccacactgc agggaagacc tcgcacgtaa ctcacgttgt gcattgtcaa agtgttacat 34200cccacactgc agggaagacc tcgcacgtaa ctcacgttgt gcattgtcaa agtgttacat 34200
tcgggcagca gcggatgatc ctccagtatg gtagcgcggg tttctgtctc aaaaggaggt 34260tcgggcagca gcggatgatc ctccagtatg gtagcgcggg tttctgtctc aaaaggaggt 34260
agacgatccc tactgtacgg agtgcgccga gacaaccgag atcgtgttgg tcgtagtgtc 34320agacgatccc tactgtacgg agtgcgccga gacaaccgag atcgtgttgg tcgtagtgtc 34320
atgccaaatg gaacgccgga cgtagtcata tttcctgaag caaaaccagg tgcgggcgtg 34380atgccaaatg gaacgccgga cgtagtcata tttcctgaag caaaaccagg tgcgggcgtg 34380
acaaacagat ctgcgtctcc ggtctcgccg cttagatcgc tctgtgtagt agttgtagta 34440acaaacagat ctgcgtctcc ggtctcgccg cttagatcgc tctgtgtagt agttgtagta 34440
tatccactct ctcaaagcat ccaggcgccc cctggcttcg ggttctatgt aaactccttc 34500tatccactct ctcaaagcat ccaggcgccc cctggcttcg ggttctatgt aaactccttc 34500
atgcgccgct gccctgataa catccaccac cgcagaataa gccacaccca gccaacctac 34560atgcgccgct gccctgataa catccaccac cgcagaataa gccacaccca gccaacctac 34560
acattcgttc tgcgagtcac acacgggagg agcgggaaga gctggaagaa ccatgttttt 34620acattcgttc tgcgagtcac acacgggagg agcgggaaga gctggaagaa ccatgttttt 34620
ttttttattc caaaagatta tccaaaacct caaaatgaag atctattaag tgaacgcgct 34680ttttttattc caaaagatta tccaaaacct caaaatgaag atctattaag tgaacgcgct 34680
cccctccggt ggcgtggtca aactctacag ccaaagaaca gataatggca tttgtaagat 34740cccctccggt ggcgtggtca aactctacag ccaaagaaca gataatggca tttgtaagat 34740
gttgcacaat ggcttccaaa aggcaaacgg ccctcacgtc caagtggacg taaaggctaa 34800gttgcacaat ggcttccaaa aggcaaacgg ccctcacgtc caagtggacg taaaggctaa 34800
acccttcagg gtgaatctcc tctataaaca ttccagcacc ttcaaccatg cccaaataat 34860acccttcagg gtgaatctcc tctataaaca ttccagcacc ttcaaccatg cccaaataat 34860
tctcatctcg ccaccttctc aatatatctc taagcaaatc ccgaatatta agtccggcca 34920tctcatctcg ccaccttctc aatatatctc taagcaaatc ccgaatatta agtccggcca 34920
ttgtaaaaat ctgctccaga gcgccctcca ccttcagcct caagcagcga atcatgattg 34980ttgtaaaaat ctgctccaga gcgccctcca ccttcagcct caagcagcga atcatgattg 34980
caaaaattca ggttcctcac agacctgtat aagattcaaa agcggaacat taacaaaaat 35040caaaaattca ggttcctcac agacctgtat aagattcaaa agcggaacat taacaaaaat 35040
accgcgatcc cgtaggtccc ttcgcagggc cagctgaaca taatcgtgca ggtctgcacg 35100accgcgatcc cgtaggtccc ttcgcagggc cagctgaaca taatcgtgca ggtctgcacg 35100
gaccagcgcg gccacttccc cgccaggaac catgacaaaa gaacccacac tgattatgac 35160gaccagcgcg gccacttccc cgccaggaac catgacaaaa gaacccacac tgattatgac 35160
acgcatactc ggagctatgc taaccagcgt agccccgatg taagcttgtt gcatgggcgg 35220acgcatactc ggagctatgc taaccagcgt agccccgatg taagcttgtt gcatgggcgg 35220
cgatataaaa tgcaaggtgc tgctcaaaaa atcaggcaaa gcctcgcgca aaaaagaaag 35280cgatataaaa tgcaaggtgc tgctcaaaaa atcaggcaaa gcctcgcgca aaaaagaaag 35280
