CN114317463A - Oncolytic adenovirus recombinant carrying TMTP1 and tBID, and construction method and application thereof - Google Patents

Abstract

The invention discloses an oncolytic adenovirus recombinant carrying TMTP1 and tBID, a construction method and application thereof, and belongs to the technical field of medical genetic engineering. According to the invention, 27 bases in a 920nt-946nt region are deleted in an E1A conserved sequence 2 region of a human 5-type adenovirus gene, a gene sequence for coding a tumor targeting peptide TMTP1 is inserted in a 19641nt-19655nt region of a Hexon hypervariable region 5, a deleted region is formed by deleting an E3 region which is positioned in a 29477nt-29714nt region of an ADP gene, a gene sequence of a mitochondrial apoptotic peptide tBod is inserted in the deleted region, and a Cla1 enzyme cutting site is introduced. The invention also discloses a construction method and application of the oncolytic adenovirus recombinant. The oncolytic adenovirus recombinant carrying TMTP1 and tBId has ideal targeting effect and strong killing effect.

Description

Oncolytic adenovirus recombinant carrying TMTP1 and tBID, and construction method and application thereof

Technical Field

The invention relates to an oncolytic adenovirus recombinant carrying TMTP1 and tBID, a construction method and application thereof, belonging 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 objects of the present invention is to provide an oncolytic adenoviral recombinant carrying TMTP1 and tBId.

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

The inventors of the present application, in order to obtain the above oncolytic adenoviral recombinants carrying TMTP1 and tBId, 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 tBId.

Apoptosis is the most important link for killing tumor cells by various anticancer drugs and is regulated by BCL-2 (B-cellymphoma-2) family. And the Bid is mitochondrial apoptotic peptide promoting apoptosis in BCL-2 family, when the cell senses damage, caspase8 is activated, activated caspase8 can cut the Bid free in cytoplasm into tBod, the tBod is gathered on the outer mitochondrial membrane again, downstream bak/bax is activated to form dimer and trimer through oligomerization, and thus, the hole is punched on the mitochondria to cause depolarization (MOMP) of the outer mitochondrial membrane, and finally the cell is apoptotic. According to the invention, a gene sequence which is shown as SEQ ID NO.22 and encodes active form tBod of mitochondrial apoptotic peptide is inserted into human type 5 adenovirus (Ad5), so that the mitochondrial apoptotic peptide tBod is specifically expressed in tumors, and tumor cell apoptosis is promoted, thereby remarkably improving the apoptosis promoting effect of oncolytic adenovirus recombinants carrying TMTP1 and tBod and the killing effect of oncolytic virus lytic cells.

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, the oncolytic adenovirus recombinant carrying TMTP1 and tBId can specifically identify tumors and metastasis thereof, has higher tumor targeted replication capacity, utilizes the dual killing effect of the apoptosis promotion effect of the tBId and the cell lysis effect of oncolytic virus, and verifies that the oncolytic adenovirus recombinant has strong killing effect on cisplatin-resistant cell lines through in vitro and in vivo experiments, and has ideal targeting effect and strong killing effect.

The oncolytic adenovirus recombinant carrying TMTP1 and tBId has the beneficial effects that:

1. the oncolytic adenovirus recombinant carrying TMTP1 and tBId 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 tBId 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 tBId has wide anticancer spectrum. The in vivo model research of the tumor animal model proves that the oncolytic adenovirus recombinant carrying TMTP1 and tBId 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 object of the present invention is to provide a method for constructing the oncolytic adenovirus recombinant carrying TMTP1 and tBId.

The technical scheme for solving the problems is as follows: the construction method of the oncolytic adenovirus recombinant carrying TMTP1 and tBId 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-tBod carrying TMTP1 and tBod

The E3 region of Ad 5/delta E1A/TMTP1 obtained in step 2 is located in the 29477nt-29714nt region of an ADP gene to form a deletion region, the gene sequence of mitochondrial apoptotic peptide tBod shown in SEQ ID NO.22 is inserted into the deletion region, and Cla1 enzyme cutting site is introduced, thus obtaining oncolytic adenovirus recombinant Ad 5/delta E1A/TMTP 1/delta ADP-tBod carrying TMTP1 and tBod shown in SEQ ID NO. 23.

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

1. the oncolytic adenovirus recombinant carrying TMTP1 and tBId is inserted with a gene sequence coding tumor targeting peptide TMTP1 and a gene sequence coding mitochondrial apoptosis peptide tBId, has the advantages of tumor specific replication, high tumor and metastatic tumor targeting property thereof, specific 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 both 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 object of the present invention is to provide the use of the oncolytic adenoviral recombinant carrying TMTP1 and tBod as described above.

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

The beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBId of the invention are as follows:

the oncolytic adenovirus recombinant carrying TMTP1 and tBId 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 tBId, and has positive pharmaceutical value and wide social significance.

Another object of the present invention is to provide another use of the above oncolytic adenoviral recombinant carrying TMTP1 and tBId.

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

The beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBId of the invention are as follows:

the oncolytic adenovirus recombinant carrying TMTP1 and tBId 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 tBId, and has positive pharmaceutical value and wide social significance.

Another object of the present invention is to provide another use of the above oncolytic adenoviral recombinant carrying TMTP1 and tBId.

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

The beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBId of the invention are as follows:

the oncolytic adenovirus recombinant carrying TMTP1 and tBId can be used for preparing drugs for improving drug resistance of the prepared anti-tumor chemotherapeutic drugs, not only develops new drugs for improving drug resistance of the prepared anti-tumor chemotherapeutic drugs, but also develops application of the oncolytic adenovirus recombinant carrying TMTP1 and tBId, 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 tBId described above.

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

The beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBId of the invention are as follows:

the oncolytic adenovirus recombinant carrying TMTP1 and tBId 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 tBId, and has positive pharmaceutical value and wide social significance.

Drawings

FIG. 1 is a structural schematic diagram of oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMTP 1/. DELTA.ADP-tBId carrying TMTP1 and tBId 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 the content of adenovirus in tissues and tumors of the control group and the experimental group in example 7 of the present invention.

FIG. 4 is a bar graph showing the relative amounts of adenovirus in the control and experimental groups in each tissue in example 7 of the present invention.

FIG. 5 is a control curve of tumor volume change in a subcutaneous tumor animal model in example 8 of the present invention.

FIG. 6 is a photograph showing the tumor volumes of the experimental group and the control group in the abdominal cavity in situ metastasis model in example 8 of the present invention.

Fig. 7 is a graph showing the results of an aspartate Aminotransferase (AST) experiment in an experimental animal of an abdominal cavity in situ metastatic tumor model in example 8 of the present invention.

FIG. 8 shows the results of alanine Aminotransferase (ALT) experiments in experimental animals of the abdominal cavity in situ metastatic tumor model in example 8 of the present invention.

FIG. 9 shows the experimental results of Urea Nitrogen (BUN) test in experimental animals of the abdominal cavity in situ metastatic tumor model in example 8 of the present invention.

FIG. 10 shows the Creatinine (Creatinine) test results of the experimental animals of the abdominal cavity in situ metastatic tumor model in example 8 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 Microbix Biosystem Inc. (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.

Fragments1, acquisition: primer 1: 5' -cgggatccgggcccccatttcc-3' (SEQ ID NO 2), corresponding to 9883-; primer 2: 5' -gtcactgggtggatcgatcacctccggtac-3' (SEQ ID NO 3), 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); 2mM dNTP, 10. mu.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 NO 5), 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 × TransStartFastFlay 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; 35 cycles of 95 ℃, 20s, -5 ℃, 20s, 72 ℃, 3 min; 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 Xcel l (TM) 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 in a culture medium10% FBS DMEM, by day two, the 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. Subjecting the plate to three cycles of freezing/thawing to release virus, collecting lysate in a 15ml tube, centrifuging at maximum speed for 10min, collecting supernatant, freezing at-80 deg.C, and collecting the supernatant as second generation virus (about 5 × 10)⁷Viral/ml. Performing secondary amplification on the above virusesAt 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; 28 cycles in total; extension at 72 ℃ for 10 min. The PCR product is 1400bp, and the PCR product is separated and purified by conventional electrophoresisAfterwards, the concentrations were detected for DNA sequencing, sequencing primers: 5'-agccggagcagagagccttg-3' (SEQ ID NO 12), and the clone with correct sequencing is selected as 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 O.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.E 1A/TMTP 1/. DELTA.ADP-tBId 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 NO21), corresponding to 30137nt-30118 nt; PCR conditions were as above, the product was 422bp (fragment 2), and after separation and purification by conventional electrophoresis, the concentration was determined forAnd carrying out 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 gene sequence of mitochondrial apoptosis peptide tBod shown in SEQ ID NO.22 is introduced into 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/tBId 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 skeleton plasmid and the E3/tBId fragment obtained in step 3.3 are electrotransferred into Escherichia coli BJ5183 electrotransferred peptide obtained in step 1.3 under the electrotransfer condition of 2.5Kv and 25 muF, and homologous recombination is carried out to obtain pAd 5/delta E1A/HVR2/TMTP 1/delta ADP-tBId plasmid, pAd 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId plasmid and pAd 5/delta E1A/HVR7/TMTP 1/delta ADP-tBId plasmid, respectively.

Example 4: obtaining, amplifying and purifying oncolytic adenovirus recombinant Ad 5/delta E1A/TMTP 1/delta ADP-tBId carrying TMTP1 and tBId

Step 4.1: obtaining oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMTP 1/. DELTA.ADP-tBId carrying TMTP1 and tBId

Step 4.1.1: respectively extracting the pAd 5/delta E1A/HVR2/TMTP 1/delta ADP-tBId plasmid, pAd 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId plasmid and pAd 5/delta E1A/HVR7/TMTP 1/delta ADP-tBId plasmid obtained in the step 3.4 in a large quantity, using PacI to digest the plasmids, extracting with phenol and chloroform, and recovering the digested 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.E 1A/TMTP 1/. DELTA.ADP-tBId obtained in step 4.1.3 were identified and E1A, Hexon and E3/ADP regions were sequenced after PCR with the corresponding primers, respectively.

Step 4.2: amplification, purification and titer determination of oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMTP 1/. DELTA.ADP-tBId carrying TMTP1 and tBId

Step 4.2.1: amplification step 4.1.4 sequencing the correct oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMTP 1/. DELTA.ADP-tBId carrying TMTP1 and tBId, detecting the titer (MOI) of the adenovirus seeds, determining the amount of virus infecting 293 cells based on 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 tBod

The purpose of this example was to determine the efficiency of oncolytic adenovirus recombinants carrying TMTP1 and tBId inserted into three different regions of the hypervariable region 2, 5, 7 of Hexon on tumor cell infection, in order to select the targeted peptide insertion region that has the best effect on tumor cell infection.

