CN114317462A

CN114317462A - Oncolytic adenovirus recombinant carrying TMVP1 and tBID, and construction method and application thereof

Info

Publication number: CN114317462A
Application number: CN202111442797.7A
Authority: CN
Inventors: 马丁; 陈世民; 杨帆; 高庆蕾; 纪腾
Original assignee: Wuhan Kaideweisi Biotechnology Co ltd
Current assignee: Wuhan Kaideweisi Biotechnology Co ltd
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-04-12

Abstract

The invention discloses an oncolytic adenovirus recombinant carrying TMVP1 and tBID, a construction method and application thereof, belonging 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 tumor targeting peptide TMVP1 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, and a gene sequence of mitochondrial apoptosis peptide tBod is inserted in the deleted region and introduced into a Cla1 enzyme cutting site. The invention also discloses a construction method and application of the oncolytic adenovirus recombinant. The oncolytic adenovirus recombinant carrying TMVP1 and tBId has ideal targeting effect and strong killing effect.

Description

Oncolytic adenovirus recombinant carrying TMVP1 and tBID, and construction method and application thereof

Technical Field

The invention relates to an oncolytic adenovirus recombinant carrying TMVP1 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 TMVP1 and tBId.

The technical scheme for solving the problems is as follows: an oncolytic adenovirus recombinant carrying TMVP1 and tBId is Ad 5/delta 27/TMVP 1/delta ADP-tBId, the nucleotide sequence of the oncolytic adenovirus recombinant carrying TMVP1 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 shown as SEQ ID NO.15 and used for coding tumor targeting peptide TMVP1 is inserted in the 19641nt-19655nt region of the Hexon hypervariable region 5, meanwhile, the deletion E3 region is located in the 29477nt-29714nt region of the ADP gene to form a deletion region, and a gene sequence shown as the mitochondrial apoptosis peptide tBId is inserted in the deletion region and introduced into Cla1 enzyme cutting site.

The inventors of the present application, in order to obtain the above oncolytic adenovirus recombinants carrying TMVP1 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 TMVP1 has the obvious advantages of targeting tumor cells with high metastatic potential, identifying tumor subclinical micrometastasis focus in early stage, being selectively swallowed by the tumor cells, having strong tumor cell toxic effect and the like. The invention obviously improves the targeting of an oncolytic adenovirus recombinant carrying TMVP1 and tBId by inserting the gene sequence of the coding tumor targeting peptide TMVP1 shown as SEQ ID NO.15 into the human type 5 adenovirus.

Apoptosis is the most important link for killing tumor cells by various anticancer drugs and is regulated by BCL-2(B-cell lymphoma-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 tBId of mitochondrial apoptotic peptide is inserted into human type 5 adenovirus (Ad5), so that the mitochondrial apoptotic peptide tBId is specifically expressed in tumors, and tumor cell apoptosis is promoted, thereby remarkably improving the apoptosis promoting effect of oncolytic adenovirus recombinants carrying TMVP1 and tBId and the killing effect of oncolytic virus lytic cells.

Furthermore, the gene sequence of the tumor targeting peptide TMVP1 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 sequence of TMVP1 gene coding for tumor targeting peptide is inserted into the hypervariable region 5 of Hexon, thereby destroying the normal expression of Hexon protein, inhibiting the combination of Fx and Hexon and preventing adenovirus from being phagocytosed by liver. The tumor targeting peptide TMVP1 can reduce the dependence of adenovirus on the Coxsackie Adenovirus Receptor (CAR) on the surface of tumor cells and increase the affinity of adenovirus to tumor cells; 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 TMVP1 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 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 TMVP1 and tBId has the beneficial effects that:

1. the oncolytic adenovirus recombinant carrying TMVP1 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 TMVP1 and tBId has complete functions, and the elimination of recombinant adenovirus by an organism is avoided to a certain extent;

5. oncolytic adenoviral recombinants carrying TMVP1 and tBid have a broad spectrum of anti-cancer. In vivo model research of tumor animal models proves that the oncolytic adenovirus recombinant carrying TMVP1 and tBId has obvious treatment effect on all tested tumor models through 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 TMVP1 and tBId.

