CN114231504A

CN114231504A - Oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and construction method and application thereof

Info

Publication number: CN114231504A
Application number: CN202111445811.9A
Authority: CN
Inventors: 马丁; 高庆蕾; 李飞; 刘晨; 何良
Original assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Current assignee: Tongji Hospital Affiliated To Tongji Medical College Of Huazhong University Of Science & Technology
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-03-25

Abstract

The invention discloses an oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, a construction method and application thereof, and belongs to the technical field of medical genetic engineering. The invention deletes 27 bases of 920-946nt region in E1A conserved sequence 2 region of human 5-type adenovirus gene, inserts gene sequence coding tumor targeting peptide TMTP1 in 19641-19655nt region of Hexon hypervariable region 5, simultaneously deletes E3 region in 29477-29714nt region of ADP gene to form deleted region, inserts full length coding sequence coding HSV-TK gene in the deleted region and introduces Cla1 enzyme cutting site. The invention also discloses a construction method and application of the oncolytic adenovirus recombinant. The oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has ideal targeting effect and strong killing effect.

Description

Oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and construction method and application thereof

Technical Field

The invention relates to an oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, a construction method and application thereof, and belongs to the technical field of medical genetic engineering.

Background

Gene therapy (gene therapy) refers to the introduction of foreign genes into target cells to correct or compensate for diseases caused by gene defects or abnormal expression of genes. As a gene therapy vector, the development prospect of the oncolytic virus for treating malignant tumor is good. Among the numerous vectors for oncolytic viral therapy, recombinant adenoviral vectors are most widely used and have been recognized for their clinical feasibility and safety. The adenovirus gene therapy vector has the following biological advantages and clinical application advantages: (1) the infection spectrum is wide, the cell which is in the division stage or the stationary stage can be effectively attacked by various tissue-derived cells, and the anti-cancer spectrum is wide; (2) after the adenovirus enters cells, the adenovirus is not integrated into host chromosomes, so that the risk of mutation and carcinogenesis is avoided, the clinical application is safe, only cold-like symptoms are generated after the adenovirus is used, and the suppression of hematopoietic function and immunologic function is avoided; (3) the clinical application is convenient, and the oral cavity (such as abdominal cavity, thoracic cavity and cranial cavity) administration, local or tumor in-vivo direct injection (one point or a plurality of points), interventional therapy and other ways are provided; (4) the adenovirus can be continuously expressed in vivo for only two to three weeks, and is particularly suitable for tumor treatment; (5) intravenous or topical application produces only a mild inflammatory response with minor side effects; (6) clinical grade quantities of adenovirus are easy to produce and purify. Based on the advantages, more and more adenovirus vectors are constructed and generated, and the good clinical application prospect is shown.

Although adenovirus has obvious advantages compared with other various gene therapy vectors, the existing adenovirus therapy vectors at home and abroad gradually make some breakthrough progress, but still have some insurmountable defects to limit the wide application of the adenovirus, which are specifically as follows: (1) liver tropism. After the adenovirus enters an organism, no matter intravenous systemic injection or intratumoral injection, local injection in thoracic cavity, abdominal cavity and the like, in the organism, the coagulation factor ten (Fx) can rapidly recognize adenovirus capsid protein Hexon, is combined with the adenovirus capsid protein Hexon and brings the adenovirus to the liver, the other side of the coagulation factor is combined with Heparan Sulfate proteoglycan (HSPGS for short) on the surface of the hepatocyte, namely, the coagulation factor forms a bridge between the adenovirus and the hepatocyte, and the adenovirus captured by the liver is gradually phagocytized by kupffer cells or macrophages in the liver. The unremoved adenovirus is largely replicated in the liver, resulting in acute liver injury, manifested by a rapid rise in transaminase. (2) Adenovirus infects tumor cells, depending on the Coxsackie Adenovirus Receptor (CAR) on the surface of the tumor cell. In most tumor cells, the receptor is in a low expression state, which is not beneficial to the adenovirus entering the tumor cells to play a killing role.

In summary, there are various drawbacks in the current tumor treatment technology and practice, and therefore, there is a need to provide a new therapeutic application approach with both targeting and potent killing, so as to solve the deficiencies in the prior art.

Disclosure of Invention

One of the purposes of the invention is to provide an oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK.

The technical scheme for solving the problems is as follows: an oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is Ad 5/delta E1A/TMTP1 delta ADP/HSV-TK, the nucleotide sequence of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is shown as SEQ ID No.23, 27 bases shown as SEQ ID No.1 in a 920nt-946nt region are deleted in an E1A conserved sequence 2 region of a human type 5 adenovirus gene, a gene sequence shown as SEQ ID No.15 and used for coding tumor targeting peptide TMTP1 is inserted in a 19641nt-19655nt region of a Hexon hypervariable region 5, the E3 region is deleted in a 29477nt-29714nt region of the ADP gene to form a deletion region, the full-length coding sequence shown as SEQ ID No.22 and used for coding the HSV-TK gene is inserted in the deletion region, and a Cla1 enzyme cutting site is introduced.

The inventors of the present application, in order to obtain the above oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, performed the following work:

human adenovirus type 5, having the name Human adenovirus type 5 and Ad5 for short.

27 bases of 920nt-946nt shown in SEQ ID NO.1 are deleted in an E1A conserved sequence 2 region (CR2) of the human type 5 adenovirus gene, and the effect of keeping E1a transcription activation characteristics as far as possible is achieved while the Rb binding characteristics of E1a protein are inactivated.

The tumor targeting peptide TMTP1 has the obvious advantages of targeting high metastatic potential tumor cells, identifying tumor subclinical micrometastasis in early stage, being selectively swallowed by the tumor cells, having stronger tumor cell toxic effect and the like. The invention inserts the gene sequence of the coding tumor targeting peptide TMTP1 shown in SEQ ID NO.15 into the human type 5 adenovirus, thereby obviously improving the targeting of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK.

HSV-TK gene and herpes simplex virus thymidine kinase gene are common suicide gene, and the thymidine kinase coded by the gene can convert the drug precursor which is nontoxic or has low toxicity to cells into strong cytotoxic substances so as to achieve the aim of killing tumor cells. The invention inserts the full-length coding sequence of the coding HSV-TK gene shown in SEQ ID NO.22 into the human type 5 adenovirus (Ad5), so that the HSV-TK gene is specifically expressed in the tumor, and the tumor cell apoptosis is promoted, thereby remarkably improving the apoptosis promotion effect of an oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK and the killing effect of an oncolytic virus cytolytic cell.

Furthermore, the gene sequence of the tumor targeting peptide TMTP1 of the embodiment of the invention is inserted into the 2, 5 and 7 regions of the Hexon hypervariable region of the human adenovirus type 5 gene, and the optimal adenovirus insertion region which can be replicated in tumor cells at high copy number is screened as the hypervariable region 5 by in vitro experiments. The insertion of a gene sequence for encoding a tumor targeting peptide TMTP1 in a hypervariable region 5 of the Hexon disrupts the normal expression of the Hexon protein, inhibits the binding of Fx and Hexon and prevents the adenovirus from being phagocytosed by the liver. The tumor targeting peptide TMTP1 can reduce the dependence of adenovirus on the Coxsackie Adenovirus Receptor (CAR) on the surface of a tumor cell and increase the affinity of the adenovirus to the tumor cell; the integrity of a high mutation region of the Hexon is damaged, the aggregation of the adenovirus in the liver is reduced, and the damage of the adenovirus to the liver is reduced.

In conclusion, as shown in fig. 1, the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK of the present invention can specifically recognize tumors and their metastases, has higher tumor targeted replication ability, can exert both apoptosis-promoting effect of HSV-TK gene and killing effect of oncolytic virus lytic cells, and has strong killing effect on cisplatin-resistant cell lines, both ideal targeting effect and strong killing effect, as proved by in vitro and in vivo experiments.

The oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has the beneficial effects that:

1. the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has strong selectivity of tumor selective replication and therapeutic gene expression;

2. the yield of the progeny oncolytic adenovirus in the tumor cell is high, a high-concentration virus treatment ring can be generated at the local part of the tumor after the cell is cracked, and a strong bystander effect can be formed by combining the advantages of treatment targets;

3. oncolytic adenovirus in tumor cells is efficiently amplified in a large amount, and simultaneously, a large amount of target therapeutic genes are transcribed, so that the tumor cells become a processing plant for synthesizing therapeutic proteins;

4. the immunoregulatory protein of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has complete functions, and the elimination of recombinant adenovirus by an organism is avoided to a certain extent;

5. the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has wide anticancer spectrum. The in vivo model research of the tumor animal model proves that the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has obvious treatment effect on all tested tumor models through the administration way of local tumor injection or intraperitoneal injection, can effectively inhibit tumor metastasis, and has no obvious treatment-related toxicity on treated animals.

The second purpose of the invention is to provide a method for constructing the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK.

The technical scheme for solving the problems is as follows: the construction method of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK comprises the following steps:

step 1: targeted deletion of human adenovirus type 5 genes

Utilizing gene synthesis and homologous recombination to directionally delete 27 bases of 920nt-946nt in the E1A conserved sequence 2 region of the human 5-type adenovirus gene as shown in SEQ ID NO.1 to obtain the directionally deleted human 5-type adenovirus gene Ad 5/delta E1A;

step 2: preparation of Ad 5/. DELTA.E 1A/TMTP1

Inserting a gene sequence which is shown as SEQ ID NO.15 and encodes tumor targeting peptide TMTP1 into the 19641nt-19655nt region of the Hexon hypervariable region 5 of the human adenovirus type 5 gene obtained in the step 1 after targeted deletion to obtain Ad 5/delta E1A/TMTP 1;

and step 3: preparation of oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMTP 1. DELTA.ADP/HSV-TK carrying TMTP1 and HSV-TK

The E3 region of Ad 5/delta E1A/TMTP1 obtained in step 2 is located in the 29477nt-29714nt region of the ADP gene to form a deletion region, the full-length coding sequence of the HSV-TK gene shown in SEQ ID NO.22 is inserted into the deletion region, and the Cla1 enzyme cutting site is introduced, so that the oncolytic adenovirus recombinant Ad 5/delta E1A/TMTP1 delta ADP/HSV-TK carrying TMTP1 and HSV-TK shown in SEQ ID NO.23 is obtained.

The construction method of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has the beneficial effects that:

1. the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK constructed by the invention is inserted with a gene sequence coding tumor targeting peptide TMTP1 and a full-length coding sequence coding HSV-TK gene, has the advantages of tumor specific replication, high tumor and metastatic tumor targeting property, specific and mass expression of exogenous therapeutic genes, strong tumor specific bystander effect and the like, has high therapeutic index, can solve the key defects in the current tumor therapy technology and practice, and provides an ideal therapeutic application path with targeting and strong killing for tumor therapy.

2. The construction method is simple, easy to operate, low in cost and wide in application prospect.

The third purpose of the invention is to provide the application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is applied to the preparation of medicines for treating tumors.

The application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK has the beneficial effects that:

the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK can be used for preparing medicaments for treating tumors, develops a novel medicament for treating tumors and the application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.

Another object of the present invention is to provide another use of the oncolytic adenoviral recombinant carrying TMTP1 and HSV-TK described above.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is applied to the preparation of a gene therapy vector.

the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK can be used for preparing gene therapy vectors, not only develops a new gene therapy vector, but also develops the application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is applied to preparation of drugs for improving drug resistance of antitumor chemotherapeutic drugs.

the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK can be used for preparing drugs for improving drug resistance of prepared antitumor chemotherapeutic drugs, not only develops new drugs for improving drug resistance of prepared antitumor chemotherapeutic drugs, but also develops application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is applied to the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.

the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK can be used for preparing an anti-tumor chemotherapeutic drug sensitizer, develops a new anti-tumor chemotherapeutic drug sensitizer and the application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.

Drawings

FIG. 1 is a schematic diagram of the structure of oncolytic adenoviral recombinant Ad5/Δ E1A/HVR5/TMTP1 Δ ADP/HSV-TK carrying TMTP1 and HSV-TK in accordance with an embodiment of the present invention.

FIG. 2 is a histogram of replication of adenovirus with different insertion regions of the hypervariable region of Hexon in tumor cells according to example 5 of the present invention.

FIG. 3 is a histogram of Ad5/Δ E1A/HVR5/TMTP1 Δ ADP/HSV-TK replicated in different cells in example 6.

FIG. 4 is a graph of tumor growth in mice in different treatment groups of example 6.

FIG. 5 shows the adenovirus content in each tissue of mice of different treatment groups in example 6.

FIG. 6 shows the results of alanine Aminotransferase (ALT) assay in animals tested for the abdominal orthotopic metastasis model in example 7 of the present invention.

Fig. 7 is a graph showing the results of an aspartate Aminotransferase (AST) experiment in an experimental animal of a abdominal cavity in situ metastasis model in example 7 of the present invention.

FIG. 8 is the result of urea nitrogen (BUN) test in experimental animals of the abdominal cavity in situ metastatic tumor model in example 7 of the present invention.

FIG. 9 shows the Creatinine (Creatinine) test results of the experimental animals of the abdominal cavity in situ metastasis model in example 7 of the present invention.

Detailed Description

The principles and features of this invention are described below in conjunction with the following detailed drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.

As shown in figure 1, the invention adopts an adenovirus recombination system AdEasy to construct the target adenovirus by a three-time homologous recombination method. Unless otherwise noted, the enzymes used in the present invention were purchased from gibco, usa, PCR primers were synthesized from biotechnology limited, beijing optimak, and cell lines were purchased from ATCC cell bank, usa, and were cultured using corresponding media, which were purchased from gibco, usa.

Example 1: construction of pAd5/Δ E1A adenovirus packaging plasmid

Step 1.1: construction of shuttle plasmid Blunt-Zero-E1A/Δ E1A

The pXC 1-delta E1A plasmid is 27 bases shown in SEQ ID NO.1 in the 920nt-946nt region deleted in the E1A conserved sequence 2 region of the human adenovirus 5 gene. The construction method comprises the following steps:

the pXC1 plasmid was purchased from MicrobixBiosystemInc. (Toronato, Ontario, Canada, Cat.: PD-01-03) and contains the human adenovirus type 5 (Ad5)22nt-5790nt sequence. The 920nt-946nt region was deleted by 3 PCR methods.