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 adenovirus recombinant Ad 5/. DELTA.E 1A/HVR5/TMTP 1/. DELTA.ADP-tBId carrying TMTP1 and tBId 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: identification of therapeutic Effect of oncolytic adenovirus recombinants carrying TMTP1 and tBod

Step 6.1: identification of oncolytic adenovirus recombinant in vitro tumor cell killing effect carrying TMTP1 and tBod

The purpose of this example is to identify the killing effect of Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBod oncolytic adenovirus on a range of tumor cells and normal cells. The cell lines selected were as shown in table 1, with different P53, Rb gene phenotypes; have different tissue origins, so that the experimental results can represent different types of tumors.

Seeding in 96-well culture plates 5X 10³The tumor cells are cultured in 10% FBS DMEM or RIPM1640, the cells are fused at about 70% by the next day, the liquid is absorbed, the quantity of oncolytic adenovirus corresponding to MOI 10 is diluted to 100 mu l by using a culture medium and added into a culture well, and the liquid is gently shakenThree times in volume at 37 ℃ with 5% CO₂After incubation in the incubator of (1) for 72h, the viral survival rate was measured by adding cck-8.

Experimental results show that when the virus MOI is 10, 44.5% of tumor cells appear to be extremely sensitive with a survival rate of less than 25%; 38.8% of the tumor cells have good response to adenovirus, and the survival rate is between 25% and 75%; however, 16.7% of the tumor cells responded slightly to adenovirus, and the results are shown in Table 1.

Step 6.2: identification of oncolytic adenovirus recombinant carrying TMTP1 and tBID on sensitization effect of chemotherapeutic drug cisplatin

Considering that oncolytic adenoviral recombinants carrying TMTP1 and tBid do not show potent killing effect on all tumor cells, it was envisaged that oncolytic adenoviral recombinants carrying TMTP1 and tBid in combination with cisplatin (DDP) kill insensitive tumor cells.

Seeded into 96-well cell culture plates at 5X 10³Tumor cells were mixed with a certain concentration of oncolytic adenovirus recombinant carrying TMTP1 and tBod and its control virus Ad 5/. DELTA.E 1A. The control virus Ad 5/. DELTA.E 1A was an oncolytic adenovirus with a 27bp deletion in E1A only, constructed and stored in the laboratory. Cell viability was measured after 48h incubation with DDP medium at different concentrations the following day, and the IC of each cell line on DDP after adenovirus incubation was calculated₅₀(i.e., the concentration of DDP that causes 50% apoptosis in tumor cells) and the synergy index CI of oncolytic adenovirus recombinant Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBod carrying TMTP1 and tBod with DDP was calculated. A CI value less than 1 indicates a synergistic effect of the two drugs, and a smaller CI value indicates a stronger synergistic effect^[1]. The specific experimental results are shown in table 1.

TABLE 1

Experimental results show that for most tumor cells, Ad 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId and DDP are combined to have a sensitizing effect, and are particularly obvious for drug-resistant cell lines such as ES2, C13K and MDA-MB-231, and the CI values are 0.2, 0.37 and 0.04 respectively. The above results fully demonstrate that oncolytic adenoviral recombinants carrying TMTP1 and tBID of the invention are sensitized to the chemotherapeutic agent cisplatin, whether in cell lines that are sensitive to or resistant to the primary DDP.

Example 7: in vivo experiments prove that oncolytic adenovirus recombinant carrying TMTP1 and tBod can escape liver uptake and can specifically target tumor cells

4-6 weeks of BALB/c nu nude mice, 2 x 10 abdominal cavity planting⁶Skov3 ovarian cancer cells, after 4 weeks, nude mice were divided into two groups (n ═ 4), and each was intraperitoneally injected with 1 × 10 cells⁸Oncolytic adenoviral recombinants Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBId with PFU carrying TMTP1 and tBId and its control virus Ad5/Δ E1A. After 48h, the liver, spleen, lung and tumor tissues of each mouse were taken out, DNA of each tissue was extracted, and the DNA content of adenovirus in 400ng of tissue DNA was detected, with the results shown in FIG. 3: the control virus Ad 5/delta E1A has aggregation in liver, spleen and lung and is distributed less in tumor tissue, and Ad 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId is mainly distributed in tumor tissue and is hardly taken up by liver; FIG. 4 shows the relative adenovirus content in each tissue of nude mice, Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBId injected mice, adenovirus concentrated mainly in tumor tissue, which was nearly 100-fold higher than that of the control virus, whereas Ad5/Δ E1A control virus was taken up by the liver in large amounts, and almost no adenovirus gene was detected in tumor tissue.

The experimental results fully show that the oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId carrying TMTP1 and tBId overcomes the hepatic tropism, is hardly taken by the liver any more, and greatly enhances the targeting to the tumor.

Example 8: in vivo experiments verify the killing effect and safety of oncolytic adenovirus recombinant carrying TMTP1 and tBId on tumors

Step 8.1: subcutaneous tumor animal model

4-6 weeks of BALB/c nu nude mice, one-sided axillary fat padPlant 2X 10⁶Four weeks later, mice were divided into six groups (n ═ 6) of tumor soybean size, six groups were: a-blank group, b-single addition of DDP, c-single injection of Ad5/Δ E1A, d-injection of Ad5/Δ E1A plus DDP, E-single injection of Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBId, f-injection of Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBId plus DDP.

Wherein, c group, d group, e group and f group are injected with adenovirus 1X 10 continuously in tumor⁸PFU, three days later, intraperitoneal injection of DDP in group b, group d and group f, 2.5mg/kg, once every other day, four times. Tumor volume was measured every three days, starting after tumor implantation, and the change in tumor volume is shown in fig. 5.

In group f, 6 mice had completely disappeared tumors, while in group E, the tumor growth was not well controlled by the single injection of Ad 5/. DELTA.E 1A/HVR5/TMTP 1/. DELTA.ADP-tBId.

Step 8.2: abdominal cavity in-situ metastatic tumor model

4-6 weeks of BALB/c nu nude mice, 2 x 10 abdominal cavity planting⁶skov3 ovarian cancer cells, four weeks later, mice were divided into six groups (n: 16-21) with the same groups and the same administration time, the virus administration was changed to intraperitoneal injection, and the rest was the same as subcutaneous tumor model, after 15d, three mice were randomly selected from each group, and the largest tumor in the abdominal cavity was taken out, as shown in fig. 6: the tumor size in the experimental group f is obviously smaller than that in each experimental group and the control group (p)<0.0001)。

Step 8.3: oncolytic adenovirus recombinant safety assay carrying TMTP1 and tBID

In the above abdominal cavity in situ metastatic tumor model, after anesthetizing the mice, blood was taken 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 (creatine), and urea nitrogen (BUN), as shown in fig. 7-10, in the control and experimental groups, renal function was not significantly impaired, but mice injected with control virus Ad5/Δ E1A had significantly elevated alanine transaminase (ALT) and aspartate transaminase (AST), while mice injected with Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBid had normal liver function.

The above results indicate that oncolytic adenovirus recombinant Ad5/Δ E1A/HVR5/TMTP1/Δ ADP-tBId carrying TMTP1 and tBId can escape liver uptake, has no damage to liver, and can specifically target tumor tissues. The tumor cell killing effect is obvious in vivo and in vitro, the sensitization effect on the chemotherapeutic drug cisplatin is particularly obvious, the tumor progress can be controlled by combining the subcutaneous tumor model and the abdominal cavity planting model, and the tumor cell sensitizing agent is safe and reliable as a gene therapy vector and has no toxic or side effect on experimental animals.

Therefore, the oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId carrying TMTP1 and tBId 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 tBId, and has positive pharmaceutical value and wide social significance.

The oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMTP 1/delta ADP-tBId carrying TMTP1 and tBId can be used for preparing a gene therapy vector, not only develops a new gene therapy vector, but also develops the application of the oncolytic adenovirus recombinant carrying TMTP1 and tBId, and has positive pharmaceutical value and wide social significance.

Reference documents:

[1].Chou TC,Motzer RJ,Tong Y,Bosl GJ(1994)Computerized quantitation of synergism and antagonism of taxol,topotecan,and cisplatin against human teratocarcinoma cell growth:A rational approach to clinical protocol design.J Natl Cancer Inst 86:1517–1524.

the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Wuhan Kaideweis Biotech Co., Ltd

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gaggaccgga acagggacct ggccactgcc ctggagcagc tgctgcaggc ctaccctaga 240

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gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180

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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

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tacgggggac ccagatatta tgtgttcgct ttgctatatg aggacctgtg gcatgtttgt 1080