The technical scheme for solving the problems is as follows: the construction method of the oncolytic adenovirus recombinant carrying TMVP1 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 27;

step 2: preparation of Ad 5/. DELTA.27/TMVP 1

Inserting a gene sequence which is shown as SEQ ID NO.15 and encodes tumor targeting peptide TMVP1 into 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 27/TMVP 1;

and step 3: preparation of oncolytic adenovirus recombinant Ad 5/. DELTA.27/TMVP 1/. DELTA.ADP-tBId carrying TMVP1 and tBId

The E3 region of Ad 5/delta 27/TMVP1 obtained in step 2 is located in the 29477nt-29714nt region of the 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 the Cla1 enzyme cutting site is introduced, so that the oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP-tBod carrying TMVP1 and tBod shown in SEQ ID NO.23 is obtained.

The construction method of the oncolytic adenovirus recombinant carrying TMVP1 and tBId has the beneficial effects that:

1. the oncolytic adenovirus recombinant carrying TMVP1 and tBID is inserted with a gene sequence coding tumor targeting peptide TMVP1 and a gene sequence coding mitochondrial apoptosis peptide tBID, has the advantages of tumor specific replication, high tumor and metastatic tumor targeting, 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 approach 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 TMVP1 and tBId as described above.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and tBId is applied to the preparation of medicines for treating tumors.

The beneficial effects of the application of the oncolytic adenovirus recombinant carrying TMVP1 and tBId of the invention are as follows:

the oncolytic adenovirus recombinant carrying TMVP1 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 TMVP1 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 TMVP1 and tBId described above.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and tBId is applied to the preparation of a gene therapy vector.

the oncolytic adenovirus recombinant carrying TMVP1 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 TMVP1 and tBId, and has positive pharmaceutical value and wide social significance.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and tBId is applied to the preparation of medicines for improving the drug resistance of anti-tumor chemotherapeutic drugs.

the oncolytic adenovirus recombinant carrying TMVP1 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 TMVP1 and tBId, and has positive pharmaceutical value and wide social significance.

The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and tBId is applied to the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.

the oncolytic adenovirus recombinant carrying TMVP1 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 TMVP1 and tBId, and has positive pharmaceutical value and wide social significance.

Drawings

FIG. 1 is a schematic structural diagram of oncolytic adenovirus recombinant Ad5/Δ 27/TMVP1/Δ ADP-tBId carrying TMVP1 and tBId according to an embodiment of the present invention.

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

FIG. 3 is a histogram of 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 control curve of tumor volume change in a subcutaneous tumor animal model in example 8 of the present invention.

Fig. 5 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. 6 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. 7 shows the results of urea nitrogen (BUN) experiments in experimental animals of the abdominal cavity in situ metastatic tumor model in example 8 of the present invention.

FIG. 8 is the Creatinine (Creatinine) test result of the experimental animal of the abdominal cavity in situ metastasis 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 pAd 5/delta 27 adenovirus packaging plasmid

Step 1.1: construction of shuttle plasmid Blunt-Zero-E1A/Δ 27

The pXC 1-delta 27 plasmid is 27 bases which are 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.

Acquisition of fragment 1: 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 27 with deletion of 121-plus 129AA 920nt-946nt through DNA sequencing, identification and screening.

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

The E1A sequence was ligated into pEASY-Blunt-Zero Blunt-end ligation plasmid (available from Beijing Quanjin Biotechnology Co., Ltd., product No. CB501) by the action of T4 ligase (available from Saimei Feishell technology Co., Ltd., product No. el0011) to obtain shuttle plasmid Blunt-Zero-E1A/delta 27.

Taking the shuttle plasmid Blunt-Zero-E1A/delta 27 as a template, carrying out PCR amplification reaction by using the primer carried in the pEASY-Blunt-Zero Blunt end connection 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 27 adenovirus packaging plasmid

The E1A shuttle fragment obtained in step 1.1 and the backbone fragment obtained in step 1.2 were electroporated into E.coli BJ5183 electroporation competence obtained in step 1.3 using a GenePulser XcellTM electroporation apparatus (available from Bio-Rad) under conditions of 2.5Kv and 25. mu.F. Positive bacteria were screened for kanamycin resistance. Selecting smaller bacterial plaques in bacterial plates, culturing in LB culture medium, extracting plasmids in small quantity, and using an upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQ ID NO.8) and the downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO.9), performing PCR amplification reaction, performing the same reaction system and reaction procedure as the step 1.1, and sequencing to determine whether the E1A region lacks the 920bp-946bp region. The extracted plasmid was transformed into high copy number bacterium DH10B (purchased from Seimer Feishale scientific Co., Ltd., cat 18290015) to obtain pAd 5-E1A/. DELTA.27 adenovirus packaging plasmid, abbreviated as pAd 5/. DELTA.27.