Acquisition of fragment 1: primer 1: 5' -cgggatccgggcccccatttcc-3' (SEQ ID NO2), corresponding to 9883-; primer 2: 5' -gtcactgggtggatcgatcacctccggtac-3' (SEQ ID NO3), corresponding to 922nt-905nt, the underlined part is the part complementary to primer 3; taking pXC1 as a template to carry out PCR reaction, wherein the total volume of the reaction system is 100 mu l and comprises: containing MgCl ₂10. mu.l of the buffer solution for PCR of (1); 2mMdNTP, 10 μ l; 10 μ M primer 1, 1 μ l; 10 μ M primer 2, 1 μ l; pXC110 ng/. mu.l, 1. mu.l; pfu high fidelity Taq enzyme, 2.5 mul; adding water to 100 μ l; the reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 45 s; 60 ℃ for 1 min; 72 ℃ for 2 min; 28 cycles in total; extension at 72 ℃ for 10 min. The PCR product is 940bp long, a fragment 1 is formed, and after separation and purification by conventional electrophoresis, the concentration is detected for subsequent PCR reaction.

Acquisition of fragment 2: primer 3: 5' -gaggtgatcgatccacccagtgacgacgag-3' (SEQ ID NO 4), corresponding to 911-947nt, the underlined part is the part complementary to primer 2; primer 4: 5' -tgctctagacacaggtgatgtcg-3' (SEQ ID NO5), corresponding to 1344nt-1325nt, with the XbaI cleavage site underlined; taking pXC1 as a template, carrying out PCR reaction under the same reaction conditions as above, wherein the product is 400bp long to form a fragment 2, and detecting the concentration for subsequent PCR reaction after conventional electrophoretic separation and purification.

Acquisition of fragment 3: mixing 50 ng/2. mu.l fragment 1 with 25 ng/1. mu.l fragment 2, and performing PCR reaction by using the mixture as a template, wherein an upstream primer is primer 1, a downstream primer is primer 4, and the reaction conditions are the same as above, and the product is about 1400bp, thereby forming fragment 3.

After purification with QIAquick 8PCR product purification kit (QIAGEN, German, Cat: 28142), the mixture was digested with BamHI and XbaI overnight, and the digested fragments were separated by electrophoresis on a 1% agarose gel and recovered for cloning. pXC1 was double digested with BamHI and XbaI overnight, and the digested products were separated by electrophoresis in 1% agarose gel to give 2 bands of approximately 1400bp and 8500bp, and the 8500bp fragment was recovered for cloning. Taking 40ng of 8500bp pXC1 enzyme-digested fragment and 90ng of fragment 3, using DNA T4 ligase to carry out ligation reaction, taking 1.5 mu l of transformed 100 mu l of DH5 alpha competent bacteria, spreading the cells on a dish for overnight culture, picking out a single colony clone the next day, extracting amplified plasmids in the colony clone, and obtaining pXC1 plasmid mutant pXC 1-delta E1A with deletion of 121-plus 129AA 920nt-946nt through DNA sequencing, identification and screening.

Taking pXC 1-delta E1A plasmid as a template, and carrying out PCR amplification reaction to obtain an E1A sequence with 27bp deletion. Wherein, the upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQ ID NO.6), downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO. 7). The PCR amplification reaction system is as follows: TransStart FastPfu Fly DNA Polymerase, 1. mu.l; 5 × TransStart FastPfu Fly Buffer, 10 μ l; 10. mu.M of the forward primer, 1. mu.l; 10. mu.M of downstream primer, 1. mu.l; 2.5mM dNTPs, 4. mu.l; adding nuclease-free water to make up the system to 50 mu l; template, 10-30 ng; a total of 50. mu.l was obtained. The procedure of PCR amplification reaction is 95 ℃ for 2 min; at 95 ℃, 20s, -5 ℃, 20s, 72 ℃, 3min, 35 cycles; 72 ℃ for 5 min; and preserving at 4 ℃.

The E1A sequence was ligated into pEASY-Blunt-Zero Blunt-end ligation plasmid (available from Beijing Quanjin Biotechnology Co., Ltd., product No. CB501) by T4 ligase (available from Saimei Feishell technology Co., Ltd., product No. el0011) to obtain shuttle plasmid Blunt-Zero-E1A/delta E1A.

And (3) carrying out PCR amplification reaction by using a shuttle plasmid Blunt-Zero-E1A/delta E1A as a template and adopting a primer carried in the pEASY-Blunt-Zero Blunt end ligation plasmid kit to obtain an E1A shuttle fragment with 27bp deletion, and purifying the shuttle fragment for homologous recombination. The PCR reaction system and the amplification reaction procedure were as described above.

Step 1.2: construction of backbone plasmid pAd-Easy-1

The pAd-Easy-1 vector (available from Agilent technologies, Inc., catalog number 240005) was cleaved with restriction enzyme pme1, and the DNA cleavage product was extracted with phenol chloroform to obtain a backbone fragment for the first homologous recombination, and the DNA concentrations of the shuttle fragment and the backbone fragment were determined.

Step 1.3: preparation of Escherichia coli BJ5183 electroconception

Escherichia coli BJ5183 (purchased from Beijing Ke Rui Si Bing Biotechnology Ltd, catalog number st10779) stored at-80 deg.C is thawed, and 1mL of the bacterial liquid is added into 1000mL of LB medium with streptomycin concentration of 30. mu.g/mL. After the bacteria are shaken at the speed of 250rpm/min and the temperature of 37 ℃ for 7h to 8h, the liquid is slightly turbid after observation every 10 min. Subpackaging the bacterial liquid into precooled centrifuge tubes, centrifuging at 4 ℃ and 3000rpm/min for 10min, and discarding the supernatant. And (4) precooling a proper amount of sterile double-distilled water washing bacterial residues twice. Precooling a proper amount of sterile 10% glycerol bacterial washing residues twice. Discarding the supernatant to obtain Escherichia coli BJ5183 electrotransformation competence, and subpackaging in 1.5mL centrifuge tubes for preservation at-80 deg.C.

Step 1.4: homologous recombination construction of pAd 5-E1A/delta E1A adenovirus packaging plasmid

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 competence obtained in step 1.3 using a GenePulser XcellTM electroporation apparatus (available from Bio-Rad) under conditions of 2.5Kv and 25. mu.F. Positive bacteria were screened for kanamycin resistance. Selecting smaller bacterial plaques in bacterial plates, culturing in LB culture medium, extracting plasmids in small quantity, and using an upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQ ID NO.8) and the downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO.9), performing PCR amplification reaction, performing the same reaction system and reaction procedure as the step 1.1, and sequencing to determine whether the E1A region lacks the 920bp-946bp region. The extracted plasmid was transformed into high copy number bacterium DH10B (purchased from Saimer Feishell science Inc., cat # 18290015) to obtain pAd 5-E1A/. DELTA.E 1A adenovirus packaging plasmid, abbreviated pAd 5/. DELTA.E 1A.

Example 2: construction of pAd 5/. DELTA.E 1A/Hexon (HVR/TMTP1) plasmid vector

Step 2.1: construction of shuttle vector pEASY-Blunt-Zero-Hexon (HVR/TMTP1)

pBHGE3 was purchased from Microbix biosystems Inc. (Toronato, Ontario, Canada, Cat: PD-01-12), and this plasmid contained the entire genomic sequence except for the Ad5 packaging signal (194- "358 nt"). pBHGE3 was obtained from Microbix Biosystem Inc. in a total amount of 10. mu.g, and was electroporated into competent bacteria, positive clones were selected, plasmids were extracted, and the obtained plasmids were purified by CsCl2-EB ultracentrifugation. The homologous recombination method is used for obtaining the delta 920-946Ad5 recombinant adenovirus construct and comprises the following steps:

in 15cm culture dish, 7.5X 10⁵293 cells, 10% FBS DMEM, by day two, cells should be 1-1.5X 10⁶Approximately 70% of the cells fused; and replacing with fresh culture solution 3-4h before transfection. Preparation of co-transfected DNA-calcium phosphate solution: 1600 μ l of sterilized 2 XHBS (280mM NaCl, 43mM HEPES, 10mM KCl, 10mM Na)₂HPO₄·7H₂O, 2% dextrose, pH 7.05-7.15); 42. mu.g each of pBHGE3 and Δ 920-946pXC 1; adding sterilized double distilled water to 2840 μ l, mixing, slowly adding 50 μ l 2.5M CaCl₂The mixture was inverted and mixed, and the DNA/CaCl was allowed to stand at room temperature₂Precipitating for 45-60min to form slightly turbid precipitate. Adding 500. mu.l of the above mixture to 293 cells in a 5ml 60mm dish at 37 ℃ with 5% CO₂Incubated for 4-6h, the liquid was aspirated and washed once with PBS. Transfection efficiency was promoted by treatment with 15% glycerol/DMEM for 1-2min, washed once with PBS and replaced with complete medium. 1.8% agarose with low melting point is prepared, autoclaved, subpackaged into 5ml, melted in boiling water before use, kept at 45 ℃, added with 4% FBS DMEM with equal amount when used, and immediately spread into a culture dish. The culture medium was aspirated off, and 5ml of the above-mentioned medium was added. Every 4-5 days, 3ml of the above liquid was added. 14-21d, plaques appeared, 6-12 plaques were selected. The plaques were transferred to serum-free DMEM medium in 1.5ml EP tubes and incubated at 37 ℃ for 24 h. Seeding in 24-well plates 1X 10⁵293 cells in 10% FBS DMEM, by day two, cells should be 2X 10⁵About 70% of the cells were fused, the liquid was aspirated, and 100. mu.l (about 10) of the above-mentioned incubation liquid was taken³Virus) was added and the liquid was gently shaken 3 times at 37 ℃ with 5% CO₂And (5) performing medium incubation for 90 min. Add complete medium to 1ml and place cells at 37 ℃ with 5% CO₂And (4) incubating for 5-10 days until complete CPE appears, namely cytotoxic effect, and the cells are rounded and float and mainly comprise nucleolus. If after 10d complete CPE was not present, it was suggested that the virus titer was too low and a second round of amplification was required. Performing three cycles of freezing/thawing on the culture plate to release virus, collecting lysate in 15ml test tube, centrifuging at maximum speed for 10min, collecting supernatant, freezing at-80 deg.C, and collecting the liquid as second generation virus,about 5 × 10⁷Viral/ml. The above viruses were re-amplified at 75cm²Seed in a Petri dish at 5X 10⁶293 cells in 10ml 10% FBS DMEM, by day two, cells should be 1X 10⁷Approximately 70% cell fusion; replacing with fresh culture solution 3-4h before transfection; adding 1ml of second-generation virus stock solution into 1ml of complete culture medium for transfection; the MOI is about 5; remove 75cm²Adding the liquid into a culture dish, and slightly shaking for three times; at 37 deg.C, 5% CO₂Incubating for 90 min; 9ml of 2% FBS DMEM was added thereto at 37 ℃ with 5% CO₂And (4) incubating for 4-7d, and extracting virus DNA for screening positive viruses. Because the 293 cell genome contains the complete E1A gene, the 293 cell DNA is easily contaminated when extracting positive virus DNA, and the identification fails, therefore, the delta 920-946Ad5 is amplified once again in the tumor cell Hela for identification, and the steps are as follows: seeding in 6-well plates 1X 10⁵Hela cells in 10% FBS DMEM, and by the next day, cells were 2X 10⁵About 70% of the cells were fused, the liquid was aspirated, and 100. mu.l (about 10) of the filtrate was taken³Virus) was added and the liquid was gently shaken 3 times at 37 ℃ with 5% CO₂And (5) performing medium incubation for 90 min. Adding complete medium to 1ml, and placing the cells at 37 deg.C and 5% CO₂The cells were scraped and collected in a 1.5ml EP tube, the supernatant was discarded by centrifugation, 300. mu.l PBS was added and three cycles of freeze/thaw were performed to release the virus, the lysate was centrifuged at maximum speed for 10min, the supernatant was collected and frozen at-80 ℃ and DNA was extracted using Qiagen kit mini DNA isolation kit, according to the kit instructions. Performing PCR reaction by using virus DNA as a template, wherein an upstream primer: 5'-cgggatccgggcccccatttcc-3' (SEQ ID NO 10), downstream primer: 5'-tgctctagacacaggtgatgtcg-3' (SEQ ID NO 11), the total reaction system volume of 100. mu.l comprising: containing MgCl ₂10. mu.l of the buffer solution for PCR of (1); 2mM dNTP, 10. mu.l; 10. mu.M of the forward primer, 1. mu.l; 10. mu.M of downstream primer, 1. mu.l; viral DNA, 10 ng; pfu high fidelity Taq enzyme, 2.5 u; water was added to 100. mu.l. The reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 45 s; 60 ℃ for 1 min; 72 ℃ for 2 min; total 28 cyclesA ring; extension at 72 ℃ for 10 min. The PCR product is 1400bp, after conventional electrophoretic separation and purification, the detection concentration is used for DNA sequencing, and a sequencing primer: 5'-agccggagcagagagccttg-3' (SEQ ID NO12), and the clone with correct sequencing is selected, namely delta 920-and 946Ad 5. Adenovirus delta 920 and 946Ad5 are used as templates, primer5.0 software is adopted to design primers, and an upstream primer: 5'-ccagagtaggtgtaataagg-3' (SEQ ID NO.13) and the downstream primer: 5'-tagaaagtcaagtggaaatg-3' (SEQ ID NO.14), a Hexon fragment was obtained by PCR amplification of 18380bp-20388bp (i.e., Hexon region) of adenovirus, and the Blunt end of the fragment was ligated into pEASY-Blunt-Zero vector (available from Beijing Kogyo Biotech Co., Ltd., Cat. No. CB501) to obtain pEASY-Blunt-Zero-Hexon plasmid.

Designing primers by using primer5.0 software, inserting gene sequences which are shown as SEQ ID NO.15 and code a tumor targeting peptide TMTP1 into

regions

2, 5 and 7 of a hypervariable region of Hexon by using a double PCR amplification reaction to obtain PCR amplification products of pEASY-Hexon (HVR/TMTP1), then, the PCR products pEASY-Hexon (HVR/TMTP1) and pEASY-Blunt-Zero-Hexon plasmids are respectively double-digested by utilizing the specific digestion sites DraIII and SacI of the Hexon region, after the digestion products are recovered by cutting the gel, the products were ligated with T4 ligase, and after the ligation was transformed into DH10B competence, positive plaques were screened to obtain pEASY-Blunt-Zero-Hexon (HVR2/TMTP1) shuttle plasmid, pEASY-Blunt-Zero-Hexon (HVR5/TMTP1) shuttle plasmid and pEASY-Blunt-Zero-Hexon (HVR7/TMTP1) shuttle plasmid, respectively.

Step 2.2: extracting a large amount of the skeleton plasmid pAd 5-E1A/delta E1A (OMEGA, product number D6692-01) constructed in the step 1.4, utilizing a specific enzyme cutting site AsisI, carrying out enzyme cutting, and then extracting phenol and chloroform to obtain a linear skeleton plasmid.