ctacagtaag tgaaaattat gggcagtggg tgatagagtg gtgggtttgg tgtggtaatt 1140

ttttttttaa tttttacagt tttgtggttt aaagaatttt gtattgtgat ttttttaaaa 1200

ggtcctgtgt ctgaacctga gcctgagccc gagccagaac cggagcctgc aagacctacc 1260

cgccgtccta aaatggcgcc tgctatcctg agacgcccga catcacctgt gtctagagaa 1320

tgcaatagta gtacggatag ctgtgactcc ggtccttcta acacacctcc tgagatacac 1380

ccggtggtcc cgctgtgccc cattaaacca gttgccgtga gagttggtgg gcgtcgccag 1440

gctgtggaat gtatcgagga cttgcttaac gagcctgggc aacctttgga cttgagctgt 1500

aaacgcccca ggccataagg tgtaaacctg tgattgcgtg tgtggttaac gcctttgttt 1560

gctgaatgag ttgatgtaag tttaataaag ggtgagataa tgtttaactt gcatggcgtg 1620

ttaaatgggg cggggcttaa agggtatata atgcgccgtg ggctaatctt ggttacatct 1680

gacctcatgg aggcttggga gtgtttggaa gatttttctg ctgtgcgtaa cttgctggaa 1740

cagagctcta acagtacctc ttggttttgg aggtttctgt ggggctcatc ccaggcaaag 1800

ttagtctgca gaattaagga ggattacaag tgggaatttg aagagctttt gaaatcctgt 1860

ggtgagctgt ttgattcttt gaatctgggt caccaggcgc ttttccaaga gaaggtcatc 1920

aagactttgg atttttccac accggggcgc gctgcggctg ctgttgcttt tttgagtttt 1980

ataaaggata aatggagcga agaaacccat ctgagcgggg ggtacctgct ggattttctg 2040

gccatgcatc tgtggagagc ggttgtgaga cacaagaatc gcctgctact gttgtcttcc 2100

gtccgcccgg cgataatacc gacggaggag cagcagcagc agcaggagga agccaggcgg 2160

cggcggcagg agcagagccc atggaacccg agagccggcc tggaccctcg ggaatgaatg 2220

ttgtacaggt ggctgaactg tatccagaac tgagacgcat tttgacaatt acagaggatg 2280

ggcaggggct aaagggggta aagagggagc ggggggcttg tgaggctaca gaggaggcta 2340

ggaatctagc ttttagctta atgaccagac accgtcctga gtgtattact tttcaacaga 2400

tcaaggataa ttgcgctaat gagcttgatc tgctggcgca gaagtattcc atagagcagc 2460

tgaccactta ctggctgcag ccaggggatg attttgagga ggctattagg gtatatgcaa 2520

aggtggcact taggccagat tgcaagtaca agatcagcaa acttgtaaat atcaggaatt 2580

gttgctacat ttctgggaac ggggccgagg tggagataga tacggaggat agggtggcct 2640

ttagatgtag catgataaat atgtggccgg gggtgcttgg catggacggg gtggttatta 2700

tgaatgtaag gtttactggc cccaatttta gcggtacggt tttcctggcc aataccaacc 2760

ttatcctaca cggtgtaagc ttctatgggt ttaacaatac ctgtgtggaa gcctggaccg 2820

atgtaagggt tcggggctgt gccttttact gctgctggaa gggggtggtg tgtcgcccca 2880

aaagcagggc ttcaattaag aaatgcctct ttgaaaggtg taccttgggt atcctgtctg 2940

agggtaactc cagggtgcgc cacaatgtgg cctccgactg tggttgcttc atgctagtga 3000

aaagcgtggc tgtgattaag cataacatgg tatgtggcaa ctgcgaggac agggcctctc 3060

agatgctgac ctgctcggac ggcaactgtc acctgctgaa gaccattcac gtagccagcc 3120

actctcgcaa ggcctggcca gtgtttgagc ataacatact gacccgctgt tccttgcatt 3180

tgggtaacag gaggggggtg ttcctacctt accaatgcaa tttgagtcac actaagatat 3240

tgcttgagcc cgagagcatg tccaaggtga acctgaacgg ggtgtttgac atgaccatga 3300

agatctggaa ggtgctgagg tacgatgaga cccgcaccag gtgcagaccc tgcgagtgtg 3360

gcggtaaaca tattaggaac cagcctgtga tgctggatgt gaccgaggag ctgaggcccg 3420

atcacttggt gctggcctgc acccgcgctg agtttggctc tagcgatgaa gatacagatt 3480

gaggtactga aatgtgtggg cgtggcttaa gggtgggaaa gaatatataa ggtgggggtc 3540

ttatgtagtt ttgtatctgt tttgcagcag ccgccgccgc catgagcacc aactcgtttg 3600

atggaagcat tgtgagctca tatttgacaa cgcgcatgcc cccatgggcc ggggtgcgtc 3660

agaatgtgat gggctccagc attgatggtc gccccgtcct gcccgcaaac tctactacct 3720

tgacctacga gaccgtgtct ggaacgccgt tggagactgc agcctccgcc gccgcttcag 3780

ccgctgcagc caccgcccgc gggattgtga ctgactttgc tttcctgagc ccgcttgcaa 3840

gcagtgcagc ttcccgttca tccgcccgcg atgacaagtt gacggctctt ttggcacaat 3900

tggattcttt gacccgggaa cttaatgtcg tttctcagca gctgttggat ctgcgccagc 3960

aggtttctgc cctgaaggct tcctcccctc ccaatgcggt ttaaaacata aataaaaaac 4020

cagactctgt ttggatttgg atcaagcaag tgtcttgctg tctttattta ggggttttgc 4080

gcgcgcggta ggcccgggac cagcggtctc ggtcgttgag ggtcctgtgt attttttcca 4140

ggacgtggta aaggtgactc tggatgttca gatacatggg cataagcccg tctctggggt 4200

ggaggtagca ccactgcaga gcttcatgct gcggggtggt gttgtagatg atccagtcgt 4260

agcaggagcg ctgggcgtgg tgcctaaaaa tgtctttcag tagcaagctg attgccaggg 4320

gcaggccctt ggtgtaagtg tttacaaagc ggttaagctg ggatgggtgc atacgtgggg 4380

atatgagatg catcttggac tgtattttta ggttggctat gttcccagcc atatccctcc 4440

ggggattcat gttgtgcaga accaccagca cagtgtatcc ggtgcacttg ggaaatttgt 4500

catgtagctt agaaggaaat gcgtggaaga acttggagac gcccttgtga cctccaagat 4560

tttccatgca ttcgtccata atgatggcaa tgggcccacg ggcggcggcc tgggcgaaga 4620

tatttctggg atcactaacg tcatagttgt gttccaggat gagatcgtca taggccattt 4680

ttacaaagcg cgggcggagg gtgccagact gcggtataat ggttccatcc ggcccagggg 4740

cgtagttacc ctcacagatt tgcatttccc acgctttgag ttcagatggg gggatcatgt 4800

ctacctgcgg ggcgatgaag aaaacggttt ccggggtagg ggagatcagc tgggaagaaa 4860

gcaggttcct gagcagctgc gacttaccgc agccggtggg cccgtaaatc acacctatta 4920

ccgggtgcaa ctggtagtta agagagctgc agctgccgtc atccctgagc aggggggcca 4980

cttcgttaag catgtccctg actcgcatgt tttccctgac caaatccgcc agaaggcgct 5040

cgccgcccag cgatagcagt tcttgcaagg aagcaaagtt tttcaacggt ttgagaccgt 5100

ccgccgtagg catgcttttg agcgtttgac caagcagttc caggcggtcc cacagctcgg 5160

tcacctgctc tacggcatct cgatccagca tatctcctcg tttcgcgggt tggggcggct 5220

ttcgctgtac ggcagtagtc ggtgctcgtc cagacgggcc agggtcatgt ctttccacgg 5280

gcgcagggtc ctcgtcagcg tagtctgggt cacggtgaag gggtgcgctc cgggctgcgc 5340

gctggccagg gtgcgcttga ggctggtcct gctggtgctg aagcgctgcc ggtcttcgcc 5400

ctgcgcgtcg gccaggtagc atttgaccat ggtgtcatag tccagcccct ccgcggcgtg 5460

gcccttggcg cgcagcttgc ccttggagga ggcgccgcac gaggggcagt gcagactttt 5520

gagggcgtag agcttgggcg cgagaaatac cgattccggg gagtaggcat ccgcgccgca 5580

ggccccgcag acggtctcgc attccacgag ccaggtgagc tctggccgtt cggggtcaaa 5640

aaccaggttt cccccatgct ttttgatgcg tttcttacct ctggtttcca tgagccggtg 5700

tccacgctcg gtgacgaaaa ggctgtccgt gtccccgtat acagacttga gaggcctgtc 5760

ctcgagcggt gttccgcggt cctcctcgta tagaaactcg gaccactctg agacaaaggc 5820

tcgcgtccag gccagcacga aggaggctaa gtgggagggg tagcggtcgt tgtccactag 5880

ggggtccact cgctccaggg tgtgaagaca catgtcgccc tcttcggcat caaggaaggt 5940

gattggtttg taggtgtagg ccacgtgacc gggtgttcct gaaggggggc tataaaaggg 6000

ggtgggggcg cgttcgtcct cactctcttc cgcatcgctg tctgcgaggg ccagctgttg 6060

gggtgagtac tccctctgaa aagcgggcat gacttctgcg ctaagattgt cagtttccaa 6120

aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg tggccgcatc 6180

catctggtca gaaaagacaa tctttttgtt gtcaagcttg gtggcaaacg acccgtagag 6240

ggcgttggac agcaacttgg cgatggagcg cagggtttgg tttttgtcgc gatcggcgcg 6300

ctccttggcc gcgatgttta gctgcacgta ttcgcgcgca acgcaccgcc attcgggaaa 6360

gacggtggtg cgctcgtcgg gcaccaggtg cacgcgccaa ccgcggttgt gcagggtgac 6420

aaggtcaacg ctggtggcta cctctccgcg taggcgctcg ttggtccagc agaggcggcc 6480

gcccttgcgc gagcagaatg gcggtagggg gtctagctgc gtctcgtccg gggggtctgc 6540

gtccacggta aagaccccgg gcagcaggcg cgcgtcgaag tagtctatct tgcatccttg 6600

caagtctagc gcctgctgcc atgcgcgggc ggcaagcgcg cgctcgtatg ggttgagtgg 6660

gggaccccat ggcatggggt gggtgagcgc ggaggcgtac atgccgcaaa tgtcgtaaac 6720

gtagaggggc tctctgagta ttccaagata tgtagggtag catcttccac cgcggatgct 6780

ggcgcgcacg taatcgtata gttcgtgcga gggagcgagg aggtcgggac cgaggttgct 6840

acgggcgggc tgctctgctc ggaagactat ctgcctgaag atggcatgtg agttggatga 6900

tatggttgga cgctggaaga cgttgaagct ggcgtctgtg agacctaccg cgtcacgcac 6960

gaaggaggcg taggagtcgc gcagcttgtt gaccagctcg gcggtgacct gcacgtctag 7020

ggcgcagtag tccagggttt ccttgatgat gtcatactta tcctgtccct tttttttcca 7080

cagctcgcgg ttgaggacaa actcttcgcg gtctttccag tactcttgga tcggaaaccc 7140

gtcggcctcc gaacggtaag agcctagcat gtagaactgg ttgacggcct ggtaggcgca 7200

gcatcccttt tctacgggta gcgcgtatgc ctgcgcggcc ttccggagcg aggtgtgggt 7260