Example 2: construction of pAd 5/. DELTA.27/Hexon (HVR/TMVP1) plasmid vector

Step 2.1: construction of shuttle vector pEASY-Blunt-Zero-Hexon (HVR/TMVP1)

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

in 15cm culture dish, 7.5X 10⁵293 cells, 10% FBS DMEM, by day two, cells should be 1-1.5X 10⁶Approximately 70% of the cells fused; and replacing with fresh culture solution 3-4h before transfection. Preparation of co-transfected DNA-calcium phosphate solution: 1600 μ l of sterilized 2 XHBS (280mM NaCl, 43mM HEPES, 10mM KCl, 10mM Na)₂HPO₄·7H₂O, 2% dextrose, pH 7.05-7.15); 42. mu.g each of pBHGE3 and Δ 920-946pXC 1; adding sterilized double distilled water to 2840 μ l, mixing, slowly adding 50 μ l 2.5M CaCl₂The mixture was inverted and mixed, and the DNA/CaCl was allowed to stand at room temperature₂Precipitating for 45-60min to form slightly turbid precipitate. Adding 500. mu.l of the above mixture to 293 cells in a 5ml 60mm dish at 37 ℃ with 5% CO₂Incubated for 4-6h, the liquid was aspirated and washed once with PBS. Transfection efficiency was promoted by treatment with 15% glycerol/DMEM for 1-2min, washed once with PBS and replaced with complete medium. 1.8% agarose with low melting point is prepared, autoclaved, subpackaged into 5ml, melted in boiling water before use, kept at 45 ℃, added with 4% FBS DMEM with equal amount when used, and immediately spread into a culture dish. The culture medium was aspirated off, and 5ml of the above-mentioned medium was added. Every 4-5 days, 3ml of the above liquid was added. 14-21d, plaques appeared, 6-12 plaques were selected. The plaques were transferred to serum-free DMEM medium in 1.5ml EP tubes and incubated at 37 ℃ for 24 h. Seeding in 24-well plates 1X 10⁵293 cells in 10% FBS DMEM, by day two, cells should be 2X 10⁵About 70% of the cells were fused, the liquid was aspirated, and 100. mu.l (about 10) of the above-mentioned incubation liquid was taken³Virus) was added and the liquid was gently shaken 3 times at 37 ℃ with 5% CO₂And (5) performing medium incubation for 90 min. Add complete medium to 1ml and place cells at 37 ℃ with 5% CO₂And (4) incubating for 5-10 days until complete CPE appears, namely cytotoxic effect, and the cells are rounded and float and mainly comprise nucleolus. If after 10d complete CPE was not present, it was suggested that the virus titer was too low and a second round of amplification was required. Subjecting the culture plate to three stepsThe virus is released by a round of freeze/thaw cycles, the lysate is collected in a 15ml tube, centrifuged at maximum speed for 10min, and the supernatant is collected, frozen at-80 ℃, and the fluid is called second-generation virus, approximately 5X 10⁷Viral/ml. The above viruses were re-amplified at 75cm²Seed in a Petri dish at 5X 10⁶293 cells in 10ml 10% FBS DMEM, by day two, cells should be 1X 10⁷Approximately 70% cell fusion; replacing with fresh culture solution 3-4h before transfection; adding 1ml of second-generation virus stock solution into 1ml of complete culture medium for transfection; the MOI is about 5; remove 75cm²Adding the liquid into a culture dish, and slightly shaking for three times; at 37 deg.C, 5% CO₂Incubating for 90 min; 9ml of 2% FBS DMEM was added thereto at 37 ℃ with 5% CO₂And (4) incubating for 4-7d, and extracting virus DNA for screening positive viruses. Because the 293 cell genome contains the complete E1A gene, the 293 cell DNA is easily contaminated when extracting positive virus DNA, and the identification fails, therefore, the delta 920-946Ad5 is amplified once again in the tumor cell Hela for identification, and the steps are as follows: seeding in 6-well plates 1X 10⁵Hela cells in 10% FBS DMEM, and by the next day, cells were 2X 10⁵About 70% of the cells were fused, the liquid was aspirated, and 100. mu.l (about 10) of the filtrate was taken³Virus) was added and the liquid was gently shaken 3 times at 37 ℃ with 5% CO₂And (5) performing medium incubation for 90 min. Adding complete medium to 1ml, and placing the cells at 37 deg.C and 5% CO₂The cells were scraped and collected in a 1.5ml EP tube, the supernatant was discarded by centrifugation, 300. mu.l PBS was added and three cycles of freeze/thaw were performed to release the virus, the lysate was centrifuged at maximum speed for 10min, the supernatant was collected and frozen at-80 ℃ and DNA was extracted using Qiagen kit mini DNA isolation kit, according to the kit instructions. Performing PCR reaction by using virus DNA as a template, wherein an upstream primer: 5'-cgggatccgggcccccatttcc-3' (SEQ ID NO 10), downstream primer: 5'-tgctctagacacaggtgatgtcg-3' (SEQ ID NO 11), the total reaction system volume of 100. mu.l comprising: containing MgCl ₂10. mu.l of the buffer solution for PCR of (1); 2mM dNTP, 10. mu.l; 10. mu.M of the forward primer, 1. mu.l; 10 muM 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, after conventional electrophoretic separation and purification, the detection concentration is used for DNA sequencing, and a sequencing primer: 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 NO.15 and code a tumor targeting peptide TMVP1 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/TMVP1), then, the PCR products pEASY-Hexon (HVR/TMVP1) 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 gel, the above 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/TMVP1) shuttle plasmid, pEASY-Blunt-Zero-Hexon (HVR5/TMVP1) shuttle plasmid and pEASY-Blunt-Zero-Hexon (HVR7/TMVP1) shuttle plasmid, respectively.