Step 2.3: the PCR product of pEASY-Hexon (HVR/TMTP1) obtained in step 2.1 was used as a homologous recombination fragment, and the fragment was electrotransferred into E.coli BJ5183 electrotransfer competence obtained in step 1.3 simultaneously with the linear backbone plasmid obtained in step 2.2 under conditions of 2.5Kv and 25. mu.F. Positive bacteria are screened by kanamycin resistance, PCR amplification reaction is carried out, and pAd 5-E1A/delta E1A-Hexon (HVR2/TMTP1) plasmid, pAd 5-E1A/delta E1A-Hexon (HVR5/TMTP1) plasmid and pAd 5-E1A/delta E1A-Hexon (HVR7/TMTP1) plasmid are identified.

Example 3: construction of Ad 5/delta E1A/TMTP1/HSV-TK adenovirus vector

Step 3.1 construction of shuttle vector for adenovirus E3 region

PCR was performed using adenovirus Δ 920-946Ad5 as template (described above, including the entire sequence of adenovirus E3 region), with the upstream primer: 5'-tgtcaccactaactgctttactcg-3' (SEQ ID NO 16), downstream primer: 5'-gctgccctgcgtctttcta-3' (SEQ ID NO 17), and a 26342-31140 fragment (i.e., the entire fragment of the E3 region) was obtained and ligated into the pEASY-Blunt-Zero vector to obtain the pEASY-Blunt-Zero-E3 plasmid.

Step 3.2: plasmid pcDNA3.1-E3/Δ ADP is a backbone plasmid, which has a fragment with EcoRI cleavage sites at both ends, into which the complete adenovirus E3 region is inserted but from which a 29477bp-29714bp (the fragment between the two EcoRI cleavage sites of the adenovirus, i.e., the ADP region) fragment is deleted. The construction method comprises the following steps:

the Ad 5E 3 region 29477-29714nt was deleted by 3 PCR methods. Acquisition of fragment 1: primer 1: 5'-atacgcgcccaccgaaac-3' (SEQ ID NO 18), corresponding to 27306nt-27323 nt; primer 2: 5' -aatctatgg atatcgatagggtgggtcgctgtagtt-3' (SEQ ID NO 19), corresponding to 29477-495 nt, the underlined part is the complementary part to primer 3, atcgat is the Cla I cleavage site; carrying out PCR reaction by using Ad5 DNA as a template, wherein the total volume of the reaction system is 100 μ l and comprises: containing MgCl ₂10. mu.l of the buffer solution for PCR of (1); 2mM dNTP, 10. mu.l; 10 μ M primer 1, 1 μ l; 10 μ M primer 2, 1 μ l; ad5 DNA 200 ng/. mu.l, 1. mu.l; pfu high fidelity Taq enzyme, 2.5 u; water was added to 100. mu.l. The reaction conditions are as follows: 30s at 94 ℃; 30s at 94 ℃; at 46 ℃ for 1 min; 72 ℃ for 1 min; 30 cycles in total; extension at 72 ℃ for 10 min. The PCR product is 2207bp (fragment 1), and after separation and purification by conventional electrophoresis, the concentration is detected and used for subsequent PCR reaction.

Acquisition of fragment 2: primer 3: 5' -cgacccaccctatcgatatccatagattggacggactg-3' (SEQ ID NO 20), corresponding to 29714nt-29734n, the underlined part being the complementary part to primer 2, atcgat being the Cla I cleavage site; primer 4: 5'-atgtctttgaggcttggagg-3' (SEQ ID NO 21), corresponding to 30137nt-30118 nt; the PCR reaction conditions are the same as above, and the product is422bp (fragment 2), and after conventional electrophoretic separation and purification, the concentration is detected for subsequent PCR reaction.

Acquisition of fragment 3: the fragment 1 and the fragment 2 were mixed in equal amounts, and used as templates for PCR reactions, the upstream primer was primer 1, the downstream primer was primer 4, the reaction conditions were the same, the product was about 2612bp (fragment 3), after purification with QIAquick 8PCR product purification kit (QIAGEN, German, Cat: 28142), the product was digested overnight with EcoRI, the digested product was separated by 1% agarose gel electrophoresis and recovered, and the digested fragments were used for subsequent ligation reactions. pcDNA3.1(Invitrogen, U.S. A., Cat: V79020) was digested with EcoRI overnight, the digested product was separated by electrophoresis on a 1% agarose gel, and the digested fragment was recovered and dephosphorylated before use in subsequent ligation reactions. And connecting the PCR reaction product fragment 3 with enzyme-digested dephosphorylated pcDNA3.1, transferring 1.5 mu l of the PCR reaction product fragment into 100 mu lDH5 alpha competent bacteria, selecting positive clones, carrying out small extraction on plasmids, and carrying out DNA sequencing, identification and screening to obtain the pcDNA3.1 plasmid mutant, namely pcDNA3.1-E3/delta ADP, of which 29477nt-29714nt is deleted.

The full-length coding sequence of the HSV-TK gene shown in SEQ ID NO.22 is introduced into the Cla1 enzyme cutting site. ClaI enzyme digestion is connected into pcDNA3.1-E3/delta ADP plasmid.

Step 3.3: and (3) carrying out EcoRI digestion on the plasmid constructed in the step (3.2), connecting the plasmid into the pEASY-Blunt-Zero-E3 plasmid constructed in the step (3.1), and carrying out PCR amplification reaction by using a primer carried in the pEASY-Blunt-Zero-E3 plasmid kit to obtain an E3/HSV-TK fragment.

Step 3.4: SpeI digestion of pAd 5-E1A/. DELTA.E 1A-Hexon (HVR2/TMTP1) plasmid, pAd 5-E1A/. DELTA.E 1A-Hexon (HVR5/TMTP1) plasmid and pAd 5-E1A/. DELTA.E 1A-Hexon (HVR7/TMTP1) plasmid constructed in step 2.3, phenol chloroform extraction and recovery of linear backbone plasmid.

The linear backbone plasmid and the E3/HSV-TK fragment obtained in step 3.3 are electrotransferred into Escherichia coli BJ5183 electrotransferred and sensitive peptide obtained in step 1.3, the electrotransferred condition is 2.5Kv and 25 muF, and homologous recombination is carried out to respectively obtain pAd 5/delta 27/HVR2-TMTP 1/delta ADP/HSV-TK plasmid, pAd 5/delta 27/HVR5-TMTP 1/delta ADP/HSV-TK plasmid and pAd 5/delta 27/HVR7-TMTP 1/delta ADP/HSV-TK plasmid.

Example 4: obtaining, amplifying and purifying oncolytic adenovirus recombinant Ad 5/delta 27/TMTP 1/delta ADP/HSV-TK carrying TMTP1 and HSV-TK

Step 4.1: obtaining oncolytic adenovirus recombinant Ad 5/delta 27/TMTP 1/delta ADP/HSV-TK carrying TMTP1 and HSV-TK

Step 4.1.1: respectively extracting the pAd 5/delta 27/HVR2-TMTP 1/delta ADP/HSV-TK plasmid, pAd 5/delta 27/HVR5-TMTP 1/delta ADP/HSV-TK plasmid and pAd 5/delta 27/HVR7-TMTP 1/delta ADP/HSV-TK plasmid obtained in the step 3.4 in a large quantity, using PacI to cut the plasmids, extracting with phenol and chloroform, and recovering the cut products.

Step 4.1.2: the product recovered in step 4.1.1 was transferred to 293 cells 24-well plates with a confluency of 30% using lipo3000 liposome (purchased from seimer feishell technologies, cat # L3000001) transfection method. Adding fresh DMEM culture medium every 2 days until the fusion degree of 293 cells reaches more than 100%, collecting the cells, repeatedly freezing and thawing to crack and release the virus into the DMEM culture medium, and centrifuging to collect supernatant virus liquid.

Step 4.1.3: transfecting the 293 cell again by adopting a DMEM medium containing the adenovirus, repeating for about 2 weeks until the 293 cell has a CPE effect, collecting the cell, and repeatedly freezing and thawing for lysis to obtain a supernatant virus solution.

Step 4.1.4: three Ad 5/. DELTA.27/TMTP 1/. DELTA.ADP/HSV-TKs obtained in step 4.1.3 were identified, and E1A, Hexon and E3/ADP regions were sequenced after PCR using the corresponding primers, respectively.

Step 4.2: amplification, purification and titer determination of oncolytic adenovirus recombinant Ad 5/delta 27/TMTP 1/delta ADP/HSV-TK carrying TMTP1 and HSV-TK

Step 4.2.1: amplification step 4.1.4 sequencing the correct oncolytic adenovirus recombinant Ad 5/delta 27/TMTP 1/delta ADP/HSV-TK carrying TMTP1 and HSV-TK, detecting the titer (MOI) of adenovirus seeds, determining the virus amount infecting 293 cells according to the MOI value, and collecting the cells when CPE effect appears within 48h-72 h. The amplification is repeated until the virus amount reaches the experimental requirement (10)⁹pfu/ml-10¹¹pfu/ml)。

Step 4.2.2: the adenovirus is purified by the traditional CsCl gradient centrifugation method, and the purified adenovirus is obtained by the dialysis of virus dialysate. Virus particle counts were measured using the UV absorbance method and MOI was measured using the Adeno-XTM Rapid Titer kit (Clonetech, cat # 632250).

Example 5: selection of optimal insertion region for oncolytic adenovirus recombinants carrying TMTP1 and HSV-TK

The purpose of this example was to determine the efficiency of oncolytic adenoviral recombinants carrying TMTP1 and HSV-TK inserted into three different regions of the

hypervariable region

2, 5, 7 of Hexon for infection of tumor cells, in order to select the targeted peptide insertion region that has the best effect on infection of tumor cells.

About 2X 10 cells were seeded in 6-well plates⁵Skov3 tumor cells in 10% FBS DMEM medium, about 70% cell fusion by the next day, aspirating the liquid, adding 2mL of fresh 10% FBS DMEM medium, diluting the oncolytic adenovirus with MOI 1 to 100 μ L in the medium, adding to the culture well, gently shaking the liquid three times, and incubating at 37 deg.C and 5% CO₂After incubation for 12h, 24h and 36h in the incubator, cell residues are collected, DNA is extracted, and the relative copy number of the adenovirus DNA of different treatment groups is identified by real-time quantitative PCR. The results are shown in FIG. 2, the oncolytic adenoviral recombinant Ad5/Δ E1A/HVR5/TMTP1 Δ ADP/HSV-TK carrying TMTP1 and HSV-TK replicated most actively in tumor cells, and its nucleotide sequence is shown in SEQ ID NO. 23. The hypervariable region 5, the HVR5, which is the optimal adenoviral insert capable of high copy number replication in tumor cells was selected as the final region by in vitro experiments.

Example 6: characterization of therapeutic Effect of oncolytic adenovirus Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK

Step 6.1: identification of oncolytic adenovirus recombinant in vitro tumor cell killing Effect carrying TMTP1 and HSV-TK

The purpose of the experiment is to identify the killing effect of the Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK novel oncolytic adenovirus on a series of tumors and normal cells, and the experiment takes wt-Ad5 as a positive control and replication-defective virus Ad5CMV-GFP as a negative control. The cell lines are selected to be a human lung cancer cell line A549, a human high-metastatic prostate cancer cell line PC3M-1E8, a human osteoblastoma cell line U-2OS, a human colon cancer cell line HCT-8, a human breast cancer cell line MCF-7, a human ovarian cancer cell line SKOV-3, a human liver cancer cell line HepG2, a human esophageal cancer cell line Eca-109, a human gastric cancer cell line MKN-45, a human brain glioma cell line SHG-44, a human head and neck squamous cell cancer cell line AGZY-973, a human pancreatic cancer cell line BxPC-3, a human endometrial cancer cell line HEC-1-A, a human rectal cancer cell line Colo320 and a human nasopharyngeal cancer cell line CNE-2. The selected cell lines have different P53, RB gene phenotypes; have different tissue origins and therefore the experimental results may represent different types of tumours. The selected normal cells are primary vascular endothelial cells, primary lung small trachea epithelial cells, prostate epithelial cells and bone marrow mononuclear cells, and the selection principle is as follows: after intravenous administration, a large number of adenovirus normal tissue cells will be contacted.

Seeding in 24-well plates 1X 10⁵Tumor or normal epithelial cells in culture medium of 10% FBS DMEM or RIPM1640, about 70% of the cells fused the next day, aspirated, diluted to 100 μ l with the MOI required to reach 70%, added by gently shaking the liquid three times at 37 deg.C and 5% CO₂The culture box of (1) was incubated for 90min, DMEM or RIPM1640 medium containing 10% FBS was added to 2ml while Ganciclovir (GCV) was added, the cells were left at 37 ℃ and 5% CO₂The culture box is incubated for 3 days, and the apoptosis rate of the cells is detected by a flow cytometer. The specific experimental results are shown in table 1.

TABLE 1

The experimental result shows that the positive control wt-Ad5 does not selectively crack tumor cells and normal cells; the killing effect of replication-defective adenovirus Ad5CMV-GFP on cells is very weak, the oncolytic adenovirus Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK selectively kills tumor cells, the effect is obviously stronger than that of wt-Ad5 on cells, and meanwhile, the recombinant adenovirus construct has no obvious influence on normal control cell lines. The results fully show that the oncolytic adenovirus Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK selectively and efficiently kills tumor cells.

Step 6.2: identification of the efficiency of tumor cell replication of oncolytic adenoviruses

To further quantify the replication efficiency of the novel oncolytic adenoviral construct Ad 5/. DELTA.E 1A/HVR5/TMTP 1/. DELTA.ADP/HSV-TK in tumor cells, the following experiments were performed:

in 24-well plates, 1X 10⁵Two tumor cells SKOV3 and C13K or normal cells HUVEC and LO2 in 10% FBS DMEM or RIPM1640, about 70% of the cells fused the next day, aspirated, added Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK 10MOI diluted to 100 μ l, gently shaken three times, at 37 deg.C, 5% CO₂Incubate for 90 min. Adding DMEM medium containing 2% FBS to 1ml, and placing the cells at 37 deg.C and 5% CO₂The culture of (4) is incubated for 1, 2, and 3 days.

Adding 300 μ l PBS solution, performing three cycles of freeze/thaw to release virus, centrifuging lysate for 10min at maximum speed, collecting supernatant, freezing at-80 deg.C, and using TCID₅₀The titer of adenovirus was measured in 293 cells.