gagcgcaaag gtgtccctga ccatgacttt gaggtactgg tatttgaagt cagtgtcgtc 7320

gcatccgccc tgctcccaga gcaaaaagtc cgtgcgcttt ttggaacgcg gatttggcag 7380

ggcgaaggtg acatcgttga agagtatctt tcccgcgcga ggcataaagt tgcgtgtgat 7440

gcggaagggt cccggcacct cggaacggtt gttaattacc tgggcggcga gcacgatctc 7500

gtcaaagccg ttgatgttgt ggcccacaat gtaaagttcc aagaagcgcg ggatgccctt 7560

gatggaaggc aattttttaa gttcctcgta ggtgagctct tcaggggagc tgagcccgtg 7620

ctctgaaagg gcccagtctg caagatgagg gttggaagcg acgaatgagc tccacaggtc 7680

acgggccatt agcatttgca ggtggtcgcg aaaggtccta aactggcgac ctatggccat 7740

tttttctggg gtgatgcagt agaaggtaag cgggtcttgt tcccagcggt cccatccaag 7800

gttcgcggct aggtctcgcg cggcagtcac tagaggctca tctccgccga acttcatgac 7860

cagcatgaag ggcacgagct gcttcccaaa ggcccccatc caagtatagg tctctacatc 7920

gtaggtgaca aagagacgct cggtgcgagg atgcgagccg atcgggaaga actggatctc 7980

ccgccaccaa ttggaggagt ggctattgat gtggtgaaag tagaagtccc tgcgacgggc 8040

cgaacactcg tgctggcttt tgtaaaaacg tgcgcagtac tggcagcggt gcacgggctg 8100

tacatcctgc acgaggttga cctgacgacc gcgcacaagg aagcagagtg ggaatttgag 8160

cccctcgcct ggcgggtttg gctggtggtc ttctacttcg gctgcttgtc cttgaccgtc 8220

tggctgctcg aggggagtta cggtggatcg gaccaccacg ccgcgcgagc ccaaagtcca 8280

gatgtccgcg cgcggcggtc ggagcttgat gacaacatcg cgcagatggg agctgtccat 8340

ggtctggagc tcccgcggcg tcaggtcagg cgggagctcc tgcaggttta cctcgcatag 8400

acgggtcagg gcgcgggcta gatccaggtg atacctaatt tccaggggct ggttggtggc 8460

ggcgtcgatg gcttgcaaga ggccgcatcc ccgcggcgcg actacggtac cgcgcggcgg 8520

gcggtgggcc gcgggggtgt ccttggatga tgcatctaaa agcggtgacg cgggcgagcc 8580

cccggaggta gggggggctc cggacccgcc gggagagggg gcaggggcac gtcggcgccg 8640

cgcgcgggca ggagctggtg ctgcgcgcgt aggttgctgg cgaacgcgac gacgcggcgg 8700

ttgatctcct gaatctggcg cctctgcgtg aagacgacgg gcccggtgag cttgagcctg 8760

aaagagagtt cgacagaatc aatttcggtg tcgttgacgg cggcctggcg caaaatctcc 8820

tgcacgtctc ctgagttgtc ttgataggcg atctcggcca tgaactgctc gatctcttcc 8880

tcctggagat ctccgcgtcc ggctcgctcc acggtggcgg cgaggtcgtt ggaaatgcgg 8940

gccatgagct gcgagaaggc gttgaggcct ccctcgttcc agacgcggct gtagaccacg 9000

cccccttcgg catcgcgggc gcgcatgacc acctgcgcga gattgagctc cacgtgccgg 9060

gcgaagacgg cgtagtttcg caggcgctga aagaggtagt tgagggtggt ggcggtgtgt 9120

tctgccacga agaagtacat aacccagcgt cgcaacgtgg attcgttgat atcccccaag 9180

gcctcaaggc gctccatggc ctcgtagaag tccacggcga agttgaaaaa ctgggagttg 9240

cgcgccgaca cggttaactc ctcctccaga agacggatga gctcggcgac agtgtcgcgc 9300

acctcgcgct caaaggctac aggggcctct tcttcttctt caatctcctc ttccataagg 9360

gcctcccctt cttcttcttc tggcggcggt gggggagggg ggacacggcg gcgacgacgg 9420

cgcaccggga ggcggtcgac aaagcgctcg atcatctccc cgcggcgacg gcgcatggtc 9480

tcggtgacgg cgcggccgtt ctcgcggggg cgcagttgga agacgccgcc cgtcatgtcc 9540

cggttatggg ttggcggggg gctgccatgc ggcagggata cggcgctaac gatgcatctc 9600

aacaattgtt gtgtaggtac tccgccgccg agggacctga gcgagtccgc atcgaccgga 9660

tcggaaaacc tctcgagaaa ggcgtctaac cagtcacagt cgcaaggtag gctgagcacc 9720

gtggcgggcg gcagcgggcg gcggtcgggg ttgtttctgg cggaggtgct gctgatgatg 9780

taattaaagt aggcggtctt gagacggcgg atggtcgaca gaagcaccat gtccttgggt 9840

ccggcctgct gaatgcgcag gcggtcggcc atgccccagg cttcgttttg acatcggcgc 9900

aggtctttgt agtagtcttg catgagcctt tctaccggca cttcttcttc tccttcctct 9960

tgtcctgcat ctcttgcatc tatcgctgcg gcggcggcgg agtttggccg taggtggcgc 10020

cctcttcctc ccatgcgtgt gaccccgaag cccctcatcg gctgaagcag ggctaggtcg 10080

gcgacaacgc gctcggctaa tatggcctgc tgcacctgcg tgagggtaga ctggaagtca 10140

tccatgtcca caaagcggtg gtatgcgccc gtgttgatgg tgtaagtgca gttggccata 10200

acggaccagt taacggtctg gtgacccggc tgcgagagct cggtgtacct gagacgcgag 10260

taagccctcg agtcaaatac gtagtcgttg caagtccgca ccaggtactg gtatcccacc 10320

aaaaagtgcg gcggcggctg gcggtagagg ggccagcgta gggtggccgg ggctccgggg 10380

gcgagatctt ccaacataag gcgatgatat ccgtagatgt acctggacat ccaggtgatg 10440

ccggcggcgg tggtggaggc gcgcggaaag tcgcggacgc ggttccagat gttgcgcagc 10500

ggcaaaaagt gctccatggt cgggacgctc tggccggtca ggcgcgcgca atcgttgacg 10560

ctctagaccg tgcaaaagga gagcctgtaa gcgggcactc ttccgtggtc tggtggataa 10620

attcgcaagg gtatcatggc ggacgaccgg ggttcgagcc ccgtatccgg ccgtccgccg 10680

tgatccatgc ggttaccgcc cgcgtgtcga acccaggtgt gcgacgtcag acaacggggg 10740

agtgctcctt ttggcttcct tccaggcgcg gcggctgctg cgctagcttt tttggccact 10800

ggccgcgcgc agcgtaagcg gttaggctgg aaagcgaaag cattaagtgg ctcgctccct 10860

gtagccggag ggttattttc caagggttga gtcgcgggac ccccggttcg agtctcggac 10920

cggccggact gcggcgaacg ggggtttgcc tccccgtcat gcaagacccc gcttgcaaat 10980

tcctccggaa acagggacga gccccttttt tgcttttccc agatgcatcc ggtgctgcgg 11040

cagatgcgcc cccctcctca gcagcggcaa gagcaagagc agcggcagac atgcagggca 11100

ccctcccctc ctcctaccgc gtcaggaggg gcgacatccg cggttgacgc ggcagcagat 11160

ggtgattacg aacccccgcg gcgccgggcc cggcactacc tggacttgga ggagggcgag 11220

ggcctggcgc ggctaggagc gccctctcct gagcggtacc caagggtgca gctgaagcgt 11280

gatacgcgtg aggcgtacgt gccgcggcag aacctgtttc gcgaccgcga gggagaggag 11340

cccgaggaga tgcgggatcg aaagttccac gcagggcgcg agctgcggca tggcctgaat 11400

cgcgagcggt tgctgcgcga ggaggacttt gagcccgacg cgcgaaccgg gattagtccc 11460

gcgcgcgcac acgtggcggc cgccgacctg gtaaccgcat acgagcagac ggtgaaccag 11520

gagattaact ttcaaaaaag ctttaacaac cacgtgcgta cgcttgtggc gcgcgaggag 11580

gtggctatag gactgatgca tctgtgggac tttgtaagcg cgctggagca aaacccaaat 11640

agcaagccgc tcatggcgca gctgttcctt atagtgcagc acagcaggga caacgaggca 11700

ttcagggatg cgctgctaaa catagtagag cccgagggcc gctggctgct cgatttgata 11760

aacatcctgc agagcatagt ggtgcaggag cgcagcttga gcctggctga caaggtggcc 11820

gccatcaact attccatgct tagcctgggc aagttttacg cccgcaagat ataccatacc 11880

ccttacgttc ccatagacaa ggaggtaaag atcgaggggt tctacatgcg catggcgctg 11940

aaggtgctta ccttgagcga cgacctgggc gtttatcgca acgagcgcat ccacaaggcc 12000

gtgagcgtga gccggcggcg cgagctcagc gaccgcgagc tgatgcacag cctgcaaagg 12060

gccctggctg gcacgggcag cggcgataga gaggccgagt cctactttga cgcgggcgct 12120

gacctgcgct gggccccaag ccgacgcgcc ctggaggcag ctggggccgg acctgggctg 12180

gcggtggcac ccgcgcgcgc tggcaacgtc ggcggcgtgg aggaatatga cgaggacgat 12240

gagtacgagc cagaggacgg cgagtactaa gcggtgatgt ttctgatcag atgatgcaag 12300

acgcaacgga cccggcggtg cgggcggcgc tgcagagcca gccgtccggc cttaactcca 12360

cggacgactg gcgccaggtc atggaccgca tcatgtcgct gactgcgcgc aatcctgacg 12420

cgttccggca gcagccgcag gccaaccggc tctccgcaat tctggaagcg gtggtcccgg 12480

cgcgcgcaaa ccccacgcac gagaaggtgc tggcgatcgt aaacgcgctg gccgaaaaca 12540

gggccatccg gcccgacgag gccggcctgg tctacgacgc gctgcttcag cgcgtggctc 12600

gttacaacag cggcaacgtg cagaccaacc tggaccggct ggtgggggat gtgcgcgagg 12660

ccgtggcgca gcgtgagcgc gcgcagcagc agggcaacct gggctccatg gttgcactaa 12720

acgccttcct gagtacacag cccgccaacg tgccgcgggg acaggaggac tacaccaact 12780

ttgtgagcgc actgcggcta atggtgactg agacaccgca aagtgaggtg taccagtctg 12840

ggccagacta ttttttccag accagtagac aaggcctgca gaccgtaaac ctgagccagg 12900

ctttcaaaaa cttgcagggg ctgtgggggg tgcgggctcc cacaggcgac cgcgcgaccg 12960

tgtctagctt gctgacgccc aactcgcgcc tgttgctgct gctaatagcg cccttcacgg 13020

acagtggcag cgtgtcccgg gacacatacc taggtcactt gctgacactg taccgcgagg 13080

ccataggtca ggcgcatgtg gacgagcata ctttccagga gattacaagt gtcagccgcg 13140

cgctggggca ggaggacacg ggcagcctgg aggcaaccct aaactacctg ctgaccaacc 13200

ggcggcagaa gatcccctcg ttgcacagtt taaacagcga ggaggagcgc attttgcgct 13260

acgtgcagca gagcgtgagc cttaacctga tgcgcgacgg ggtaacgccc agcgtggcgc 13320

tggacatgac cgcgcgcaac atggaaccgg gcatgtatgc ctcaaaccgg ccgtttatca 13380

accgcctaat ggactacttg catcgcgcgg ccgccgtgaa ccccgagtat ttcaccaatg 13440