Step 2.2: extracting a large amount of the skeleton plasmid pAd 5-E1A/delta 27 (OMEGA, product number D6692-01) constructed in the step 1.4, carrying out enzyme digestion by utilizing a specific enzyme digestion site AsisI, and then extracting with phenol and chloroform to obtain a linear skeleton plasmid.

Step 2.3: the PCR product of pEASY-Hexon (HVR/TMVP1) 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 27-Hexon (HVR2/TMVP1) plasmid, pAd 5-E1A/delta 27-Hexon (HVR5/TMVP1) plasmid and pAd 5-E1A/delta 27-Hexon (HVR7/TMVP1) plasmid are identified.

Example 3: construction of Ad 5/. DELTA.27/TMVP 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 Ad5E3 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 Ad5DNA as a template, 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; ad5DNA 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 isPrimer 2 complementary part, atcgat is Cla I restriction site; primer 4: 5'-atgtctttgaggcttggagg-3' (SEQ ID NO 21), corresponding to 30137nt-30118 nt; the PCR reaction conditions are the same as above, the product is 422bp (fragment 2), and after the conventional electrophoresis separation and purification, the concentration is detected for the 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 l of DH5 alpha competent bacteria, selecting positive clones, carrying out small extraction on plasmids, and carrying out DNA sequencing, identification and screening to obtain pcDNA3.1 plasmid mutant-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.27-Hexon (HVR2/TMVP1), pAd 5-E1A/. DELTA.27-Hexon (HVR5/TMVP1) and pAd 5-E1A/. DELTA.27-Hexon (HVR7/TMVP1) plasmids constructed in step 2.3. Phenol chloroform extraction and recovery of linear skeleton plasmid.

The linear backbone plasmid and the E3/tBId fragment obtained in step 3.3 were electrotransferred into E.coli BJ5183 electrotransferase peptide obtained in step 1.3 under conditions of 2.5Kv and 25. mu.F, and homologous recombination yielded pAd5/Δ 27/HVR2-TMVP1/Δ ADP-tBId plasmid, pAd5/Δ 27/HVR5-TMVP1/Δ ADP-tBId plasmid and pAd5/Δ 27/HVR7-TMVP1/Δ ADP-tBId plasmid, respectively.