As shown in FIG. 3, the potency of Ad 5/. DELTA.E 1A/HVR5/TMTP 1/. DELTA.ADP/HSV-TK after replication in tumor cells was 4-5 times stronger than that of the original, while the infectious potency did not change significantly in normal cells, and the results are sufficient to indicate that the oncolytic adenovirus has the property of highly efficient specific replication in tumor cells.

Step 6.3: characterization of in vivo antitumor Effect of oncolytic adenovirus Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK

Tumor cell animal models: selecting BALB/c nude mice of 4-6 weeks of age, taking gastric cancer cell line MKN-45 cells of logarithmic growth phase, and adjusting cell density to be 1 multiplied by 10⁶100 μ l, 200 μ l subcutaneously in the right dorsal side of each nude mouse, when the average tumor diameter reached 0.4-0.6cm, the nude mice were divided into 4 groups at random according to the tumor size, i.e., a medium control group, a wt-Ad5 group, an Ad5CMV-GFP group and an Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK group, and 5 nude mice in each group were injected intratumorally with PBS, wt-Ad5, Ad5CMV-GFP and Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK, 2 × 10 each⁸pfu/(only)Times) once daily for 5 consecutive days. Intraperitoneal injection of GCV 125 mg/kg on days 2-18^-1·d^–1. Tumor volumes were observed and measured 2 times per week until the end of the experiment (50 days or tumor volumes greater than 2 cm)³)。

The experimental results are shown in fig. 4, and at the end point of the experiment, the mean tumor inhibition rate of the wt-Ad5 group is 26% +/-6%; the tumor inhibition rate of the Ad5CMV-GFP group is-7% +/-4%; three nude mice in Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK group completely regressed tumors, the average tumor inhibition rate is 91% + -8%, and the results fully indicate that the novel oncolytic adenovirus Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK has a determined in vivo anti-tumor effect.

Step 6.4: in vivo identification of oncolytic adenoviral recombinants carrying TMTP1 and HSV-TK specifically targeting tumor cells

The specific experimental method comprises the following steps:

4-6 weeks of BALB/c nu nude mice, 2 x 10 abdominal cavity planting⁶skov3 ovarian cancer cells, 4 weeks later, nude mice were divided into two groups (n ═ 4), and each was intraperitoneally injected with 1 × 10 cells⁸pfu novel oncolytic adenovirus Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK and its control virus Ad5/Δ E1A. After 48h, the liver, spleen, kidney and tumor tissues of each mouse were taken out, DNA of each tissue was extracted, and the DNA content of adenovirus in 400ng of tissue DNA was detected, with the results shown in FIG. 5: the control virus Ad 5/delta E1A has aggregation in liver, lung and spleen and is distributed less in tumor tissues, the Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK is mainly distributed in tumor tissues, and the liver uptake is obviously reduced; the content in tumor tissues is nearly 100 times that of the control virus Ad 5/delta E1A.

Example 7: identification of oncolytic adenovirus Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK for hepatorenal toxicity

The specific experimental method comprises the following steps: 4-6 weeks of BALB/c nu nude mice, 2 x 10 abdominal cavity planting⁶skov3 ovarian cancer cells, 4 weeks later, nude mice were divided into three groups (n-4) of: a-blank group, b-single injection Ad 5/delta E1A, c-single injection Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK, wherein the b group and the c group are respectively injected into 1 × 10 parts of the abdomen continuously⁸PFU contrast virus Ad 5/delta E1A and corresponding novel oncolytic adenovirus Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK. After 48 hours, blood was drawn from the orbit and the liver and kidney functions of the experimental mice in each group were measured, including: alanine transaminase (ALT), aspartate transaminase (AST), Creatinine (Creatinine) and urea nitrogen (BUN) were obtained, as shown in FIGS. 6-9, but in the control and experimental groups, renal function was not significantly impaired, but in mice injected with control virus Ad5/Δ E1A, alanine transaminase (ALT) and aspartate transaminase (AST) were significantly elevated, whereas in mice injected with Ad5/Δ E1A/HVR5/TMTP1/Δ ADP/HSV-TK, liver function was normal.

The results show that the oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK carrying TMTP1 and HSV-TK overcomes the hepatotropism, remarkably enhances the targeting property to the tumor, has low toxicity to the liver and the kidney and has higher safety.

Therefore, the oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMTP 1/delta ADP/HSV-TK carrying TMTP1 and HSV-TK can be used for preparing medicaments for treating tumors, not only develops a novel medicament for treating tumors, but also develops the application of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.

Although the present invention has been described with respect to the preferred embodiments, it is not intended to be limited to the embodiments disclosed, and many modifications and variations are possible to those skilled in the art without departing from the spirit of the invention.

Sequence listing

<110> affiliated Tongji hospital of Tongji medical college of Huazhong university of science and technology

<120> oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK, and construction method and application thereof

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gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240

gtacccgagc cgatgactta ctggcgggtg ctgggggctt ccgagacaat cgcgaacatc 300

tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360

atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420

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ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcggta ccttatgggc 540

agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600

acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660

cagcgccccg gtgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720

ctacttgcca atacggtgcg gtatctgcag tgcggcgggt cgtggcggga ggattgggga 780

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gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240

taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300

agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360

gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420

cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 480

tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc 540

tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga 600

aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc 660

tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc 720

cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt 780

gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca 840

cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa 900

ccttgtaccg gaggtgatcn tccacccagt gacgacgagg atgaagaggg tgaggagttt 960

gtgttagatt atgtggagca ccccgggcac ggttgcaggt cttgtcatta tcaccggagg 1020

aatacggggg acccagatat tatgtgttcg ctttgctata tgaggacctg tggcatgttt 1080

gtctacagta agtgaaaatt atgggcagtg ggtgatagag tggtgggttt ggtgtggtaa 1140

tttttttttt aatttttaca gttttgtggt ttaaagaatt ttgtattgtg atttttttaa 1200

aaggtcctgt gtctgaacct gagcctgagc ccgagccaga accggagcct gcaagaccta 1260

cccgccgtcc taaaatggcg cctgctatcc tgagacgccc gacatcacct gtgtctagag 1320

aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct cctgagatac 1380

acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt gggcgtcgcc 1440

aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg gacttgagct 1500

gtaaacgccc caggccataa ggtgtaaacc tgtgattgcg tgtgtggtta acgcctttgt 1560

ttgctgaatg agttgatgta agtttaataa agggtgagat aatgtttaac ttgcatggcg 1620

tgttaaatgg ggcggggctt aaagggtata taatgcgccg tgggctaatc ttggttacat 1680

ctgacctcat ggaggcttgg gagtgtttgg aagatttttc tgctgtgcgt aacttgctgg 1740

aacagagctc taacagtacc tcttggtttt ggaggtttct gtggggctca tcccaggcaa 1800

agttagtctg cagaattaag gaggattaca agtgggaatt tgaagagctt ttgaaatcct 1860

gtggtgagct gtttgattct ttgaatctgg gtcaccaggc gcttttccaa gagaaggtca 1920

tcaagacttt ggatttttcc acaccggggc gcgctgcggc tgctgttgct tttttgagtt 1980

ttataaagga taaatggagc gaagaaaccc atctgagcgg ggggtacctg ctggattttc 2040

tggccatgca tctgtggaga gcggttgtga gacacaagaa tcgcctgcta ctgttgtctt 2100

ccgtccgccc ggcgataata ccgacggagg agcagcagca gcagcaggag gaagccaggc 2160

ggcggcggca ggagcagagc ccatggaacc cgagagccgg cctggaccct cgggaatgaa 2220

tgttgtacag gtggctgaac tgtatccaga actgagacgc attttgacaa ttacagagga 2280

tgggcagggg ctaaaggggg taaagaggga gcggggggct tgtgaggcta cagaggaggc 2340

taggaatcta gcttttagct taatgaccag acaccgtcct gagtgtatta cttttcaaca 2400

gatcaaggat aattgcgcta atgagcttga tctgctggcg cagaagtatt ccatagagca 2460

gctgaccact tactggctgc agccagggga tgattttgag gaggctatta gggtatatgc 2520

aaaggtggca cttaggccag attgcaagta caagatcagc aaacttgtaa atatcaggaa 2580

ttgttgctac atttctggga acggggccga ggtggagata gatacggagg atagggtggc 2640

ctttagatgt agcatgataa atatgtggcc gggggtgctt ggcatggacg gggtggttat 2700

tatgaatgta aggtttactg gccccaattt tagcggtacg gttttcctgg ccaataccaa 2760

ccttatccta cacggtgtaa gcttctatgg gtttaacaat acctgtgtgg aagcctggac 2820

cgatgtaagg gttcggggct gtgcctttta ctgctgctgg aagggggtgg tgtgtcgccc 2880

caaaagcagg gcttcaatta agaaatgcct ctttgaaagg tgtaccttgg gtatcctgtc 2940

tgagggtaac tccagggtgc gccacaatgt ggcctccgac tgtggttgct tcatgctagt 3000

gaaaagcgtg gctgtgatta agcataacat ggtatgtggc aactgcgagg acagggcctc 3060

tcagatgctg acctgctcgg acggcaactg tcacctgctg aagaccattc acgtagccag 3120

ccactctcgc aaggcctggc cagtgtttga gcataacata ctgacccgct gttccttgca 3180

tttgggtaac aggagggggg tgttcctacc ttaccaatgc aatttgagtc acactaagat 3240

attgcttgag cccgagagca tgtccaaggt gaacctgaac ggggtgtttg acatgaccat 3300

gaagatctgg aaggtgctga ggtacgatga gacccgcacc aggtgcagac cctgcgagtg 3360

tggcggtaaa catattagga accagcctgt gatgctggat gtgaccgagg agctgaggcc 3420

cgatcacttg gtgctggcct gcacccgcgc tgagtttggc tctagcgatg aagatacaga 3480

ttgaggtact gaaatgtgtg ggcgtggctt aagggtggga aagaatatat aaggtggggg 3540

tcttatgtag ttttgtatct gttttgcagc agccgccgcc gccatgagca ccaactcgtt 3600

tgatggaagc attgtgagct catatttgac aacgcgcatg cccccatggg ccggggtgcg 3660

tcagaatgtg atgggctcca gcattgatgg tcgccccgtc ctgcccgcaa actctactac 3720

cttgacctac gagaccgtgt ctggaacgcc gttggagact gcagcctccg ccgccgcttc 3780

agccgctgca gccaccgccc gcgggattgt gactgacttt gctttcctga gcccgcttgc 3840

aagcagtgca gcttcccgtt catccgcccg cgatgacaag ttgacggctc ttttggcaca 3900

attggattct ttgacccggg aacttaatgt cgtttctcag cagctgttgg atctgcgcca 3960

gcaggtttct gccctgaagg cttcctcccc tcccaatgcg gtttaaaaca taaataaaaa 4020

accagactct gtttggattt ggatcaagca agtgtcttgc tgtctttatt taggggtttt 4080

gcgcgcgcgg taggcccggg accagcggtc tcggtcgttg agggtcctgt gtattttttc 4140

caggacgtgg taaaggtgac tctggatgtt cagatacatg ggcataagcc cgtctctggg 4200

gtggaggtag caccactgca gagcttcatg ctgcggggtg gtgttgtaga tgatccagtc 4260

gtagcaggag cgctgggcgt ggtgcctaaa aatgtctttc agtagcaagc tgattgccag 4320

gggcaggccc ttggtgtaag tgtttacaaa gcggttaagc tgggatgggt gcatacgtgg 4380

ggatatgaga tgcatcttgg actgtatttt taggttggct atgttcccag ccatatccct 4440

ccggggattc atgttgtgca gaaccaccag cacagtgtat ccggtgcact tgggaaattt 4500

gtcatgtagc ttagaaggaa atgcgtggaa gaacttggag acgcccttgt gacctccaag 4560

attttccatg cattcgtcca taatgatggc aatgggccca cgggcggcgg cctgggcgaa 4620

gatatttctg ggatcactaa cgtcatagtt gtgttccagg atgagatcgt cataggccat 4680

ttttacaaag cgcgggcgga gggtgccaga ctgcggtata atggttccat ccggcccagg 4740

ggcgtagtta ccctcacaga tttgcatttc ccacgctttg agttcagatg gggggatcat 4800

gtctacctgc ggggcgatga agaaaacggt ttccggggta ggggagatca gctgggaaga 4860

aagcaggttc ctgagcagct gcgacttacc gcagccggtg ggcccgtaaa tcacacctat 4920

taccgggtgc aactggtagt taagagagct gcagctgccg tcatccctga gcaggggggc 4980

cacttcgtta agcatgtccc tgactcgcat gttttccctg accaaatccg ccagaaggcg 5040

ctcgccgccc agcgatagca gttcttgcaa ggaagcaaag tttttcaacg gtttgagacc 5100

gtccgccgta ggcatgcttt tgagcgtttg accaagcagt tccaggcggt cccacagctc 5160

ggtcacctgc tctacggcat ctcgatccag catatctcct cgtttcgcgg gttggggcgg 5220

ctttcgctgt acggcagtag tcggtgctcg tccagacggg ccagggtcat gtctttccac 5280

gggcgcaggg tcctcgtcag cgtagtctgg gtcacggtga aggggtgcgc tccgggctgc 5340

gcgctggcca gggtgcgctt gaggctggtc ctgctggtgc tgaagcgctg ccggtcttcg 5400

ccctgcgcgt cggccaggta gcatttgacc atggtgtcat agtccagccc ctccgcggcg 5460

tggcccttgg cgcgcagctt gcccttggag gaggcgccgc acgaggggca gtgcagactt 5520

ttgagggcgt agagcttggg cgcgagaaat accgattccg gggagtaggc atccgcgccg 5580

caggccccgc agacggtctc gcattccacg agccaggtga gctctggccg ttcggggtca 5640

aaaaccaggt ttcccccatg ctttttgatg cgtttcttac ctctggtttc catgagccgg 5700

tgtccacgct cggtgacgaa aaggctgtcc gtgtccccgt atacagactt gagaggcctg 5760

tcctcgagcg gtgttccgcg gtcctcctcg tatagaaact cggaccactc tgagacaaag 5820

gctcgcgtcc aggccagcac gaaggaggct aagtgggagg ggtagcggtc gttgtccact 5880

agggggtcca ctcgctccag ggtgtgaaga cacatgtcgc cctcttcggc atcaaggaag 5940

gtgattggtt tgtaggtgta ggccacgtga ccgggtgttc ctgaaggggg gctataaaag 6000

ggggtggggg cgcgttcgtc ctcactctct tccgcatcgc tgtctgcgag ggccagctgt 6060

tggggtgagt actccctctg aaaagcgggc atgacttctg cgctaagatt gtcagtttcc 6120

aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag ggtggccgca 6180

tccatctggt cagaaaagac aatctttttg ttgtcaagct tggtggcaaa cgacccgtag 6240

agggcgttgg acagcaactt ggcgatggag cgcagggttt ggtttttgtc gcgatcggcg 6300

cgctccttgg ccgcgatgtt tagctgcacg tattcgcgcg caacgcaccg ccattcggga 6360

aagacggtgg tgcgctcgtc gggcaccagg tgcacgcgcc aaccgcggtt gtgcagggtg 6420

acaaggtcaa cgctggtggc tacctctccg cgtaggcgct cgttggtcca gcagaggcgg 6480

ccgcccttgc gcgagcagaa tggcggtagg gggtctagct gcgtctcgtc cggggggtct 6540

gcgtccacgg taaagacccc gggcagcagg cgcgcgtcga agtagtctat cttgcatcct 6600

tgcaagtcta gcgcctgctg ccatgcgcgg gcggcaagcg cgcgctcgta tgggttgagt 6660

gggggacccc atggcatggg gtgggtgagc gcggaggcgt acatgccgca aatgtcgtaa 6720

acgtagaggg gctctctgag tattccaaga tatgtagggt agcatcttcc accgcggatg 6780

ctggcgcgca cgtaatcgta tagttcgtgc gagggagcga ggaggtcggg accgaggttg 6840

ctacgggcgg gctgctctgc tcggaagact atctgcctga agatggcatg tgagttggat 6900

gatatggttg gacgctggaa gacgttgaag ctggcgtctg tgagacctac cgcgtcacgc 6960

acgaaggagg cgtaggagtc gcgcagcttg ttgaccagct cggcggtgac ctgcacgtct 7020

agggcgcagt agtccagggt ttccttgatg atgtcatact tatcctgtcc cttttttttc 7080

cacagctcgc ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac 7140

ccgtcggcct ccgaacggta agagcctagc atgtagaact ggttgacggc ctggtaggcg 7200

cagcatccct tttctacggg tagcgcgtat gcctgcgcgg ccttccggag cgaggtgtgg 7260

gtgagcgcaa aggtgtccct gaccatgact ttgaggtact ggtatttgaa gtcagtgtcg 7320

tcgcatccgc cctgctccca gagcaaaaag tccgtgcgct ttttggaacg cggatttggc 7380

agggcgaagg tgacatcgtt gaagagtatc tttcccgcgc gaggcataaa gttgcgtgtg 7440

atgcggaagg gtcccggcac ctcggaacgg ttgttaatta cctgggcggc gagcacgatc 7500

tcgtcaaagc cgttgatgtt gtggcccaca atgtaaagtt ccaagaagcg cgggatgccc 7560

ttgatggaag gcaatttttt aagttcctcg taggtgagct cttcagggga gctgagcccg 7620

tgctctgaaa gggcccagtc tgcaagatga gggttggaag cgacgaatga gctccacagg 7680

tcacgggcca ttagcatttg caggtggtcg cgaaaggtcc taaactggcg acctatggcc 7740

attttttctg gggtgatgca gtagaaggta agcgggtctt gttcccagcg gtcccatcca 7800

aggttcgcgg ctaggtctcg cgcggcagtc actagaggct catctccgcc gaacttcatg 7860

accagcatga agggcacgag ctgcttccca aaggccccca tccaagtata ggtctctaca 7920

tcgtaggtga caaagagacg ctcggtgcga ggatgcgagc cgatcgggaa gaactggatc 7980

tcccgccacc aattggagga gtggctattg atgtggtgaa agtagaagtc cctgcgacgg 8040

gccgaacact cgtgctggct tttgtaaaaa cgtgcgcagt actggcagcg gtgcacgggc 8100

tgtacatcct gcacgaggtt gacctgacga ccgcgcacaa ggaagcagag tgggaatttg 8160

agcccctcgc ctggcgggtt tggctggtgg tcttctactt cggctgcttg tccttgaccg 8220

tctggctgct cgaggggagt tacggtggat cggaccacca cgccgcgcga gcccaaagtc 8280

cagatgtccg cgcgcggcgg tcggagcttg atgacaacat cgcgcagatg ggagctgtcc 8340

atggtctgga gctcccgcgg cgtcaggtca ggcgggagct cctgcaggtt tacctcgcat 8400

agacgggtca gggcgcgggc tagatccagg tgatacctaa tttccagggg ctggttggtg 8460

gcggcgtcga tggcttgcaa gaggccgcat ccccgcggcg cgactacggt accgcgcggc 8520

gggcggtggg ccgcgggggt gtccttggat gatgcatcta aaagcggtga cgcgggcgag 8580

cccccggagg tagggggggc tccggacccg ccgggagagg gggcaggggc acgtcggcgc 8640

cgcgcgcggg caggagctgg tgctgcgcgc gtaggttgct ggcgaacgcg acgacgcggc 8700

ggttgatctc ctgaatctgg cgcctctgcg tgaagacgac gggcccggtg agcttgagcc 8760

tgaaagagag ttcgacagaa tcaatttcgg tgtcgttgac ggcggcctgg cgcaaaatct 8820

cctgcacgtc tcctgagttg tcttgatagg cgatctcggc catgaactgc tcgatctctt 8880

cctcctggag atctccgcgt ccggctcgct ccacggtggc ggcgaggtcg ttggaaatgc 8940

gggccatgag ctgcgagaag gcgttgaggc ctccctcgtt ccagacgcgg ctgtagacca 9000

cgcccccttc ggcatcgcgg gcgcgcatga ccacctgcgc gagattgagc tccacgtgcc 9060

gggcgaagac ggcgtagttt cgcaggcgct gaaagaggta gttgagggtg gtggcggtgt 9120

gttctgccac gaagaagtac ataacccagc gtcgcaacgt ggattcgttg atatccccca 9180

aggcctcaag gcgctccatg gcctcgtaga agtccacggc gaagttgaaa aactgggagt 9240

tgcgcgccga cacggttaac tcctcctcca gaagacggat gagctcggcg acagtgtcgc 9300

gcacctcgcg ctcaaaggct acaggggcct cttcttcttc ttcaatctcc tcttccataa 9360

gggcctcccc ttcttcttct tctggcggcg gtgggggagg ggggacacgg cggcgacgac 9420

ggcgcaccgg gaggcggtcg acaaagcgct cgatcatctc cccgcggcga cggcgcatgg 9480

tctcggtgac ggcgcggccg ttctcgcggg ggcgcagttg gaagacgccg cccgtcatgt 9540

cccggttatg ggttggcggg gggctgccat gcggcaggga tacggcgcta acgatgcatc 9600

tcaacaattg ttgtgtaggt actccgccgc cgagggacct gagcgagtcc gcatcgaccg 9660

gatcggaaaa cctctcgaga aaggcgtcta accagtcaca gtcgcaaggt aggctgagca 9720

ccgtggcggg cggcagcggg cggcggtcgg ggttgtttct ggcggaggtg ctgctgatga 9780

tgtaattaaa gtaggcggtc ttgagacggc ggatggtcga cagaagcacc atgtccttgg 9840

gtccggcctg ctgaatgcgc aggcggtcgg ccatgcccca ggcttcgttt tgacatcggc 9900

gcaggtcttt gtagtagtct tgcatgagcc tttctaccgg cacttcttct tctccttcct 9960

cttgtcctgc atctcttgca tctatcgctg cggcggcggc ggagtttggc cgtaggtggc 10020

gccctcttcc tcccatgcgt gtgaccccga agcccctcat cggctgaagc agggctaggt 10080

cggcgacaac gcgctcggct aatatggcct gctgcacctg cgtgagggta gactggaagt 10140

catccatgtc cacaaagcgg tggtatgcgc ccgtgttgat ggtgtaagtg cagttggcca 10200

taacggacca gttaacggtc tggtgacccg gctgcgagag ctcggtgtac ctgagacgcg 10260

agtaagccct cgagtcaaat acgtagtcgt tgcaagtccg caccaggtac tggtatccca 10320

ccaaaaagtg cggcggcggc tggcggtaga ggggccagcg tagggtggcc ggggctccgg 10380

gggcgagatc ttccaacata aggcgatgat atccgtagat gtacctggac atccaggtga 10440

tgccggcggc ggtggtggag gcgcgcggaa agtcgcggac gcggttccag atgttgcgca 10500

gcggcaaaaa gtgctccatg gtcgggacgc tctggccggt caggcgcgcg caatcgttga 10560

cgctctagac cgtgcaaaag gagagcctgt aagcgggcac tcttccgtgg tctggtggat 10620

aaattcgcaa gggtatcatg gcggacgacc ggggttcgag ccccgtatcc ggccgtccgc 10680

cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt gtgcgacgtc agacaacggg 10740