ccatcttgaa cccgcactgg ctaccgcccc ctggtttcta caccggggga ttcgaggtgc 13500

ccgagggtaa cgatggattc ctctgggacg acatagacga cagcgtgttt tccccgcaac 13560

cgcagaccct gctagagttg caacagcgcg agcaggcaga ggcggcgctg cgaaaggaaa 13620

gcttccgcag gccaagcagc ttgtccgatc taggcgctgc ggccccgcgg tcagatgcta 13680

gtagcccatt tccaagcttg atagggtctc ttaccagcac tcgcaccacc cgcccgcgcc 13740

tgctgggcga ggaggagtac ctaaacaact cgctgctgca gccgcagcgc gaaaaaaacc 13800

tgcctccggc atttcccaac aacgggatag agagcctagt ggacaagatg agtagatgga 13860

agacgtacgc gcaggagcac agggacgtgc caggcccgcg cccgcccacc cgtcgtcaaa 13920

ggcacgaccg tcagcggggt ctggtgtggg aggacgatga ctcggcagac gacagcagcg 13980

tcctggattt gggagggagt ggcaacccgt ttgcgcacct tcgccccagg ctggggagaa 14040

tgttttaaaa aaaaaaaagc atgatgcaaa ataaaaaact caccaaggcc atggcaccga 14100

gcgttggttt tcttgtattc cccttagtat gcggcgcgcg gcgatgtatg aggaaggtcc 14160

tcctccctcc tacgagagtg tggtgagcgc ggcgccagtg gcggcggcgc tgggttctcc 14220

cttcgatgct cccctggacc cgccgtttgt gcctccgcgg tacctgcggc ctaccggggg 14280

gagaaacagc atccgttact ctgagttggc acccctattc gacaccaccc gtgtgtacct 14340

ggtggacaac aagtcaacgg atgtggcatc cctgaactac cagaacgacc acagcaactt 14400

tctgaccacg gtcattcaaa acaatgacta cagcccgggg gaggcaagca cacagaccat 14460

caatcttgac gaccggtcgc actggggcgg cgacctgaaa accatcctgc ataccaacat 14520

gccaaatgtg aacgagttca tgtttaccaa taagtttaag gcgcgggtga tggtgtcgcg 14580

cttgcctact aaggacaatc aggtggagct gaaatacgag tgggtggagt tcacgctgcc 14640

cgagggcaac tactccgaga ccatgaccat agaccttatg aacaacgcga tcgtggagca 14700

ctacttgaaa gtgggcagac agaacggggt tctggaaagc gacatcgggg taaagtttga 14760

cacccgcaac ttcagactgg ggtttgaccc cgtcactggt cttgtcatgc ctggggtata 14820

tacaaacgaa gccttccatc cagacatcat tttgctgcca ggatgcgggg tggacttcac 14880

ccacagccgc ctgagcaact tgttgggcat ccgcaagcgg caacccttcc aggagggctt 14940

taggatcacc tacgatgatc tggagggtgg taacattccc gcactgttgg atgtggacgc 15000

ctaccaggcg agcttgaaag atgacaccga acagggcggg ggtggcgcag gcggcagcaa 15060

cagcagtggc agcggcgcgg aagagaactc caacgcggca gccgcggcaa tgcagccggt 15120

ggaggacatg aacgatcatg ccattcgcgg cgacaccttt gccacacggg ctgaggagaa 15180

gcgcgctgag gccgaagcag cggccgaagc tgccgccccc gctgcgcaac ccgaggtcga 15240

gaagcctcag aagaaaccgg tgatcaaacc cctgacagag gacagcaaga aacgcagtta 15300

caacctaata agcaatgaca gcaccttcac ccagtaccgc agctggtacc ttgcatacaa 15360

ctacggcgac cctcagaccg gaatccgctc atggaccctg ctttgcactc ctgacgtaac 15420

ctgcggctcg gagcaggtct actggtcgtt gccagacatg atgcaagacc ccgtgacctt 15480

ccgctccacg cgccagatca gcaactttcc ggtggtgggc gccgagctgt tgcccgtgca 15540

ctccaagagc ttctacaacg accaggccgt ctactcccaa ctcatccgcc agtttacctc 15600

tctgacccac gtgttcaatc gctttcccga gaaccagatt ttggcgcgcc cgccagcccc 15660

caccatcacc accgtcagtg aaaacgttcc tgctctcaca gatcacggga cgctaccgct 15720

gcgcaacagc atcggaggag tccagcgagt gaccattact gacgccagac gccgcacctg 15780

cccctacgtt tacaaggccc tgggcatagt ctcgccgcgc gtcctatcga gccgcacttt 15840

ttgagcaagc atgtccatcc ttatatcgcc cagcaataac acaggctggg gcctgcgctt 15900

cccaagcaag atgtttggcg gggccaagaa gcgctccgac caacacccag tgcgcgtgcg 15960

cgggcactac cgcgcgccct ggggcgcgca caaacgcggc cgcactgggc gcaccaccgt 16020

cgatgacgcc atcgacgcgg tggtggagga ggcgcgcaac tacacgccca cgccgccacc 16080

agtgtccaca gtggacgcgg ccattcagac cgtggtgcgc ggagcccggc gctatgctaa 16140

aatgaagaga cggcggaggc gcgtagcacg tcgccaccgc cgccgacccg gcactgccgc 16200

ccaacgcgcg gcggcggccc tgcttaaccg cgcacgtcgc accggccgac gggcggccat 16260

gcgggccgct cgaaggctgg ccgcgggtat tgtcactgtg ccccccaggt ccaggcgacg 16320

agcggccgcc gcagcagccg cggccattag tgctatgact cagggtcgca ggggcaacgt 16380

gtattgggtg cgcgactcgg ttagcggcct gcgcgtgccc gtgcgcaccc gccccccgcg 16440

caactagatt gcaagaaaaa actacttaga ctcgtactgt tgtatgtatc cagcggcggc 16500

ggcgcgcaac gaagctatgt ccaagcgcaa aatcaaagaa gagatgctcc aggtcatcgc 16560

gccggagatc tatggccccc cgaagaagga agagcaggat tacaagcccc gaaagctaaa 16620

gcgggtcaaa aagaaaaaga aagatgatga tgatgaactt gacgacgagg tggaactgct 16680

gcacgctacc gcgcccaggc gacgggtaca gtggaaaggt cgacgcgtaa aacgtgtttt 16740

gcgacccggc accaccgtag tctttacgcc cggtgagcgc tccacccgca cctacaagcg 16800

cgtgtatgat gaggtgtacg gcgacgagga cctgcttgag caggccaacg agcgcctcgg 16860

ggagtttgcc tacggaaagc ggcataagga catgctggcg ttgccgctgg acgagggcaa 16920

cccaacacct agcctaaagc ccgtaacact gcagcaggtg ctgcccgcgc ttgcaccgtc 16980

cgaagaaaag cgcggcctaa agcgcgagtc tggtgacttg gcacccaccg tgcagctgat 17040

ggtacccaag cgccagcgac tggaagatgt cttggaaaaa atgaccgtgg aacctgggct 17100

ggagcccgag gtccgcgtgc ggccaatcaa gcaggtggcg ccgggactgg gcgtgcagac 17160

cgtggacgtt cagataccca ctaccagtag caccagtatt gccaccgcca cagagggcat 17220

ggagacacaa acgtccccgg ttgcctcagc ggtggcggat gccgcggtgc aggcggtcgc 17280

tgcggccgcg tccaagacct ctacggaggt gcaaacggac ccgtggatgt ttcgcgtttc 17340

agccccccgg cgcccgcgcg gttcgaggaa gtacggcgcc gccagcgcgc tactgcccga 17400

atatgcccta catccttcca ttgcgcctac ccccggctat cgtggctaca cctaccgccc 17460

cagaagacga gcaactaccc gacgccgaac caccactgga acccgccgcc gccgtcgccg 17520

tcgccagccc gtgctggccc cgatttccgt gcgcagggtg gctcgcgaag gaggcaggac 17580

cctggtgctg ccaacagcgc gctaccaccc cagcatcgtt taaaagccgg tctttgtggt 17640

tcttgcagat atggccctca cctgccgcct ccgtttcccg gtgccgggat tccgaggaag 17700

aatgcaccgt aggaggggca tggccggcca cggcctgacg ggcggcatgc gtcgtgcgca 17760

ccaccggcgg cggcgcgcgt cgcaccgtcg catgcgcggc ggtatcctgc ccctccttat 17820

tccactgatc gccgcggcga ttggcgccgt gcccggaatt gcatccgtgg ccttgcaggc 17880

gcagagacac tgattaaaaa caagttgcat gtggaaaaat caaaataaaa agtctggact 17940

ctcacgctcg cttggtcctg taactatttt gtagaatgga agacatcaac tttgcgtctc 18000

tggccccgcg acacggctcg cgcccgttca tgggaaactg gcaagatatc ggcaccagca 18060

atatgagcgg tggcgccttc agctggggct cgctgtggag cggcattaaa aatttcggtt 18120

ccaccgttaa gaactatggc agcaaggcct ggaacagcag cacaggccag atgctgaggg 18180

ataagttgaa agagcaaaat ttccaacaaa aggtggtaga tggcctggcc tctggcatta 18240

gcggggtggt ggacctggcc aaccaggcag tgcaaaataa gattaacagt aagcttgatc 18300

cccgccctcc cgtagaggag cctccaccgg ccgtggagac agtgtctcca gaggggcgtg 18360

gcgaaaagcg tccgcgcccc gacagggaag aaactctggt gacgcaaata gacgagcctc 18420

cctcgtacga ggaggcacta aagcaaggcc tgcccaccac ccgtcccatc gcgcccatgg 18480

ctaccggagt gctgggccag cacacacccg taacgctgga cctgcctccc cccgccgaca 18540

cccagcagaa acctgtgctg ccaggcccga ccgccgttgt tgtaacccgt cctagccgcg 18600

cgtccctgcg ccgcgccgcc agcggtccgc gatcgttgcg gcccgtagcc agtggcaact 18660

ggcaaagcac actgaacagc atcgtgggtc tgggggtgca atccctgaag cgccgacgat 18720

gcttctgaat agctaacgtg tcgtatgtgt gtcatgtatg cgtccatgtc gccgccagag 18780

gagctgctga gccgccgcgc gcccgctttc caagatggct accccttcga tgatgccgca 18840

gtggtcttac atgcacatct cgggccagga cgcctcggag tacctgagcc ccgggctggt 18900

gcagtttgcc cgcgccaccg agacgtactt cagcctgaat aacaagttta gaaaccccac 18960

ggtggcgcct acgcacgacg tgaccacaga ccggtcccag cgtttgacgc tgcggttcat 19020

ccctgtggac cgtgaggata ctgcgtactc gtacaaggcg cggttcaccc tagctgtggg 19080

tgataaccgt gtgctggaca tggcttccac gtactttgac atccgcggcg tgctggacag 19140

gggccctact tttaagccct actctggcac tgcctacaac gccctggctc ccaagggtgc 19200

cccaaatcct tgcgaatggg atgaagctgc tactgctctt gaaataaacc tagaagaaga 19260

ggacgatgac aacgaagacg aagtagacga gcaagctgag cagcaaaaaa ctcacgtatt 19320

tgggcaggcg ccttattctg gtataaatat tacaaaggag ggtattcaaa taggtgtcgg 19380

aggaggagga tcaaacgtgg tgcgtcaagg tggtggtggt tctcctaaat atgccgataa 19440

aacatttcaa cctgaacctc aaataggaga atctcagtgg tacgaaactg aaattaatca 19500

tgcagctggg agagtcctta aaaagactac cccaatgaaa ccatgttacg gttcatatgc 19560