Example 4: obtaining, amplifying and purifying oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP-tBId carrying TMVP1 and tBId

Step 4.1: acquisition of oncolytic adenovirus recombinant Ad 5/. DELTA.27/TMVP 1/. DELTA.ADP-tBId carrying TMVP1 and tBId

Step 4.1.1: the plasmid pAd 5/delta 27/HVR2-TMVP 1/delta ADP-tBId, plasmid pAd 5/delta 27/HVR5-TMVP 1/delta ADP-tBId and plasmid pAd 5/delta 27/HVR7-TMVP 1/delta ADP-tBId obtained in step 3.4 were extracted in bulk, respectively, the plasmid was digested with PacI, phenol chloroform was extracted, and the digested product was recovered.

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

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

Step 4.1.4: three Ad 5/. DELTA.27/TMVP 1/. DELTA.ADP-tBId obtained in step 4.1.3 were identified and E1A, Hexon and E3/ADP regions were post-PCR sequenced with the corresponding primers, respectively.

Step 4.2: amplification, purification and titre determination of oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP-tBId carrying TMVP1 and tBId

Step 4.2.1: amplification step 4.1.4 sequencing the correct oncolytic adenovirus recombinant Ad 5/. DELTA.27/TMVP 1/. DELTA.ADP-tBId carrying TMVP1 and tBId, detecting the titer (MOI) of 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 TMVP1 and tBod

The purpose of this example was to determine the efficiency of oncolytic adenovirus recombinants carrying TMVP1 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 Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBId carrying TMVP1 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 TMVP1 and tBod

Step 6.1: identification of oncolytic adenovirus recombinant in vitro tumor cell killing effect carrying TMVP1 and tBod

The purpose of this example is to identify the killing effect of Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBod oncolytic adenovirus on a range of tumor cells as well as 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 10³Tumor cells in 10% FBS DMEM or RIPM1640 medium, about 70% cell fusion by the next day, aspirating the medium, diluting the oncolytic adenovirus with MOI 10 to 100 μ l in the medium, adding to the culture well, shaking the medium gently three times, and incubating at 37 deg.C and 5% CO₂After incubation in the incubator of (1) for 72h, the viral survival rate was measured by adding cck-8.

Experimental results show that 42.8% of tumor cells appear to be extremely sensitive with a survival rate of less than 25% when the virus MOI is 10; 42.8% of the tumor cells have good response to adenovirus, and the survival rate is between 25% and 75%; however, 14.3% of the tumor cells responded slightly to adenovirus, and the specific experimental results are shown in Table 1.

Step 6.2: identification of oncolytic adenovirus recombinant carrying TMVP1 and tBID on sensitization effect of chemotherapeutic drug cisplatin

Whereas oncolytic adenoviral recombinants carrying TMVP1 and tBid do not show a potent killing effect on all tumor cells, it is envisaged that oncolytic adenoviral recombinants carrying TMVP1 and tBid in combination with cisplatin (DDP) kill insensitive tumor cells.

Seeded into 96-well cell culture plates at 5X 10³Tumor cells were simultaneously mixed with a concentration of oncolytic adenoviral recombinants carrying TMVP1 and tBod and its control virus Ad5/Δ 27. This control virus Ad 5/. DELTA.27 was an oncolytic adenovirus with a 27bp deletion in E1A only, constructed and stored in this 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 at which 50% of the tumor cells are apoptotic) and the synergy index CI of oncolytic adenovirus recombinant Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBod carrying TMVP1 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

The experimental result shows that for most tumor cells, the combination of Ad 5/delta 27/HVR5/TMVP 1/delta ADP-tBId and DDP has a sensitization effect, and is particularly obvious for drug-resistant cell lines such as ES2 and MDA-MB-231, and the CI values are 0.18 and 0.03 respectively. The above results fully demonstrate that oncolytic adenoviral recombinants carrying TMVP1 and tBID of the invention are sensitized to the chemotherapeutic cisplatin, whether in cell lines sensitive to DDP or resistant to DDP.