ggagtgctcc ttttggcttc cttccaggcg cggcggctgc tgcgctagct tttttggcca 10800

ctggccgcgc gcagcgtaag cggttaggct ggaaagcgaa agcattaagt ggctcgctcc 10860

ctgtagccgg agggttattt tccaagggtt gagtcgcggg acccccggtt cgagtctcgg 10920

accggccgga ctgcggcgaa cgggggtttg cctccccgtc atgcaagacc ccgcttgcaa 10980

attcctccgg aaacagggac gagccccttt tttgcttttc ccagatgcat ccggtgctgc 11040

ggcagatgcg cccccctcct cagcagcggc aagagcaaga gcagcggcag acatgcaggg 11100

caccctcccc tcctcctacc gcgtcaggag gggcgacatc cgcggttgac gcggcagcag 11160

atggtgatta cgaacccccg cggcgccggg cccggcacta cctggacttg gaggagggcg 11220

agggcctggc gcggctagga gcgccctctc ctgagcggta cccaagggtg cagctgaagc 11280

gtgatacgcg tgaggcgtac gtgccgcggc agaacctgtt tcgcgaccgc gagggagagg 11340

agcccgagga gatgcgggat cgaaagttcc acgcagggcg cgagctgcgg catggcctga 11400

atcgcgagcg gttgctgcgc gaggaggact ttgagcccga cgcgcgaacc gggattagtc 11460

ccgcgcgcgc acacgtggcg gccgccgacc tggtaaccgc atacgagcag acggtgaacc 11520

aggagattaa ctttcaaaaa agctttaaca accacgtgcg tacgcttgtg gcgcgcgagg 11580

aggtggctat aggactgatg catctgtggg actttgtaag cgcgctggag caaaacccaa 11640

atagcaagcc gctcatggcg cagctgttcc ttatagtgca gcacagcagg gacaacgagg 11700

cattcaggga tgcgctgcta aacatagtag agcccgaggg ccgctggctg ctcgatttga 11760

taaacatcct gcagagcata gtggtgcagg agcgcagctt gagcctggct gacaaggtgg 11820

ccgccatcaa ctattccatg cttagcctgg gcaagtttta cgcccgcaag atataccata 11880

ccccttacgt tcccatagac aaggaggtaa agatcgaggg gttctacatg cgcatggcgc 11940

tgaaggtgct taccttgagc gacgacctgg gcgtttatcg caacgagcgc atccacaagg 12000

ccgtgagcgt gagccggcgg cgcgagctca gcgaccgcga gctgatgcac agcctgcaaa 12060

gggccctggc tggcacgggc agcggcgata gagaggccga gtcctacttt gacgcgggcg 12120

ctgacctgcg ctgggcccca agccgacgcg ccctggaggc agctggggcc ggacctgggc 12180

tggcggtggc acccgcgcgc gctggcaacg tcggcggcgt ggaggaatat gacgaggacg 12240

atgagtacga gccagaggac ggcgagtact aagcggtgat gtttctgatc agatgatgca 12300

agacgcaacg gacccggcgg tgcgggcggc gctgcagagc cagccgtccg gccttaactc 12360

cacggacgac tggcgccagg tcatggaccg catcatgtcg ctgactgcgc gcaatcctga 12420

cgcgttccgg cagcagccgc aggccaaccg gctctccgca attctggaag cggtggtccc 12480

ggcgcgcgca aaccccacgc acgagaaggt gctggcgatc gtaaacgcgc tggccgaaaa 12540

cagggccatc cggcccgacg aggccggcct ggtctacgac gcgctgcttc agcgcgtggc 12600

tcgttacaac agcggcaacg tgcagaccaa cctggaccgg ctggtggggg atgtgcgcga 12660

ggccgtggcg cagcgtgagc gcgcgcagca gcagggcaac ctgggctcca tggttgcact 12720

aaacgccttc ctgagtacac agcccgccaa cgtgccgcgg ggacaggagg actacaccaa 12780

ctttgtgagc gcactgcggc taatggtgac tgagacaccg caaagtgagg tgtaccagtc 12840

tgggccagac tattttttcc agaccagtag acaaggcctg cagaccgtaa acctgagcca 12900

ggctttcaaa aacttgcagg ggctgtgggg ggtgcgggct cccacaggcg accgcgcgac 12960

cgtgtctagc ttgctgacgc ccaactcgcg cctgttgctg ctgctaatag cgcccttcac 13020

ggacagtggc agcgtgtccc gggacacata cctaggtcac ttgctgacac tgtaccgcga 13080

ggccataggt caggcgcatg tggacgagca tactttccag gagattacaa gtgtcagccg 13140

cgcgctgggg caggaggaca cgggcagcct ggaggcaacc ctaaactacc tgctgaccaa 13200

ccggcggcag aagatcccct cgttgcacag tttaaacagc gaggaggagc gcattttgcg 13260

ctacgtgcag cagagcgtga gccttaacct gatgcgcgac ggggtaacgc ccagcgtggc 13320

gctggacatg accgcgcgca acatggaacc gggcatgtat gcctcaaacc ggccgtttat 13380

caaccgccta atggactact tgcatcgcgc ggccgccgtg aaccccgagt atttcaccaa 13440

tgccatcttg aacccgcact ggctaccgcc ccctggtttc tacaccgggg gattcgaggt 13500

gcccgagggt aacgatggat tcctctggga cgacatagac gacagcgtgt tttccccgca 13560

accgcagacc ctgctagagt tgcaacagcg cgagcaggca gaggcggcgc tgcgaaagga 13620

aagcttccgc aggccaagca gcttgtccga tctaggcgct gcggccccgc ggtcagatgc 13680

tagtagccca tttccaagct tgatagggtc tcttaccagc actcgcacca cccgcccgcg 13740

cctgctgggc gaggaggagt acctaaacaa ctcgctgctg cagccgcagc gcgaaaaaaa 13800

cctgcctccg gcatttccca acaacgggat agagagccta gtggacaaga tgagtagatg 13860

gaagacgtac gcgcaggagc acagggacgt gccaggcccg cgcccgccca cccgtcgtca 13920

aaggcacgac cgtcagcggg gtctggtgtg ggaggacgat gactcggcag acgacagcag 13980

cgtcctggat ttgggaggga gtggcaaccc gtttgcgcac cttcgcccca ggctggggag 14040

aatgttttaa aaaaaaaaaa gcatgatgca aaataaaaaa ctcaccaagg ccatggcacc 14100

gagcgttggt tttcttgtat tccccttagt atgcggcgcg cggcgatgta tgaggaaggt 14160

cctcctccct cctacgagag tgtggtgagc gcggcgccag tggcggcggc gctgggttct 14220

cccttcgatg ctcccctgga cccgccgttt gtgcctccgc ggtacctgcg gcctaccggg 14280

gggagaaaca gcatccgtta ctctgagttg gcacccctat tcgacaccac ccgtgtgtac 14340

ctggtggaca acaagtcaac ggatgtggca tccctgaact accagaacga ccacagcaac 14400

tttctgacca cggtcattca aaacaatgac tacagcccgg gggaggcaag cacacagacc 14460

atcaatcttg acgaccggtc gcactggggc ggcgacctga aaaccatcct gcataccaac 14520

atgccaaatg tgaacgagtt catgtttacc aataagttta aggcgcgggt gatggtgtcg 14580

cgcttgccta ctaaggacaa tcaggtggag ctgaaatacg agtgggtgga gttcacgctg 14640

cccgagggca actactccga gaccatgacc atagacctta tgaacaacgc gatcgtggag 14700

cactacttga aagtgggcag acagaacggg gttctggaaa gcgacatcgg ggtaaagttt 14760

gacacccgca acttcagact ggggtttgac cccgtcactg gtcttgtcat gcctggggta 14820

tatacaaacg aagccttcca tccagacatc attttgctgc caggatgcgg ggtggacttc 14880

acccacagcc gcctgagcaa cttgttgggc atccgcaagc ggcaaccctt ccaggagggc 14940

tttaggatca cctacgatga tctggagggt ggtaacattc ccgcactgtt ggatgtggac 15000

gcctaccagg cgagcttgaa agatgacacc gaacagggcg ggggtggcgc aggcggcagc 15060

aacagcagtg gcagcggcgc ggaagagaac tccaacgcgg cagccgcggc aatgcagccg 15120

gtggaggaca tgaacgatca tgccattcgc ggcgacacct ttgccacacg ggctgaggag 15180

aagcgcgctg aggccgaagc agcggccgaa gctgccgccc ccgctgcgca acccgaggtc 15240

gagaagcctc agaagaaacc ggtgatcaaa cccctgacag aggacagcaa gaaacgcagt 15300

tacaacctaa taagcaatga cagcaccttc acccagtacc gcagctggta ccttgcatac 15360

aactacggcg accctcagac cggaatccgc tcatggaccc tgctttgcac tcctgacgta 15420

acctgcggct cggagcaggt ctactggtcg ttgccagaca tgatgcaaga ccccgtgacc 15480

ttccgctcca cgcgccagat cagcaacttt ccggtggtgg gcgccgagct gttgcccgtg 15540

cactccaaga gcttctacaa cgaccaggcc gtctactccc aactcatccg ccagtttacc 15600

tctctgaccc acgtgttcaa tcgctttccc gagaaccaga ttttggcgcg cccgccagcc 15660

cccaccatca ccaccgtcag tgaaaacgtt cctgctctca cagatcacgg gacgctaccg 15720

ctgcgcaaca gcatcggagg agtccagcga gtgaccatta ctgacgccag acgccgcacc 15780

tgcccctacg tttacaaggc cctgggcata gtctcgccgc gcgtcctatc gagccgcact 15840

ttttgagcaa gcatgtccat ccttatatcg cccagcaata acacaggctg gggcctgcgc 15900

ttcccaagca agatgtttgg cggggccaag aagcgctccg accaacaccc agtgcgcgtg 15960

cgcgggcact accgcgcgcc ctggggcgcg cacaaacgcg gccgcactgg gcgcaccacc 16020

gtcgatgacg ccatcgacgc ggtggtggag gaggcgcgca actacacgcc cacgccgcca 16080

ccagtgtcca cagtggacgc ggccattcag accgtggtgc gcggagcccg gcgctatgct 16140

aaaatgaaga gacggcggag gcgcgtagca cgtcgccacc gccgccgacc cggcactgcc 16200

gcccaacgcg cggcggcggc cctgcttaac cgcgcacgtc gcaccggccg acgggcggcc 16260

atgcgggccg ctcgaaggct ggccgcgggt attgtcactg tgccccccag gtccaggcga 16320

cgagcggccg ccgcagcagc cgcggccatt agtgctatga ctcagggtcg caggggcaac 16380

gtgtattggg tgcgcgactc ggttagcggc ctgcgcgtgc ccgtgcgcac ccgccccccg 16440

cgcaactaga ttgcaagaaa aaactactta gactcgtact gttgtatgta tccagcggcg 16500

gcggcgcgca acgaagctat gtccaagcgc aaaatcaaag aagagatgct ccaggtcatc 16560

gcgccggaga tctatggccc cccgaagaag gaagagcagg attacaagcc ccgaaagcta 16620

aagcgggtca aaaagaaaaa gaaagatgat gatgatgaac ttgacgacga ggtggaactg 16680

ctgcacgcta ccgcgcccag gcgacgggta cagtggaaag gtcgacgcgt aaaacgtgtt 16740

ttgcgacccg gcaccaccgt agtctttacg cccggtgagc gctccacccg cacctacaag 16800

cgcgtgtatg atgaggtgta cggcgacgag gacctgcttg agcaggccaa cgagcgcctc 16860

ggggagtttg cctacggaaa gcggcataag gacatgctgg cgttgccgct ggacgagggc 16920

aacccaacac ctagcctaaa gcccgtaaca ctgcagcagg tgctgcccgc gcttgcaccg 16980

tccgaagaaa agcgcggcct aaagcgcgag tctggtgact tggcacccac cgtgcagctg 17040

atggtaccca agcgccagcg actggaagat gtcttggaaa aaatgaccgt ggaacctggg 17100

ctggagcccg aggtccgcgt gcggccaatc aagcaggtgg cgccgggact gggcgtgcag 17160

accgtggacg ttcagatacc cactaccagt agcaccagta ttgccaccgc cacagagggc 17220

atggagacac aaacgtcccc ggttgcctca gcggtggcgg atgccgcggt gcaggcggtc 17280

gctgcggccg cgtccaagac ctctacggag gtgcaaacgg acccgtggat gtttcgcgtt 17340

tcagcccccc ggcgcccgcg cggttcgagg aagtacggcg ccgccagcgc gctactgccc 17400

gaatatgccc tacatccttc cattgcgcct acccccggct atcgtggcta cacctaccgc 17460

cccagaagac gagcaactac ccgacgccga accaccactg gaacccgccg ccgccgtcgc 17520

cgtcgccagc ccgtgctggc cccgatttcc gtgcgcaggg tggctcgcga aggaggcagg 17580

accctggtgc tgccaacagc gcgctaccac cccagcatcg tttaaaagcc ggtctttgtg 17640

gttcttgcag atatggccct cacctgccgc ctccgtttcc cggtgccggg attccgagga 17700

agaatgcacc gtaggagggg catggccggc cacggcctga cgggcggcat gcgtcgtgcg 17760

caccaccggc ggcggcgcgc gtcgcaccgt cgcatgcgcg gcggtatcct gcccctcctt 17820

attccactga tcgccgcggc gattggcgcc gtgcccggaa ttgcatccgt ggccttgcag 17880

gcgcagagac actgattaaa aacaagttgc atgtggaaaa atcaaaataa aaagtctgga 17940

ctctcacgct cgcttggtcc tgtaactatt ttgtagaatg gaagacatca actttgcgtc 18000

tctggccccg cgacacggct cgcgcccgtt catgggaaac tggcaagata tcggcaccag 18060

caatatgagc ggtggcgcct tcagctgggg ctcgctgtgg agcggcatta aaaatttcgg 18120

ttccaccgtt aagaactatg gcagcaaggc ctggaacagc agcacaggcc agatgctgag 18180

ggataagttg aaagagcaaa atttccaaca aaaggtggta gatggcctgg cctctggcat 18240

tagcggggtg gtggacctgg ccaaccaggc agtgcaaaat aagattaaca gtaagcttga 18300

tccccgccct cccgtagagg agcctccacc ggccgtggag acagtgtctc cagaggggcg 18360

tggcgaaaag cgtccgcgcc ccgacaggga agaaactctg gtgacgcaaa tagacgagcc 18420

tccctcgtac gaggaggcac taaagcaagg cctgcccacc acccgtccca tcgcgcccat 18480

ggctaccgga gtgctgggcc agcacacacc cgtaacgctg gacctgcctc cccccgccga 18540

cacccagcag aaacctgtgc tgccaggccc gaccgccgtt gttgtaaccc gtcctagccg 18600

cgcgtccctg cgccgcgccg ccagcggtcc gcgatcgttg cggcccgtag ccagtggcaa 18660

ctggcaaagc acactgaaca gcatcgtggg tctgggggtg caatccctga agcgccgacg 18720

atgcttctga atagctaacg tgtcgtatgt gtgtcatgta tgcgtccatg tcgccgccag 18780

aggagctgct gagccgccgc gcgcccgctt tccaagatgg ctaccccttc gatgatgccg 18840

cagtggtctt acatgcacat ctcgggccag gacgcctcgg agtacctgag ccccgggctg 18900

gtgcagtttg cccgcgccac cgagacgtac ttcagcctga ataacaagtt tagaaacccc 18960

acggtggcgc ctacgcacga cgtgaccaca gaccggtccc agcgtttgac gctgcggttc 19020

atccctgtgg accgtgagga tactgcgtac tcgtacaagg cgcggttcac cctagctgtg 19080

ggtgataacc gtgtgctgga catggcttcc acgtactttg acatccgcgg cgtgctggac 19140

aggggcccta cttttaagcc ctactctggc actgcctaca acgccctggc tcccaagggt 19200

gccccaaatc cttgcgaatg ggatgaagct gctactgctc ttgaaataaa cctagaagaa 19260

gaggacgatg acaacgaaga cgaagtagac gagcaagctg agcagcaaaa aactcacgta 19320

tttgggcagg cgccttattc tggtataaat attacaaagg agggtattca aataggtgtc 19380

gaaggtcaaa cacctaaata tgccgataaa acatttcaac ctgaacctca aataggagaa 19440

tctcagtggt acgaaactga aattaatcat gcagctggga gagtccttaa aaagactacc 19500

ccaatgaaac catgttacgg ttcatatgca aaacccacaa atgaaaatgg agggcaaggc 19560

attcttgtaa agcaacaaaa tggaaagcta gaaagtcaag tggaaatgca attttaacgt 19620

ggtgcgtcaa tctcaactac tgaggcagcc gcaggcaatg gtgataactt gactcctaaa 19680

gtggtattgt acagtgaaga tgtagatata gaaaccccag acactcatat ttcttacatg 19740

cccactatta aggaaggtaa ctcacgagaa ctaatgggcc aacaatctat gcccaacagg 19800

cctaattaca ttgcttttag ggacaatttt attggtctaa tgtattacaa cagcacgggt 19860

aatatgggtg ttctggcggg ccaagcatcg cagttgaatg ctgttgtaga tttgcaagac 19920