aaaacccaca aatgaaaatg gagggcaagg cattcttgta aagcaacaaa atggaaagct 19620

agaaagtcaa gtggaaatgc aatttttctc aactactgag gcgaccgcag gcaatggtga 19680

taacttgact cctaaagtgg tattgtacag tgaagatgta gatatagaaa ccccagacac 19740

tcatatttct tacatgccca ctattaagga aggtaactca cgagaactaa tgggccaaca 19800

atctatgccc aacaggccta attacattgc ttttagggac aattttattg gtctaatgta 19860

ttacaacagc acgggtaata tgggtgttct ggcgggccaa gcatcgcagt tgaatgctgt 19920

tgtagatttg caagacagaa acacagagct ttcataccag cttttgcttg attccattgg 19980

tgatagaacc aggtactttt ctatgtggaa tcaggctgtt gacagctatg atccagatgt 20040

tagaattatt gaaaatcatg gaactgaaga tgaacttcca aattactgct ttccactggg 20100

aggtgtgatt aatacagaga ctcttaccaa ggtaaaacct aaaacaggtc aggaaaatgg 20160

atgggaaaaa gatgctacag aattttcaga taaaaatgaa ataagagttg gaaataattt 20220

tgccatggaa atcaatctaa atgccaacct gtggagaaat ttcctgtact ccaacatagc 20280

gctgtatttg cccgacaagc taaagtacag tccttccaac gtaaaaattt ctgataaccc 20340

aaacacctac gactacatga acaagcgagt ggtggctccc gggttagtgg actgctacat 20400

taaccttgga gcacgctggt cccttgacta tatggacaac gtcaacccat ttaaccacca 20460

ccgcaatgct ggcctgcgct accgctcaat gttgctgggc aatggtcgct atgtgccctt 20520

ccacatccag gtgcctcaga agttctttgc cattaaaaac ctccttctcc tgccgggctc 20580

atacacctac gagtggaact tcaggaagga tgttaacatg gttctgcaga gctccctagg 20640

aaatgaccta agggttgacg gagccagcat taagtttgat agcatttgcc tttacgccac 20700

cttcttcccc atggcccaca acaccgcctc cacgcttgag gccatgctta gaaacgacac 20760

caacgaccag tcctttaacg actatctctc cgccgccaac atgctctacc ctatacccgc 20820

caacgctacc aacgtgccca tatccatccc ctcccgcaac tgggcggctt tccgcggctg 20880

ggccttcacg cgccttaaga ctaaggaaac cccatcactg ggctcgggct acgaccctta 20940

ttacacctac tctggctcta taccctacct agatggaacc ttttacctca accacacctt 21000

taagaaggtg gccattacct ttgactcttc tgtcagctgg cctggcaatg accgcctgct 21060

tacccccaac gagtttgaaa ttaagcgctc agttgacggg gagggttaca acgttgccca 21120

gtgtaacatg accaaagact ggttcctggt acaaatgcta gctaactaca acattggcta 21180

ccagggcttc tatatcccag agagctacaa ggaccgcatg tactccttct ttagaaactt 21240

ccagcccatg agccgtcagg tggtggatga tactaaatac aaggactacc aacaggtggg 21300

catcctacac caacacaaca actctggatt tgttggctac cttgccccca ccatgcgcga 21360

aggacaggcc taccctgcta acttccccta tccgcttata ggcaagaccg cagttgacag 21420

cattacccag aaaaagtttc tttgcgatcg caccctttgg cgcatcccat tctccagtaa 21480

ctttatgtcc atgggcgcac tcacagacct gggccaaaac cttctctacg ccaactccgc 21540

ccacgcgcta gacatgactt ttgaggtgga tcccatggac gagcccaccc ttctttatgt 21600

tttgtttgaa gtctttgacg tggtccgtgt gcaccggccg caccgcggcg tcatcgaaac 21660

cgtgtacctg cgcacgccct tctcggccgg caacgccaca acataaagaa gcaagcaaca 21720

tcaacaacag ctgccgccat gggctccagt gagcaggaac tgaaagccat tgtcaaagat 21780

cttggttgtg ggccatattt tttgggcacc tatgacaagc gctttccagg ctttgtttct 21840

ccacacaagc tcgcctgcgc catagtcaat acggccggtc gcgagactgg gggcgtacac 21900

tggatggcct ttgcctggaa cccgcactca aaaacatgct acctctttga gccctttggc 21960

ttttctgacc agcgactcaa gcaggtttac cagtttgagt acgagtcact cctgcgccgt 22020

agcgccattg cttcttcccc cgaccgctgt ataacgctgg aaaagtccac ccaaagcgta 22080

caggggccca actcggccgc ctgtggacta ttctgctgca tgtttctcca cgcctttgcc 22140

aactggcccc aaactcccat ggatcacaac cccaccatga accttattac cggggtaccc 22200

aactccatgc tcaacagtcc ccaggtacag cccaccctgc gtcgcaacca ggaacagctc 22260

tacagcttcc tggagcgcca ctcgccctac ttccgcagcc acagtgcgca gattaggagc 22320

gccacttctt tttgtcactt gaaaaacatg taaaaataat gtactagaga cactttcaat 22380

aaaggcaaat gcttttattt gtacactctc gggtgattat ttacccccac ccttgccgtc 22440

tgcgccgttt aaaaatcaaa ggggttctgc cgcgcatcgc tatgcgccac tggcagggac 22500

acgttgcgat actggtgttt agtgctccac ttaaactcag gcacaaccat ccgcggcagc 22560

tcggtgaagt tttcactcca caggctgcgc accatcacca acgcgtttag caggtcgggc 22620

gccgatatct tgaagtcgca gttggggcct ccgccctgcg cgcgcgagtt gcgatacaca 22680

gggttgcagc actggaacac tatcagcgcc gggtggtgca cgctggccag cacgctcttg 22740

tcggagatca gatccgcgtc caggtcctcc gcgttgctca gggcgaacgg agtcaacttt 22800

ggtagctgcc ttcccaaaaa gggcgcgtgc ccaggctttg agttgcactc gcaccgtagt 22860

ggcatcaaaa ggtgaccgtg cccggtctgg gcgttaggat acagcgcctg cataaaagcc 22920

ttgatctgct taaaagccac ctgagccttt gcgccttcag agaagaacat gccgcaagac 22980

ttgccggaaa actgattggc cggacaggcc gcgtcgtgca cgcagcacct tgcgtcggtg 23040

ttggagatct gcaccacatt tcggccccac cggttcttca cgatcttggc cttgctagac 23100

tgctccttca gcgcgcgctg cccgttttcg ctcgtcacat ccatttcaat cacgtgctcc 23160

ttatttatca taatgcttcc gtgtagacac ttaagctcgc cttcgatctc agcgcagcgg 23220

tgcagccaca acgcgcagcc cgtgggctcg tgatgcttgt aggtcacctc tgcaaacgac 23280

tgcaggtacg cctgcaggaa tcgccccatc atcgtcacaa aggtcttgtt gctggtgaag 23340

gtcagctgca acccgcggtg ctcctcgttc agccaggtct tgcatacggc cgccagagct 23400

tccacttggt caggcagtag tttgaagttc gcctttagat cgttatccac gtggtacttg 23460

tccatcagcg cgcgcgcagc ctccatgccc ttctcccacg cagacacgat cggcacactc 23520

agcgggttca tcaccgtaat ttcactttcc gcttcgctgg gctcttcctc ttcctcttgc 23580

gtccgcatac cacgcgccac tgggtcgtct tcattcagcc gccgcactgt gcgcttacct 23640

cctttgccat gcttgattag caccggtggg ttgctgaaac ccaccatttg tagcgccaca 23700

tcttctcttt cttcctcgct gtccacgatt acctctggtg atggcgggcg ctcgggcttg 23760

ggagaagggc gcttcttttt cttcttgggc gcaatggcca aatccgccgc cgaggtcgat 23820

ggccgcgggc tgggtgtgcg cggcaccagc gcgtcttgtg atgagtcttc ctcgtcctcg 23880

gactcgatac gccgcctcat ccgctttttt gggggcgccc ggggaggcgg cggcgacggg 23940

gacggggacg acacgtcctc catggttggg ggacgtcgcg ccgcaccgcg tccgcgctcg 24000

ggggtggttt cgcgctgctc ctcttcccga ctggccattt ccttctccta taggcagaaa 24060

aagatcatgg agtcagtcgg aagaaggaca gcctaaccgc cccctctgag ttcgccacca 24120

ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg 24180

aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggacc 24240

gctcagtacc aacagaggat aaaaagcaag accaggacaa cgcagaggca aacgaggaac 24300

aagtcgggcg gggggacgaa aggcatggcg actacctaga tgtgggagac gacgtgctgt 24360

tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg 24420

tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctattc tcaccgcgcg 24480

taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc 24540

ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga 24600

tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg 24660

gcgctgtcat acctgatatc gcctcgctca acgaagtgcc aaaaatcttt gagggtcttg 24720

gacgcgacga gaagcgcgcg gcaaacgctc tgcaacagga aaacagcgaa aatgaaagtc 24780

actctggagt gttggtggaa ctcgagggtg acaacgcgcg cctagccgta ctaaaacgca 24840

gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gtcatgagca 24900

cagtcatgag tgagctgatc gtgcgccgtg cgcagcccct ggagagggat gcaaatttgc 24960

aagaacaaac agaggagggc ctacccgcag ttggcgacga gcagctagcg cgctggcttc 25020

aaacgcgcga gcctgccgac ttggaggagc gacgcaaact aatgatggcc gcagtgctcg 25080

ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc 25140

tagaggaaac attgcactac acctttcgac agggctacgt acgccaggcc tgcaagatct 25200

ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgccttg 25260

ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact 25320

gcgtttactt atttctatgc tacacctggc agacggccat gggcgtttgg cagcagtgct 25380

tggaggagtg caacctcaag gagctgcaga aactgctaaa gcaaaacttg aaggacctat 25440

ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacatcatt ttccccgaac 25500

gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaga 25560

actttaggaa ctttatccta gagcgctcag gaatcttgcc cgccacctgc tgtgcacttc 25620

ctagcgactt tgtgcccatt aagtaccgcg aatgccctcc gccgctttgg ggccactgct 25680

accttctgca gctagccaac taccttgcct accactctga cataatggaa gacgtgagcg 25740