cacatcgtag tcatgctcat gcagataaag gcaggtaagc tccggaacca ccacagaaaa 35340cacatcgtag tcatgctcat gcagataaag gcaggtaagc tccggaacca ccacagaaaa 35340
agacaccatt tttctctcaa acatgtctgc gggtttctgc ataaacacaa aataaaataa 35400agacaccatt tttctctcaa acatgtctgc gggtttctgc ataaacacaa aataaaataa 35400
caaaaaaaca tttaaacatt agaagcctgt cttacaacag gaaaaacaac ccttataagc 35460caaaaaaaca tttaaacatt agaagcctgt cttacaacag gaaaaacaac ccttataagc 35460
ataagacgga ctacggccat gccggcgtga ccgtaaaaaa actggtcacc gtgattaaaa 35520ataagacgga ctacggccat gccggcgtga ccgtaaaaaa actggtcacc gtgattaaaa 35520
agcaccaccg acagctcctc ggtcatgtcc ggagtcataa tgtaagactc ggtaaacaca 35580agcaccaccg acagctcctc ggtcatgtcc ggagtcataa tgtaagactc ggtaaacaca 35580
tcaggttgat tcacatcggt cagtgctaaa aagcgaccga aatagcccgg gggaatacat 35640tcaggttgat tcacatcggt cagtgctaaa aagcgaccga aatagcccgg gggaatacat 35640
acccgcaggc gtagagacaa cattacagcc cccataggag gtataacaaa attaatagga 35700acccgcaggc gtagagacaa cattacagcc cccataggag gtataacaaa attaatagga 35700
gagaaaaaca cataaacacc tgaaaaaccc tcctgcctag gcaaaatagc accctcccgc 35760gagaaaaaca cataaacacc tgaaaaaccc tcctgcctag gcaaaatagc accctcccgc 35760
tccagaacaa catacagcgc ttccacagcg gcagccataa cagtcagcct taccagtaaa 35820tccagaacaa catacagcgc ttccacagcg gcagccataa cagtcagcct taccagtaaa 35820
aaagaaaacc tattaaaaaa acaccactcg acacggcacc agctcaatca gtcacagtgt 35880aaagaaaacc tattaaaaaa acaccactcg acacggcacc agctcaatca gtcacagtgt 35880
aaaaaagggc caagtgcaga gcgagtatat ataggactaa aaaatgacgt aacggttaaa 35940aaaaaagggc caagtgcaga gcgagtatat ataggactaa aaaatgacgt aacggttaaa 35940
gtccacaaaa aacacccaga aaaccgcacg cgaacctacg cccagaaacg aaagccaaaa 36000gtccacaaaa aacacccaga aaaccgcacg cgaacctacg cccagaaacg aaagccaaaa 36000
aacccacaac ttcctcaaat cgtcacttcc gttttcccac gttacgtaac ttcccatttt 36060aacccacaac ttcctcaaat cgtcacttcc gttttcccac gttacgtaac ttcccatttt 36060
aagaaaacta caattcccaa cacatacaag ttactccgcc ctaaaaccta cgtcacccgc 36120aagaaaacta caattcccaa cacatacaag ttactccgcc ctaaaaccta cgtcacccgc 36120
cccgttccca cgccccgcgc cacgtcacaa actccacccc ctcattatca tattggcttc 36180cccgttccca cgccccgcgc cacgtcacaa actccacccc ctcattatca tattggcttc 36180
aatccaaaat aaggtatatt attgatgatg 36210aatccaaaat aaggtatatt attgatgatg 36210
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| CN117568405A (en) * | 2023-11-14 | 2024-02-20 | 武汉凯德维斯生物技术有限公司 | Oncolytic adenovirus recombinant vector, construction method and application thereof |
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| CN117568405A (en) * | 2023-11-14 | 2024-02-20 | 武汉凯德维斯生物技术有限公司 | Oncolytic adenovirus recombinant vector, construction method and application thereof |
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