agaaacacag agctttcata ccagcttttg cttgattcca ttggtgatag aaccaggtac 19980

ttttctatgt ggaatcaggc tgttgacagc tatgatccag atgttagaat tattgaaaat 20040

catggaactg aagatgaact tccaaattac tgctttccac tgggaggtgt gattaataca 20100

gagactctta ccaaggtaaa acctaaaaca ggtcaggaaa atggatggga aaaagatgct 20160

acagaatttt cagataaaaa tgaaataaga gttggaaata attttgccat ggaaatcaat 20220

ctaaatgcca acctgtggag aaatttcctg tactccaaca tagcgctgta tttgcccgac 20280

aagctaaagt acagtccttc caacgtaaaa atttctgata acccaaacac ctacgactac 20340

atgaacaagc gagtggtggc tcccgggtta gtggactgct acattaacct tggagcacgc 20400

tggtcccttg actatatgga caacgtcaac ccatttaacc accaccgcaa tgctggcctg 20460

cgctaccgct caatgttgct gggcaatggt cgctatgtgc ccttccacat ccaggtgcct 20520

cagaagttct ttgccattaa aaacctcctt ctcctgccgg gctcatacac ctacgagtgg 20580

aacttcagga aggatgttaa catggttctg cagagctccc taggaaatga cctaagggtt 20640

gacggagcca gcattaagtt tgatagcatt tgcctttacg ccaccttctt ccccatggcc 20700

cacaacaccg cctccacgct tgaggccatg cttagaaacg acaccaacga ccagtccttt 20760

aacgactatc tctccgccgc caacatgctc taccctatac ccgccaacgc taccaacgtg 20820

cccatatcca tcccctcccg caactgggcg gctttccgcg gctgggcctt cacgcgcctt 20880

aagactaagg aaaccccatc actgggctcg ggctacgacc cttattacac ctactctggc 20940

tctataccct acctagatgg aaccttttac ctcaaccaca cctttaagaa ggtggccatt 21000

acctttgact cttctgtcag ctggcctggc aatgaccgcc tgcttacccc caacgagttt 21060

gaaattaagc gctcagttga cggggagggt tacaacgttg cccagtgtaa catgaccaaa 21120

gactggttcc tggtacaaat gctagctaac tacaacattg gctaccaggg cttctatatc 21180

ccagagagct acaaggaccg catgtactcc ttctttagaa acttccagcc catgagccgt 21240

caggtggtgg atgatactaa atacaaggac taccaacagg tgggcatcct acaccaacac 21300

aacaactctg gatttgttgg ctaccttgcc cccaccatgc gcgaaggaca ggcctaccct 21360

gctaacttcc cctatccgct tataggcaag accgcagttg acagcattac ccagaaaaag 21420

tttctttgcg atcgcaccct ttggcgcatc ccattctcca gtaactttat gtccatgggc 21480

gcactcacag acctgggcca aaaccttctc tacgccaact ccgcccacgc gctagacatg 21540

acttttgagg tggatcccat ggacgagccc acccttcttt atgttttgtt tgaagtcttt 21600

gacgtggtcc gtgtgcaccg gccgcaccgc ggcgtcatcg aaaccgtgta cctgcgcacg 21660

cccttctcgg ccggcaacgc cacaacataa agaagcaagc aacatcaaca acagctgccg 21720

ccatgggctc cagtgagcag gaactgaaag ccattgtcaa agatcttggt tgtgggccat 21780

attttttggg cacctatgac aagcgctttc caggctttgt ttctccacac aagctcgcct 21840

gcgccatagt caatacggcc ggtcgcgaga ctgggggcgt acactggatg gcctttgcct 21900

ggaacccgca ctcaaaaaca tgctacctct ttgagccctt tggcttttct gaccagcgac 21960

tcaagcaggt ttaccagttt gagtacgagt cactcctgcg ccgtagcgcc attgcttctt 22020

cccccgaccg ctgtataacg ctggaaaagt ccacccaaag cgtacagggg cccaactcgg 22080

ccgcctgtgg actattctgc tgcatgtttc tccacgcctt tgccaactgg ccccaaactc 22140

ccatggatca caaccccacc atgaacctta ttaccggggt acccaactcc atgctcaaca 22200

gtccccaggt acagcccacc ctgcgtcgca accaggaaca gctctacagc ttcctggagc 22260

gccactcgcc ctacttccgc agccacagtg cgcagattag gagcgccact tctttttgtc 22320

acttgaaaaa catgtaaaaa taatgtacta gagacacttt caataaaggc aaatgctttt 22380

atttgtacac tctcgggtga ttatttaccc ccacccttgc cgtctgcgcc gtttaaaaat 22440

caaaggggtt ctgccgcgca tcgctatgcg ccactggcag ggacacgttg cgatactggt 22500

gtttagtgct ccacttaaac tcaggcacaa ccatccgcgg cagctcggtg aagttttcac 22560

tccacaggct gcgcaccatc accaacgcgt ttagcaggtc gggcgccgat atcttgaagt 22620

cgcagttggg gcctccgccc tgcgcgcgcg agttgcgata cacagggttg cagcactgga 22680

acactatcag cgccgggtgg tgcacgctgg ccagcacgct cttgtcggag atcagatccg 22740

cgtccaggtc ctccgcgttg ctcagggcga acggagtcaa ctttggtagc tgccttccca 22800

aaaagggcgc gtgcccaggc tttgagttgc actcgcaccg tagtggcatc aaaaggtgac 22860

cgtgcccggt ctgggcgtta ggatacagcg cctgcataaa agccttgatc tgcttaaaag 22920

ccacctgagc ctttgcgcct tcagagaaga acatgccgca agacttgccg gaaaactgat 22980

tggccggaca ggccgcgtcg tgcacgcagc accttgcgtc ggtgttggag atctgcacca 23040

catttcggcc ccaccggttc ttcacgatct tggccttgct agactgctcc ttcagcgcgc 23100

gctgcccgtt ttcgctcgtc acatccattt caatcacgtg ctccttattt atcataatgc 23160

ttccgtgtag acacttaagc tcgccttcga tctcagcgca gcggtgcagc cacaacgcgc 23220

agcccgtggg ctcgtgatgc ttgtaggtca cctctgcaaa cgactgcagg tacgcctgca 23280

ggaatcgccc catcatcgtc acaaaggtct tgttgctggt gaaggtcagc tgcaacccgc 23340

ggtgctcctc gttcagccag gtcttgcata cggccgccag agcttccact tggtcaggca 23400

gtagtttgaa gttcgccttt agatcgttat ccacgtggta cttgtccatc agcgcgcgcg 23460

cagcctccat gcccttctcc cacgcagaca cgatcggcac actcagcggg ttcatcaccg 23520

taatttcact ttccgcttcg ctgggctctt cctcttcctc ttgcgtccgc ataccacgcg 23580

ccactgggtc gtcttcattc agccgccgca ctgtgcgctt acctcctttg ccatgcttga 23640

ttagcaccgg tgggttgctg aaacccacca tttgtagcgc cacatcttct ctttcttcct 23700

cgctgtccac gattacctct ggtgatggcg ggcgctcggg cttgggagaa gggcgcttct 23760

ttttcttctt gggcgcaatg gccaaatccg ccgccgaggt cgatggccgc gggctgggtg 23820

tgcgcggcac cagcgcgtct tgtgatgagt cttcctcgtc ctcggactcg atacgccgcc 23880

tcatccgctt ttttgggggc gcccggggag gcggcggcga cggggacggg gacgacacgt 23940

cctccatggt tgggggacgt cgcgccgcac cgcgtccgcg ctcgggggtg gtttcgcgct 24000

gctcctcttc ccgactggcc atttccttct cctataggca gaaaaagatc atggagtcag 24060

tcgagaagaa ggacagccta accgccccct ctgagttcgc caccaccgcc tccaccgatg 24120

ccgccaacgc gcctaccacc ttccccgtcg aggcaccccc gcttgaggag gaggaagtga 24180

ttatcgagca ggacccaggt tttgtaagcg aagacgacga ggaccgctca gtaccaacag 24240

aggataaaaa gcaagaccag gacaacgcag aggcaaacga ggaacaagtc gggcgggggg 24300

acgaaaggca tggcgactac ctagatgtgg gagacgacgt gctgttgaag catctgcagc 24360

gccagtgcgc cattatctgc gacgcgttgc aagagcgcag cgatgtgccc ctcgccatag 24420

cggatgtcag ccttgcctac gaacgccacc tattctcacc gcgcgtaccc cccaaacgcc 24480

aagaaaacgg cacatgcgag cccaacccgc gcctcaactt ctaccccgta tttgccgtgc 24540

cagaggtgct tgccacctat cacatctttt tccaaaactg caagataccc ctatcctgcc 24600

gtgccaaccg cagccgagcg gacaagcagc tggccttgcg gcagggcgct gtcatacctg 24660

atatcgcctc gctcaacgaa gtgccaaaaa tctttgaggg tcttggacgc gacgagaagc 24720

gcgcggcaaa cgctctgcaa caggaaaaca gcgaaaatga aagtcactct ggagtgttgg 24780

tggaactcga gggtgacaac gcgcgcctag ccgtactaaa acgcagcatc gaggtcaccc 24840

actttgccta cccggcactt aacctacccc ccaaggtcat gagcacagtc atgagtgagc 24900

tgatcgtgcg ccgtgcgcag cccctggaga gggatgcaaa tttgcaagaa caaacagagg 24960

agggcctacc cgcagttggc gacgagcagc tagcgcgctg gcttcaaacg cgcgagcctg 25020

ccgacttgga ggagcgacgc aaactaatga tggccgcagt gctcgttacc gtggagcttg 25080

agtgcatgca gcggttcttt gctgacccgg agatgcagcg caagctagag gaaacattgc 25140

actacacctt tcgacagggc tacgtacgcc aggcctgcaa gatctccaac gtggagctct 25200

gcaacctggt ctcctacctt ggaattttgc acgaaaaccg ccttgggcaa aacgtgcttc 25260

attccacgct caagggcgag gcgcgccgcg actacgtccg cgactgcgtt tacttatttc 25320

tatgctacac ctggcagacg gccatgggcg tttggcagca gtgcttggag gagtgcaacc 25380

tcaaggagct gcagaaactg ctaaagcaaa acttgaagga cctatggacg gccttcaacg 25440

agcgctccgt ggccgcgcac ctggcggaca tcattttccc cgaacgcctg cttaaaaccc 25500

tgcaacaggg tctgccagac ttcaccagtc aaagcatgtt gcagaacttt aggaacttta 25560

tcctagagcg ctcaggaatc ttgcccgcca cctgctgtgc acttcctagc gactttgtgc 25620

ccattaagta ccgcgaatgc cctccgccgc tttggggcca ctgctacctt ctgcagctag 25680

ccaactacct tgcctaccac tctgacataa tggaagacgt gagcggtgac ggtctactgg 25740

agtgtcactg tcgctgcaac ctatgcaccc cgcaccgctc cctggtttgc aattcgcagc 25800

tgcttaacga aagtcaaatt atcggtacct ttgagctgca gggtccctcg cctgacgaaa 25860

agtccgcggc tccggggttg aaactcactc cggggctgtg gacgtcggct taccttcgca 25920

aatttgtacc tgaggactac cacgcccacg agattaggtt ctacgaagac caatcccgcc 25980

cgccaaatgc ggagcttacc gcctgcgtca ttacccaggg ccacattctt ggccaattgc 26040

aagccatcaa caaagcccgc caagagtttc tgctacgaaa gggacggggg gtttacttgg 26100

acccccagtc cggcgaggag ctcaacccaa tccccccgcc gccgcagccc tatcagcagc 26160

agccgcgggc ccttgcttcc caggatggca cccaaaaaga agctgcagct gccgccgcca 26220

cccacggacg aggaggaata ctgggacagt caggcagagg aggttttgga cgaggaggag 26280

gaggacatga tggaagactg ggagagccta gacgaggaag cttccgaggt cgaagaggtg 26340

tcagacgaaa caccgtcacc ctcggtcgca ttcccctcgc cggcgcccca gaaatcggca 26400

accggttcca gcatggctac aacctccgct cctcaggcgc cgccggcact gcccgttcgc 26460

cgacccaacc gtagatggga caccactgga accagggccg gtaagtccaa gcagccgccg 26520

ccgttagccc aagagcaaca acagcgccaa ggctaccgct catggcgcgg gcacaagaac 26580

gccatagttg cttgcttgca agactgtggg ggcaacatct ccttcgcccg ccgctttctt 26640

ctctaccatc acggcgtggc cttcccccgt aacatcctgc attactaccg tcatctctac 26700

agcccatact gcaccggcgg cagcggcagc ggcagcaaca gcagcggcca cacagaagca 26760

aaggcgaccg gatagcaaga ctctgacaaa gcccaagaaa tccacagcgg cggcagcagc 26820

aggaggagga gcgctgcgtc tggcgcccaa cgaacccgta tcgacccgcg agcttagaaa 26880

caggattttt cccactctgt atgctatatt tcaacagagc aggggccaag aacaagagct 26940

gaaaataaaa aacaggtctc tgcgatccct cacccgcagc tgcctgtatc acaaaagcga 27000

agatcagctt cggcgcacgc tggaagacgc ggaggctctc ttcagtaaat actgcgcgct 27060

gactcttaag gactagtttc gcgccctttc tcaaatttaa gcgcgaaaac tacgtcatct 27120

ccagcggcca cacccggcgc cagcacctgt cgtcagcgcc attatgagca aggaaattcc 27180

cacgccctac atgtggagtt accagccaca aatgggactt gcggctggag ctgcccaaga 27240

ctactcaacc cgaataaact acatgagcgc gggaccccac atgatatccc gggtcaacgg 27300

aatccgcgcc caccgaaacc gaattctctt ggaacaggcg gctattacca ccacacctcg 27360

taataacctt aatccccgta gttggcccgc tgccctggtg taccaggaaa gtcccgctcc 27420

caccactgtg gtacttccca gagacgccca ggccgaagtt cagatgacta actcaggggc 27480

gcagcttgcg ggcggctttc gtcacagggt gcggtcgccc gggcagggta taactcacct 27540

gacaatcaga gggcgaggta ttcagctcaa cgacgagtcg gtgagctcct cgcttggtct 27600

ccgtccggac gggacatttc agatcggcgg cgccggccgt ccttcattca cgcctcgtca 27660

ggcaatccta actctgcaga cctcgtcctc tgagccgcgc tctggaggca ttggaactct 27720

gcaatttatt gaggagtttg tgccatcggt ctactttaac cccttctcgg gacctcccgg 27780

ccactatccg gatcaattta ttcctaactt tgacgcggta aaggactcgg cggacggcta 27840

cgactgaatg ttaagtggag aggcagagca actgcgcctg aaacacctgg tccactgtcg 27900

ccgccacaag tgctttgccc gcgactccgg tgagttttgc tactttgaat tgcccgagga 27960

tcatatcgag ggcccggcgc acggcgtccg gcttaccgcc cagggagagc ttgcccgtag 28020

cctgattcgg gagtttaccc agcgccccct gctagttgag cgggacaggg gaccctgtgt 28080

tctcactgtg atttgcaact gtcctaacct tggattacat caagatcttt gttgccatct 28140

ctgtgctgag tataataaat acagaaatta aaatatactg gggctcctat cgccatcctg 28200

taaacgccac cgtcttcacc cgcccaagca aaccaaggcg aaccttacct ggtactttta 28260

acatctctcc ctctgtgatt tacaacagtt tcaacccaga cggagtgagt ctacgagaga 28320

acctctccga gctcagctac tccatcagaa aaaacaccac cctccttacc tgccgggaac 28380

gtacgagtgc gtcaccggcc gctgcaccac acctaccgcc tgaccgtaaa ccagactttt 28440

tccggacaga cctcaataac tctgtttacc agaacaggag gtgagcttag aaaaccctta 28500

gggtattagg ccaaaggcgc agctactgtg gggtttatga acaattcaag caactctacg 28560

ggctattcta attcaggttt ctctagaatc ggggttgggg ttattctctg tcttgtgatt 28620

ctctttattc ttatactaac gcttctctgc ctaaggctcg ccgcctgctg tgtgcacatt 28680

tgcatttatt gtcagctttt taaacgctgg ggtcgccacc caagatgatt aggtacataa 28740

tcctaggttt actcaccctt gcgtcagccc acggtaccac ccaaaaggtg gattttaagg 28800

agccagcctg taatgttaca ttcgcagctg aagctaatga gtgcaccact cttataaaat 28860

gcaccacaga acatgaaaag ctgcttattc gccacaaaaa caaaattggc aagtatgctg 28920

tttatgctat ttggcagcca ggtgacacta cagagtataa tgttacagtt ttccagggta 28980

aaagtcataa aacttttatg tatacttttc cattttatga aatgtgcgac attaccatgt 29040

acatgagcaa acagtataag ttgtggcccc cacaaaattg tgtggaaaac actggcactt 29100