gtgacggtct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg 25800

tttgcaattc gcagctgctt aacgaaagtc aaattatcgg tacctttgag ctgcagggtc 25860

cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt 25920

cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg 25980

aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca 26040

ttcttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac 26100

ggggggttta cttggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc 26160

agccctatca gcagcagccg cgggcccttg cttcccagga tggcacccaa aaagaagctg 26220

cagctgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt 26280

ttggacgagg aggaggagga catgatggaa gactgggaga gcctagacga ggaagcttcc 26340

gaggtcgaag aggtgtcaga cgaaacaccg tcaccctcgg tcgcattccc ctcgccggcg 26400

ccccagaaat cggcaaccgg ttccagcatg gctacaacct ccgctcctca ggcgccgccg 26460

gcactgcccg ttcgccgacc caaccgtaga tgggacacca ctggaaccag ggccggtaag 26520

tccaagcagc cgccgccgtt agcccaagag caacaacagc gccaaggcta ccgctcatgg 26580

cgcgggcaca agaacgccat agttgcttgc ttgcaagact gtgggggcaa catctccttc 26640

gcccgccgct ttcttctcta ccatcacggc gtggccttcc cccgtaacat cctgcattac 26700

taccgtcatc tctacagccc atactgcacc ggcggcagcg gcagcggcag caacagcagc 26760

ggccacacag aagcaaaggc gaccggatag caagactctg acaaagccca agaaatccac 26820

agcggcggca gcagcaggag gaggagcgct gcgtctggcg cccaacgaac ccgtatcgac 26880

ccgcgagctt agaaacagga tttttcccac tctgtatgct atatttcaac agagcagggg 26940

ccaagaacaa gagctgaaaa taaaaaacag gtctctgcga tccctcaccc gcagctgcct 27000

gtatcacaaa agcgaagatc agcttcggcg cacgctggaa gacgcggagg ctctcttcag 27060

taaatactgc gcgctgactc ttaaggacta gtttcgcgcc ctttctcaaa tttaagcgcg 27120

aaaactacgt catctccagc ggccacaccc ggcgccagca cctgtcgtca gcgccattat 27180

gagcaaggaa attcccacgc cctacatgtg gagttaccag ccacaaatgg gacttgcggc 27240

tggagctgcc caagactact caacccgaat aaactacatg agcgcgggac cccacatgat 27300

atcccgggtc aacggaatcc gcgcccaccg aaaccgaatt ctcttggaac aggcggctat 27360

taccaccaca cctcgtaata accttaatcc ccgtagttgg cccgctgccc tggtgtacca 27420

ggaaagtccc gctcccacca ctgtggtact tcccagagac gcccaggccg aagttcagat 27480

gactaactca ggggcgcagc ttgcgggcgg ctttcgtcac agggtgcggt cgcccgggca 27540

gggtataact cacctgacaa tcagagggcg aggtattcag ctcaacgacg agtcggtgag 27600

ctcctcgctt ggtctccgtc cggacgggac atttcagatc ggcggcgccg gccgtccttc 27660

attcacgcct cgtcaggcaa tcctaactct gcagacctcg tcctctgagc cgcgctctgg 27720

aggcattgga actctgcaat ttattgagga gtttgtgcca tcggtctact ttaacccctt 27780

ctcgggacct cccggccact atccggatca atttattcct aactttgacg cggtaaagga 27840

ctcggcggac ggctacgact gaatgttaag tggagaggca gagcaactgc gcctgaaaca 27900

cctggtccac tgtcgccgcc acaagtgctt tgcccgcgac tccggtgagt tttgctactt 27960

tgaattgccc gaggatcata tcgagggccc ggcgcacggc gtccggctta ccgcccaggg 28020

agagcttgcc cgtagcctga ttcgggagtt tacccagcgc cccctgctag ttgagcggga 28080

caggggaccc tgtgttctca ctgtgatttg caactgtcct aaccttggat tacatcaaga 28140

tctttgttgc catctctgtg ctgagtataa taaatacaga aattaaaata tactggggct 28200

cctatcgcca tcctgtaaac gccaccgtct tcacccgccc aagcaaacca aggcgaacct 28260

tacctggtac ttttaacatc tctccctctg tgatttacaa cagtttcaac ccagacggag 28320

tgagtctacg agagaacctc tccgagctca gctactccat cagaaaaaac accaccctcc 28380

ttacctgccg ggaacgtacg agtgcgtcac cggccgctgc accacaccta ccgcctgacc 28440

gtaaaccaga ctttttccgg acagacctca ataactctgt ttaccagaac aggaggtgag 28500

cttagaaaac ccttagggta ttaggccaaa ggcgcagcta ctgtggggtt tatgaacaat 28560

tcaagcaact ctacgggcta ttctaattca ggtttctcta gaatcggggt tggggttatt 28620

ctctgtcttg tgattctctt tattcttata ctaacgcttc tctgcctaag gctcgccgcc 28680

tgctgtgtgc acatttgcat ttattgtcag ctttttaaac gctggggtcg ccacccaaga 28740

tgattaggta cataatccta ggtttactca cccttgcgtc agcccacggt accacccaaa 28800

aggtggattt taaggagcca gcctgtaatg ttacattcgc agctgaagct aatgagtgca 28860

ccactcttat aaaatgcacc acagaacatg aaaagctgct tattcgccac aaaaacaaaa 28920

ttggcaagta tgctgtttat gctatttggc agccaggtga cactacagag tataatgtta 28980

cagttttcca gggtaaaagt cataaaactt ttatgtatac ttttccattt tatgaaatgt 29040

gcgacattac catgtacatg agcaaacagt ataagttgtg gcccccacaa aattgtgtgg 29100

aaaacactgg cactttctgc tgcactgcta tgctaattac agtgctcgct ttggtctgta 29160

ccctactcta tattaaatac aaaagcagac gcagctttat tgaggaaaag aaaatgcctt 29220

aatttactaa gttacaaagc taatgtcacc actaactgct ttactcgctg cttgcaaaac 29280

aaattcaaaa agttagcatt ataattagaa taggatttaa accccccggt catttcctgc 29340

tcaataccat tcccctgaac aattgactct atgtgggata tgctccagcg ctacaacctt 29400

gaagtcaggc ttcctggatg tcagcatctg actttggcca gcacctgtcc cgcggatttg 29460

ttccagtcca actacagcga cccaccctaa cagagatgac caacacaacc aacgcggccg 29520

ccgctaccgg acttacatct accacaaata caccccaagt ttctgccttt gtcaataact 29580

gggatgcaac cgcagcagcc actcccgctt gggaagaata gaggcagatt ctgaaagtca 29640

agaagacatc atccggaata ttgccaggca cctcgcccag gtcggggaca gcatggaccg 29700

tagcatccct ccgggcctgg tgaacggcct ggccctgcag ctcaggaaca ccagccggtc 29760

ggaggaggac cggaacaggg acctggccac tgccctggag cagctgctgc aggcctaccc 29820

tagagacatg gagaaggaga agaccatgct ggtgctggcc ctgctgctgg ccaagaaggt 29880

ggccagtcac acgccgtcct tgctccgtga tgtctttcac acaacagtga attttattaa 29940

ccagaaccta cgcacctacg tgaggagctt agccagaaat gggatggatg caaatccata 30000

gattggacgg actgaaacac atgttctttt ctcttacagt atgattaaat gagacatgat 30060

tcctcgagtt tttatattac tgacccttgt tgcgcttttt tgtgcgtgct ccacattggc 30120

tgcggtttct cacatcgaag tagactgcat tccagccttc acagtctatt tgctttacgg 30180

atttgtcacc ctcacgctca tctgcagcct catcactgtg gtcatcgcct ttatccagtg 30240

cattgactgg gtctgtgtgc gctttgcata tctcagacac catccccagt acagggacag 30300

gactatagct gagcttctta gaattcttta attatgaaat ttactgtgac ttttctgctg 30360

attatttgca ccctatctgc gttttgttcc ccgacctcca agcctcaaag acatatatca 30420

tgcagattca ctcgtatatg gaatattcca agttgctaca atgaaaaaag cgatctttcc 30480

gaagcctggt tatatgcaat catctctgtt atggtgttct gcagtaccat cttagcccta 30540

gctatatatc cctaccttga cattggctgg aaacgaatag atgccatgaa ccacccaact 30600

ttccccgcgc ccgctatgct tccactgcaa caagttgttg ccggcggctt tgtcccagcc 30660

aatcagcctc gccccacttc tcccaccccc actgaaatca gctactttaa tctaacagga 30720

ggagatgact gacaccctag atctagaaat ggacggaatt attacagagc agcgcctgct 30780

agaaagacgc agggcagcgg ccgagcaaca gcgcatgaat caagagctcc aagacatggt 30840

taacttgcac cagtgcaaaa ggggtatctt ttgtctggta aagcaggcca aagtcaccta 30900

cgacagtaat accaccggac accgccttag ctacaagttg ccaaccaagc gtcagaaatt 30960

ggtggtcatg gtgggagaaa agcccattac cataactcag cactcggtag aaaccgaagg 31020

ctgcattcac tcaccttgtc aaggacctga ggatctctgc acccttatta agaccctgtg 31080

cggtctcaaa gatcttattc cctttaacta ataaaaaaaa ataataaagc atcacttact 31140

taaaatcagt tagcaaattt ctgtccagtt tattcagcag cacctccttg ccctcctccc 31200

agctctggta ttgcagcttc ctcctggctg caaactttct ccacaatcta aatggaatgt 31260

cagtttcctc ctgttcctgt ccatccgcac ccactatctt catgttgttg cagatgaagc 31320

gcgcaagacc gtctgaagat accttcaacc ccgtgtatcc atatgacacg gaaaccggtc 31380

ctccaactgt gccttttctt actcctccct ttgtatcccc caatgggttt caagagagtc 31440

cccctggggt actctctttg cgcctatccg aacctctagt tacctccaat ggcatgcttg 31500

cgctcaaaat gggcaacggc ctctctctgg acgaggccgg caaccttacc tcccaaaatg 31560

taaccactgt gagcccacct ctcaaaaaaa ccaagtcaaa cataaacctg gaaatatctg 31620

cacccctcac agttacctca gaagccctaa ctgtggctgc cgccgcacct ctaatggtcg 31680

cgggcaacac actcaccatg caatcacagg ccccgctaac cgtgcacgac tccaaactta 31740

gcattgccac ccaaggaccc ctcacagtgt cagaaggaaa gctagccctg caaacatcag 31800

gccccctcac caccaccgat agcagtaccc ttactatcac tgcctcaccc cctctaacta 31860

ctgccactgg tagcttgggc attgacttga aagagcccat ttatacacaa aatggaaaac 31920

taggactaaa gtacggggct cctttgcatg taacagacga cctaaacact ttgaccgtag 31980

caactggtcc aggtgtgact attaataata cttccttgca aactaaagtt actggagcct 32040

tgggttttga ttcacaaggc aatatgcaac ttaatgtagc aggaggacta aggattgatt 32100

ctcaaaacag acgccttata cttgatgtta gttatccgtt tgatgctcaa aaccaactaa 32160

atctaagact aggacagggc cctcttttta taaactcagc ccacaacttg gatattaact 32220

acaacaaagg cctttacttg tttacagctt caaacaattc caaaaagctt gaggttaacc 32280

taagcactgc caaggggttg atgtttgacg ctacagccat agccattaat gcaggagatg 32340

ggcttgaatt tggttcacct aatgcaccaa acacaaatcc cctcaaaaca aaaattggcc 32400

atggcctaga atttgattca aacaaggcta tggttcctaa actaggaact ggccttagtt 32460

ttgacagcac aggtgccatt acagtaggaa acaaaaataa tgataagcta actttgtgga 32520

ccacaccagc tccatctcct aactgtagac taaatgcaga gaaagatgct aaactcactt 32580

tggtcttaac aaaatgtggc agtcaaatac ttgctacagt ttcagttttg gctgttaaag 32640

gcagtttggc tccaatatct ggaacagttc aaagtgctca tcttattata agatttgacg 32700

aaaatggagt gctactaaac aattccttcc tggacccaga atattggaac tttagaaatg 32760

gagatcttac tgaaggcaca gcctatacaa acgctgttgg atttatgcct aacctatcag 32820

cttatccaaa atctcacggt aaaactgcca aaagtaacat tgtcagtcaa gtttacttaa 32880

acggagacaa aactaaacct gtaacactaa ccattacact aaacggtaca caggaaacag 32940

gagacacaac tccaagtgca tactctatgt cattttcatg ggactggtct ggccacaact 33000

acattaatga aatatttgcc acatcctctt acactttttc atacattgcc caagaataaa 33060

gaatcgtttg tgttatgttt caacgtgttt atttttcaat tgcagaaaat ttcaagtcat 33120

ttttcattca gtagtatagc cccaccacca catagcttat acagatcacc gtaccttaat 33180

caaactcaca gaaccctagt attcaacctg ccacctccct cccaacacac agagtacaca 33240

gtcctttctc cccggctggc cttaaaaagc atcatatcat gggtaacaga catattctta 33300

ggtgttatat tccacacggt ttcctgtcga gccaaacgct catcagtgat attaataaac 33360

tccccgggca gctcacttaa gttcatgtcg ctgtccagct gctgagccac aggctgctgt 33420

ccaacttgcg gttgcttaac gggcggcgaa ggagaagtcc acgcctacat gggggtagag 33480

tcataatcgt gcatcaggat agggcggtgg tgctgcagca gcgcgcgaat aaactgctgc 33540

cgccgccgct ccgtcctgca ggaatacaac atggcagtgg tctcctcagc gatgattcgc 33600

accgcccgca gcataaggcg ccttgtcctc cgggcacagc agcgcaccct gatctcactt 33660

aaatcagcac agtaactgca gcacagcacc acaatattgt tcaaaatccc acagtgcaag 33720

gcgctgtatc caaagctcat ggcggggacc acagaaccca cgtggccatc ataccacaag 33780

cgcaggtaga ttaagtggcg acccctcata aacacgctgg acataaacat tacctctttt 33840

ggcatgttgt aattcaccac ctcccggtac catataaacc tctgattaaa catggcgcca 33900

tccaccacca tcctaaacca gctggccaaa acctgcccgc cggctataca ctgcagggaa 33960

ccgggactgg aacaatgaca gtggagagcc caggactcgt aaccatggat catcatgctc 34020

gtcatgatat caatgttggc acaacacagg cacacgtgca tacacttcct caggattaca 34080

agctcctccc gcgttagaac catatcccag ggaacaaccc attcctgaat cagcgtaaat 34140

cccacactgc agggaagacc tcgcacgtaa ctcacgttgt gcattgtcaa agtgttacat 34200

tcgggcagca gcggatgatc ctccagtatg gtagcgcggg tttctgtctc aaaaggaggt 34260

agacgatccc tactgtacgg agtgcgccga gacaaccgag atcgtgttgg tcgtagtgtc 34320

atgccaaatg gaacgccgga cgtagtcata tttcctgaag caaaaccagg tgcgggcgtg 34380

acaaacagat ctgcgtctcc ggtctcgccg cttagatcgc tctgtgtagt agttgtagta 34440

tatccactct ctcaaagcat ccaggcgccc cctggcttcg ggttctatgt aaactccttc 34500

atgcgccgct gccctgataa catccaccac cgcagaataa gccacaccca gccaacctac 34560

acattcgttc tgcgagtcac acacgggagg agcgggaaga gctggaagaa ccatgttttt 34620

ttttttattc caaaagatta tccaaaacct caaaatgaag atctattaag tgaacgcgct 34680

cccctccggt ggcgtggtca aactctacag ccaaagaaca gataatggca tttgtaagat 34740

gttgcacaat ggcttccaaa aggcaaacgg ccctcacgtc caagtggacg taaaggctaa 34800

acccttcagg gtgaatctcc tctataaaca ttccagcacc ttcaaccatg cccaaataat 34860

tctcatctcg ccaccttctc aatatatctc taagcaaatc ccgaatatta agtccggcca 34920

ttgtaaaaat ctgctccaga gcgccctcca ccttcagcct caagcagcga atcatgattg 34980

caaaaattca ggttcctcac agacctgtat aagattcaaa agcggaacat taacaaaaat 35040

accgcgatcc cgtaggtccc ttcgcagggc cagctgaaca taatcgtgca ggtctgcacg 35100

gaccagcgcg gccacttccc cgccaggaac catgacaaaa gaacccacac tgattatgac 35160

acgcatactc ggagctatgc taaccagcgt agccccgatg taagcttgtt gcatgggcgg 35220

cgatataaaa tgcaaggtgc tgctcaaaaa atcaggcaaa gcctcgcgca aaaaagaaag 35280

cacatcgtag tcatgctcat gcagataaag gcaggtaagc tccggaacca ccacagaaaa 35340

agacaccatt tttctctcaa acatgtctgc gggtttctgc ataaacacaa aataaaataa 35400

caaaaaaaca tttaaacatt agaagcctgt cttacaacag gaaaaacaac ccttataagc 35460

ataagacgga ctacggccat gccggcgtga ccgtaaaaaa actggtcacc gtgattaaaa 35520

agcaccaccg acagctcctc ggtcatgtcc ggagtcataa tgtaagactc ggtaaacaca 35580

tcaggttgat tcacatcggt cagtgctaaa aagcgaccga aatagcccgg gggaatacat 35640

acccgcaggc gtagagacaa cattacagcc cccataggag gtataacaaa attaatagga 35700

gagaaaaaca cataaacacc tgaaaaaccc tcctgcctag gcaaaatagc accctcccgc 35760

tccagaacaa catacagcgc ttccacagcg gcagccataa cagtcagcct taccagtaaa 35820

aaagaaaacc tattaaaaaa acaccactcg acacggcacc agctcaatca gtcacagtgt 35880

aaaaaagggc caagtgcaga gcgagtatat ataggactaa aaaatgacgt aacggttaaa 35940

gtccacaaaa aacacccaga aaaccgcacg cgaacctacg cccagaaacg aaagccaaaa 36000

aacccacaac ttcctcaaat cgtcacttcc gttttcccac gttacgtaac ttcccatttt 36060

aagaaaacta caattcccaa cacatacaag ttactccgcc ctaaaaccta cgtcacccgc 36120

cccgttccca cgccccgcgc cacgtcacaa actccacccc ctcattatca tattggcttc 36180

aatccaaaat aaggtatatt attgatgatg 36210

Claims (6)

1. An oncolytic adenovirus recombinant carrying TMTP1 and tBId is Ad 5/delta E1A/TMTP 1/delta ADP-tBId, the nucleotide sequence of the oncolytic adenovirus recombinant carrying TMTP1 and tBId is shown as SEQ ID No.23, 27 bases shown as SEQ ID No.1 in the 920nt-946nt region are deleted in the E1A conserved sequence 2 region of a human type 5 adenovirus gene, a gene sequence coding tumor targeting peptide TMTP1 shown as SEQ ID No.15 is inserted in the 19641nt-19655nt region of a Hexon hypervariable region 5, meanwhile, the E3 region is deleted in the 29477nt-29714nt region of the ADP gene to form a deletion region, the deletion region is inserted in the gene sequence of mitochondrial apoptosis peptide tBId shown as SEQ ID No.22, and a Cla1 site is introduced.

2. The method for constructing an oncolytic adenoviral recombinant carrying TMTP1 and tBId according to claim 1, comprising the steps of:

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-tBod carrying TMTP1 and tBod

The E3 region of Ad 5/delta E1A/TMTP1 obtained in step 2 is located in the 29477nt-29714nt region of an ADP gene to form a deletion region, the gene sequence of mitochondrial apoptotic peptide tBod shown in SEQ ID NO.22 is inserted into the deletion region, and Cla1 enzyme cutting site is introduced, thus obtaining oncolytic adenovirus recombinant Ad 5/delta E1A/TMTP 1/delta ADP-tBod carrying TMTP1 and tBod shown in SEQ ID NO. 23.

3. Use of the oncolytic adenoviral recombinant carrying TMTP1 and tBid according to claim 1 for the preparation of a medicament for the treatment of tumors.

4. Use of the oncolytic adenoviral recombinant carrying TMTP1 and tBid according to claim 1 for the preparation of a gene therapy vector.

5. Use of the oncolytic adenoviral recombinant carrying TMTP1 and tBid according to claim 1 for the preparation of a medicament for improving resistance against tumor chemotherapeutic drugs.

6. The use of the oncolytic adenoviral recombinant carrying TMTP1 and tBId of claim 1 in the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.

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