tctgctgcac tgctatgcta attacagtgc tcgctttggt ctgtacccta ctctatatta 29160

aatacaaaag cagacgcagc tttattgagg aaaagaaaat gccttaattt actaagttac 29220

aaagctaatg tcaccactaa ctgctttact cgctgcttgc aaaacaaatt caaaaagtta 29280

gcattataat tagaatagga tttaaacccc ccggtcattt cctgctcaat accattcccc 29340

tgaacaattg actctatgtg ggatatgctc cagcgctaca accttgaagt caggcttcct 29400

ggatgtcagc atctgacttt ggccagcacc tgtcccgcgg atttgttcca gtccaactac 29460

agcgacatcg atatggcttc gtatcccggc catcagcacg cgtctgcgtt cgaccaggct 29520

gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg gcagcaagaa 29580

gccacggaag tccgcccgga gcagaaaatg cccacgctac tgcgggttta tatagacggt 29640

ccccacggga tggggaaaac caccaccacg caactgctgg tggccctggg ttcgcgcgac 29700

gatatcgtct acgtacccga gccgatgact tactggcggg tgctgggggc ttccgagaca 29760

atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc ggccggggac 29820

gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc cgtgaccgac 29880

gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc gcccccggcc 29940

ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc gcccgcgcga 30000

taccttatgg gcagcatgac cccccaggcc gtgccggcgt tcgtggccct catcccgccg 30060

accttgcccg gcacaaacat cgtgttgggg gcccttccgg aggacagaca catcgaccgc 30120

ctggccaaac gccagcgccc cggcgagcgg cttgacctgg ctatgctggc cgcgattcgc 30180

cgcgtttacg ggctgcttgc caatacggtg cggtatctgc agggcggcgg gtcgtggcgg 30240

gaggattggg gacagctttc ggggacggcc gtgccgcccc agggtgccga gccccagagc 30300

aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg ggcccccgag 30360

ttgctggccc ccaacggcga cctgtacaac gtgtttgcct gggccttgga cgtcttggcc 30420

aaacgcctcc gtcccatgca cgtccttatc ctggattacg accaatcgcc cgccggctgc 30480

cgggacgccc tgctgcaact tacctccggg atgatccaga cccacgtcac caccccaggc 30540

tccataccga cgatctgcga cctggcgcgc acgtttgccc gggagatggg ggcggctcac 30600

tgaatcgatt gatggaatcc atagattgga cggactgaaa cacatgttct tttctcttac 30660

agtatgatta aatgagacat gattcctcga gtttttatat tactgaccct tgttgcgctt 30720

ttttgtgcgt gctccacatt ggctgcggtt tctcacatcg aagtagactg cattccagcc 30780

ttcacagtct atttgcttta cggatttgtc accctcacgc tcatctgcag cctcatcact 30840

gtggtcatcg cctttatcca gtgcattgac tgggtctgtg tgcgctttgc atatctcaga 30900

caccatcccc agtacaggga caggactata gctgagcttc ttagaattct ttaattatga 30960

aatttactgt gacttttctg ctgattattt gcaccctatc tgcgttttgt tccccgacct 31020

ccaagcctca aagacatata tcatgcagat tcactcgtat atggaatatt ccaagttgct 31080

acaatgaaaa aagcgatctt tccgaagcct ggttatatgc aatcatctct gttatggtgt 31140

tctgcagtac catcttagcc ctagctatat atccctacct tgacattggc tggaaacgaa 31200

tagatgccat gaaccaccca actttccccg cgcccgctat gcttccactg caacaagttg 31260

ttgccggcgg ctttgtccca gccaatcagc ctcgccccac ttctcccacc cccactgaaa 31320

tcagctactt taatctaaca ggaggagatg actgacaccc tagatctaga aatggacgga 31380

attattacag agcagcgcct gctagaaaga cgcagggcag cggccgagca acagcgcatg 31440

aatcaagagc tccaagacat ggttaacttg caccagtgca aaaggggtat cttttgtctg 31500

gtaaagcagg ccaaagtcac ctacgacagt aataccaccg gacaccgcct tagctacaag 31560

ttgccaacca agcgtcagaa attggtggtc atggtgggag aaaagcccat taccataact 31620

cagcactcgg tagaaaccga aggctgcatt cactcacctt gtcaaggacc tgaggatctc 31680

tgcaccctta ttaagaccct gtgcggtctc aaagatctta ttccctttaa ctaataaaaa 31740

aaaataataa agcatcactt acttaaaatc agttagcaaa tttctgtcca gtttattcag 31800

cagcacctcc ttgccctcct cccagctctg gtattgcagc ttcctcctgg ctgcaaactt 31860

tctccacaat ctaaatggaa tgtcagtttc ctcctgttcc tgtccatccg cacccactat 31920

cttcatgttg ttgcagatga agcgcgcaag accgtctgaa gataccttca accccgtgta 31980

tccatatgac acggaaaccg gtcctccaac tgtgcctttt cttactcctc cctttgtatc 32040

ccccaatggg tttcaagaga gtccccctgg ggtactctct ttgcgcctat ccgaacctct 32100

agttacctcc aatggcatgc ttgcgctcaa aatgggcaac ggcctctctc tggacgaggc 32160

cggcaacctt acctcccaaa atgtaaccac tgtgagccca cctctcaaaa aaaccaagtc 32220

aaacataaac ctggaaatat ctgcacccct cacagttacc tcagaagccc taactgtggc 32280

tgccgccgca cctctaatgg tcgcgggcaa cacactcacc atgcaatcac aggccccgct 32340

aaccgtgcac gactccaaac ttagcattgc cacccaagga cccctcacag tgtcagaagg 32400

aaagctagcc ctgcaaacat caggccccct caccaccacc gatagcagta cccttactat 32460

cactgcctca ccccctctaa ctactgccac tggtagcttg ggcattgact tgaaagagcc 32520

catttataca caaaatggaa aactaggact aaagtacggg gctcctttgc atgtaacaga 32580

cgacctaaac actttgaccg tagcaactgg tccaggtgtg actattaata atacttcctt 32640

gcaaactaaa gttactggag ccttgggttt tgattcacaa ggcaatatgc aacttaatgt 32700

agcaggagga ctaaggattg attctcaaaa cagacgcctt atacttgatg ttagttatcc 32760

gtttgatgct caaaaccaac taaatctaag actaggacag ggccctcttt ttataaactc 32820

agcccacaac ttggatatta actacaacaa aggcctttac ttgtttacag cttcaaacaa 32880

ttccaaaaag cttgaggtta acctaagcac tgccaagggg ttgatgtttg acgctacagc 32940

catagccatt aatgcaggag atgggcttga atttggttca cctaatgcac caaacacaaa 33000

tcccctcaaa acaaaaattg gccatggcct agaatttgat tcaaacaagg ctatggttcc 33060

taaactagga actggcctta gttttgacag cacaggtgcc attacagtag gaaacaaaaa 33120

taatgataag ctaactttgt ggaccacacc agctccatct cctaactgta gactaaatgc 33180

agagaaagat gctaaactca ctttggtctt aacaaaatgt ggcagtcaaa tacttgctac 33240

agtttcagtt ttggctgtta aaggcagttt ggctccaata tctggaacag ttcaaagtgc 33300

tcatcttatt ataagatttg acgaaaatgg agtgctacta aacaattcct tcctggaccc 33360

agaatattgg aactttagaa atggagatct tactgaaggc acagcctata caaacgctgt 33420

tggatttatg cctaacctat cagcttatcc aaaatctcac ggtaaaactg ccaaaagtaa 33480

cattgtcagt caagtttact taaacggaga caaaactaaa cctgtaacac taaccattac 33540

actaaacggt acacaggaaa caggagacac aactccaagt gcatactcta tgtcattttc 33600

atgggactgg tctggccaca actacattaa tgaaatattt gccacatcct cttacacttt 33660

ttcatacatt gcccaagaat aaagaatcgt ttgtgttatg tttcaacgtg tttatttttc 33720

aattgcagaa aatttcaagt catttttcat tcagtagtat agccccacca ccacatagct 33780

tatacagatc accgtacctt aatcaaactc acagaaccct agtattcaac ctgccacctc 33840

cctcccaaca cacagagtac acagtccttt ctccccggct ggccttaaaa agcatcatat 33900

catgggtaac agacatattc ttaggtgtta tattccacac ggtttcctgt cgagccaaac 33960

gctcatcagt gatattaata aactccccgg gcagctcact taagttcatg tcgctgtcca 34020

gctgctgagc cacaggctgc tgtccaactt gcggttgctt aacgggcggc gaaggagaag 34080

tccacgccta catgggggta gagtcataat cgtgcatcag gatagggcgg tggtgctgca 34140

gcagcgcgcg aataaactgc tgccgccgcc gctccgtcct gcaggaatac aacatggcag 34200

tggtctcctc agcgatgatt cgcaccgccc gcagcataag gcgccttgtc ctccgggcac 34260

agcagcgcac cctgatctca cttaaatcag cacagtaact gcagcacagc accacaatat 34320

tgttcaaaat cccacagtgc aaggcgctgt atccaaagct catggcgggg accacagaac 34380

ccacgtggcc atcataccac aagcgcaggt agattaagtg gcgacccctc ataaacacgc 34440

tggacataaa cattacctct tttggcatgt tgtaattcac cacctcccgg taccatataa 34500

acctctgatt aaacatggcg ccatccacca ccatcctaaa ccagctggcc aaaacctgcc 34560

cgccggctat acactgcagg gaaccgggac tggaacaatg acagtggaga gcccaggact 34620

cgtaaccatg gatcatcatg ctcgtcatga tatcaatgtt ggcacaacac aggcacacgt 34680

gcatacactt cctcaggatt acaagctcct cccgcgttag aaccatatcc cagggaacaa 34740

cccattcctg aatcagcgta aatcccacac tgcagggaag acctcgcacg taactcacgt 34800

tgtgcattgt caaagtgtta cattcgggca gcagcggatg atcctccagt atggtagcgc 34860

gggtttctgt ctcaaaagga ggtagacgat ccctactgta cggagtgcgc cgagacaacc 34920

gagatcgtgt tggtcgtagt gtcatgccaa atggaacgcc ggacgtagtc atatttcctg 34980

aagcaaaacc aggtgcgggc gtgacaaaca gatctgcgtc tccggtctcg ccgcttagat 35040

cgctctgtgt agtagttgta gtatatccac tctctcaaag catccaggcg ccccctggct 35100

tcgggttcta tgtaaactcc ttcatgcgcc gctgccctga taacatccac caccgcagaa 35160

taagccacac ccagccaacc tacacattcg ttctgcgagt cacacacggg aggagcggga 35220

agagctggaa gaaccatgtt ttttttttta ttccaaaaga ttatccaaaa cctcaaaatg 35280

aagatctatt aagtgaacgc gctcccctcc ggtggcgtgg tcaaactcta cagccaaaga 35340

acagataatg gcatttgtaa gatgttgcac aatggcttcc aaaaggcaaa cggccctcac 35400

gtccaagtgg acgtaaaggc taaacccttc agggtgaatc tcctctataa acattccagc 35460

accttcaacc atgcccaaat aattctcatc tcgccacctt ctcaatatat ctctaagcaa 35520

atcccgaata ttaagtccgg ccattgtaaa aatctgctcc agagcgccct ccaccttcag 35580

cctcaagcag cgaatcatga ttgcaaaaat tcaggttcct cacagacctg tataagattc 35640

aaaagcggaa cattaacaaa aataccgcga tcccgtaggt cccttcgcag ggccagctga 35700

acataatcgt gcaggtctgc acggaccagc gcggccactt ccccgccagg aaccttgaca 35760

aaagaaccca cactgattat gacacgcata ctcggagcta tgctaaccag cgtagccccg 35820

atgtaagctt tgttgcatgg gcggcgatat aaaatgcaag gtgctgctca aaaaatcagg 35880

caaagcctcg cgcaaaaaag aaagcacatc gtagtcatgc tcatgcagat aaaggcaggt 35940

aagctccgga accaccacag aaaaagacac catttttctc tcaaacatgt ctgcgggttt 36000

ctgcataaac acaaaataaa ataacaaaaa aacatttaaa cattagaagc ctgtcttaca 36060

acaggaaaaa caacccttat aagcataaga cggactacgg ccatgccggc gtgaccgtaa 36120

aaaaactggt caccgtgatt aaaaagcacc accgacagct cctcggtcat gtccggagtc 36180

ataatgtaag actcggtaaa cacatcaggt tgattcatcg gtcagtgcta aaaagcgacc 36240

gaaatagccc gggggaatac atacccgcag gcgtagagac aacattacag cccccatagg 36300

aggtataaca aaattaatag gagagaaaaa cacataaaca cctgaaaaac cctcctgcct 36360

aggcaaaata gcaccctccc gctccagaac aacatacagc gcttcacagc ggcagcctaa 36420

cagtcagcct taccagtaaa aaagaaaacc tattaaaaaa acaccactcg acacggcacc 36480

agctcaatca gtcacagtgt aaaaaagggc caagtgcaga gcgagtatat ataggactaa 36540

aaaatgacgt aacggttaaa gtccacaaaa aacacccaga aaaccgcacg cgaacctacg 36600

cccagaaacg aaagccaaaa aacccacaac ttcctcaaat cgtcacttcc gttttcccac 36660

gttacgtaac ttcccatttt aagaaaacta caattcccaa cacatacaag ttactccgcc 36720

ctaaaaccta cgtcacccgc cccgttccca cgccccgcgc cacgtcacaa actccacccc 36780

ctcattatca tattggcttc aatccaaaat aaggtatatt attgatgatg 36830

Claims

1. An oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is Ad 5/delta E1A/TMTP1 delta ADP/HSV-TK, the nucleotide sequence of the oncolytic adenovirus recombinant carrying TMTP1 and HSV-TK is shown as SEQ ID No.23, 27 basic groups shown as SEQ ID No.1 in a 920nt-946nt region are deleted in an E1A conserved sequence 2 region of a human 5-type adenovirus gene, a gene sequence shown as SEQ ID No.15 and used for coding tumor targeting peptide TMTP1 is inserted in a 19641nt-19655nt region of a Hexon hypervariable region 5, meanwhile, an E3 region is deleted in a 29477nt-29714 region of the ADP gene to form a deletion region, the deletion region is inserted in the full-length enzyme digestion coding sequence shown as SEQ ID No.22 and used for coding the HSV-TK gene, and Cla1 sites are introduced.

2. The method of constructing an oncolytic adenoviral recombinant harboring TMTP1 and HSV-TK according to claim 1, comprising the steps of:

step 1: targeted deletion of human adenovirus type 5 genes

step 2: preparation of Ad 5/. DELTA.E 1A/TMTP1

3. Use of an oncolytic adenoviral recombinant carrying TMTP1 and HSV-TK according to claim 1 for the manufacture of a medicament for the treatment of a tumour.

4. Use of the oncolytic adenoviral recombinant carrying TMTP1 and HSV-TK of claim 1 for the preparation of a gene therapy vector.

5. Use of an oncolytic adenoviral recombinant carrying TMTP1 and HSV-TK according to claim 1 for the preparation of a medicament for the amelioration of resistance to anti-tumour chemotherapeutic agents.

6. Use of the oncolytic adenoviral recombinant carrying TMTP1 and HSV-TK of claim 1 for the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.