Example 7: in vivo experiments prove that oncolytic adenovirus recombinant carrying TMVP1 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 27/HVR5/TMVP1/Δ ADP-tBId with PFU carrying TMVP1 and tBId and its control virus Ad5/Δ 27. 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 27 is gathered in the liver, spleen and lung and is distributed less in tumor tissues, and Ad 5/delta 27/HVR5/TMVP 1/delta ADP-tBId is mainly distributed in the tumor tissues and is hardly taken up by the liver; FIG. 4 shows the relative adenovirus content in each tissue of nude mice, and in mice injected with Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBId, adenovirus was mainly concentrated in tumor tissue, which was nearly 100-fold higher than that of the control virus, whereas Ad5/Δ 27, which was a control virus, was taken up by the liver in large amounts, and adenovirus genes were hardly detected in tumor tissue.

The experimental results fully show that the oncolytic adenovirus recombinant Ad 5/delta 27/HVR5/TMVP 1/delta ADP-tBId carrying TMVP1 and tBId overcomes the liver tropism, is hardly taken by the liver any more, and greatly enhances the targeting of the oncolytic adenovirus recombinant Ad 5/delta 27/HVR5/TMVP 1/delta ADP-tBId to the tumor.

Example 8: in vivo experiments verify the killing effect and safety of oncolytic adenovirus recombinant carrying TMVP1 and tBId on tumor

Step 8.1: subcutaneous tumor animal model

BALB/c nu nude mouse with 4-6 weeks old and unilateral axillary fat padBottom planting of 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 27, d-injection of Ad5/Δ 27 plus DDP, e-single injection of Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBId, f-injection of Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBId plus DDP.

Wherein, c and d groups are injected with 1 × 10⁸PFU three days later, DDP was intraperitoneally injected into groups b and d at a concentration of 2.5mg/kg once every other day for four times. Tumor volume was measured every three days, starting after tumor implantation, and the change in tumor volume is shown in fig. 4. Ad 5/delta 27/TMVP 1/delta ADP-tBId alone controlled tumor progression, while in combination with DDP, further reduced tumor volume significantly.

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 assays carrying TMVP1 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: the results of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminase (AST), Creatinine (creatine), and urea nitrogen (BUN) are shown in fig. 5-8, but in the control group and the experimental group, the renal function was not significantly impaired, but in the mice injected with the control virus Ad5/Δ 27, the glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminase (AST) were significantly elevated, while in the mice injected with Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBid, the liver function was normal.

The above results indicate that oncolytic adenovirus recombinant Ad5/Δ 27/HVR5/TMVP1/Δ ADP-tBId carrying TMVP1 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 obvious, the tumor progress can be controlled by combining the two in a subcutaneous tumor 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 27/HVR5/TMVP 1/delta ADP-tBId carrying TMVP1 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 TMVP1 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

<120> oncolytic adenovirus recombinant carrying TMVP1 and tBID, construction method and application thereof

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gatccacata atctaacaca aactc 25

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cgggatccgg gcccccattt cc 22

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tgctctagac acaggtgatg tcg 23

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ccagagtagg tgtaataagg 20

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tagaaagtca agtggaaatg 20

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cttgctcgtg gacgt 15

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tgtcaccact aactgcttta ctcg 24

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gctgccctgc gtctttcta 19

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atacgcgccc accgaaac 18

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cgacccaccc tatcgatatc catagattgg acggactg 38

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atgtctttga ggcttggagg 20

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atccctccgg gcctggtgaa cggcctggcc ctgcagctca ggaacaccag ccggtcggag 180

gaggaccgga acagggacct ggccactgcc ctggagcagc tgctgcaggc ctaccctaga 240

gacatggaga aggagaagac catgctggtg ctggccctgc tgctggccaa gaaggtggcc 300

agtcacacgc cgtccttgct ccgtgatgtc tttcacacaa cagtgaattt tattaaccag 360

aacctacgca cctacgtgag gagcttagcc agaaatggga tggatgaa 408

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ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120

gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180

gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240

taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300

agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggccgcggg 360

gactttgacc gtttacgtgg agactcgccc aggtgttttt ctcaggtgtt ttccgcgttc 420

cgggtcaaag ttggcgtttt attattatag tcagctgacg tgtagtgtat ttatacccgg 480

tgagttcctc aagaggccac tcttgagtgc cagcgagtag agttttctcc tccgagccgc 540

tccgacaccg ggactgaaaa tgagacatat tatctgccac ggaggtgtta ttaccgaaga 600

aatggccgcc agtcttttgg accagctgat cgaagaggta ctggctgata atcttccacc 660

tcctagccat tttgaaccac ctacccttca cgaactgtat gatttagacg tgacggcccc 720

cgaagatccc aacgaggagg cggtttcgca gatttttccc gactctgtaa tgttggcggt 780

gcaggaaggg attgacttac tcacttttcc gccggcgccc ggttctccgg agccgcctca 840

cctttcccgg cagcccgagc agccggagca gagagccttg ggtccggttt ctatgccaaa 900

ccttgtaccg gaggtgatcc cacccagtga cgacgaggat gaagagggtg aggagtttgt 960

gttagattat gtggagcacc ccgggcacgg ttgcaggtct tgtcattatc accggaggaa 1020

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 taggtgtcga 19380

aggtcaaaca cctaaatatg ccgataaaac atttcaacct gaacctcaaa taggagaatc 19440

tcagtggtac gaaactgaaa ttaatcatgc agctgggaga gtccttaaaa agactacccc 19500

aatgaaacca tgttacggtt catatgcaaa acccacaaat gaaaatggag ggcaaggcat 19560

tcttgtaaag caacaaaatg gaaagctaga aagtcaagtg gaaatgcaat tttcttgctc 19620

gtggacgttc tcaactactg aggcgaccgc aggcaatggt gataacttga ctcctaaagt 19680

ggtattgtac agtgaagatg tagatataga aaccccagac actcatattt cttacatgcc 19740

cactattaag gaaggtaact cacgagaact aatgggccaa caatctatgc ccaacaggcc 19800

taattacatt gcttttaggg acaattttat tggtctaatg tattacaaca gcacgggtaa 19860

tatgggtgtt ctggcgggcc aagcatcgca gttgaatgct gttgtagatt tgcaagacag 19920

aaacacagag ctttcatacc agcttttgct tgattccatt ggtgatagaa ccaggtactt 19980

ttctatgtgg aatcaggctg ttgacagcta tgatccagat gttagaatta ttgaaaatca 20040

tggaactgaa gatgaacttc caaattactg ctttccactg ggaggtgtga ttaatacaga 20100

gactcttacc aaggtaaaac ctaaaacagg tcaggaaaat ggatgggaaa aagatgctac 20160

agaattttca gataaaaatg aaataagagt tggaaataat tttgccatgg aaatcaatct 20220

aaatgccaac ctgtggagaa atttcctgta ctccaacata gcgctgtatt tgcccgacaa 20280

gctaaagtac agtccttcca acgtaaaaat ttctgataac ccaaacacct acgactacat 20340

gaacaagcga gtggtggctc ccgggttagt ggactgctac attaaccttg gagcacgctg 20400

gtcccttgac tatatggaca acgtcaaccc atttaaccac caccgcaatg ctggcctgcg 20460

ctaccgctca atgttgctgg gcaatggtcg ctatgtgccc ttccacatcc aggtgcctca 20520

gaagttcttt gccattaaaa acctccttct cctgccgggc tcatacacct acgagtggaa 20580

cttcaggaag gatgttaaca tggttctgca gagctcccta ggaaatgacc taagggttga 20640

cggagccagc attaagtttg atagcatttg cctttacgcc accttcttcc ccatggccca 20700

caacaccgcc tccacgcttg aggccatgct tagaaacgac accaacgacc agtcctttaa 20760

cgactatctc tccgccgcca acatgctcta ccctataccc gccaacgcta ccaacgtgcc 20820

catatccatc ccctcccgca actgggcggc tttccgcggc tgggccttca cgcgccttaa 20880

gactaaggaa accccatcac tgggctcggg ctacgaccct tattacacct actctggctc 20940

tataccctac ctagatggaa ccttttacct caaccacacc tttaagaagg tggccattac 21000

ctttgactct tctgtcagct ggcctggcaa tgaccgcctg cttaccccca acgagtttga 21060

aattaagcgc tcagttgacg gggagggtta caacgttgcc cagtgtaaca tgaccaaaga 21120

ctggttcctg gtacaaatgc tagctaacta caacattggc taccagggct tctatatccc 21180

agagagctac aaggaccgca tgtactcctt ctttagaaac ttccagccca tgagccgtca 21240

ggtggtggat gatactaaat acaaggacta ccaacaggtg ggcatcctac accaacacaa 21300

caactctgga tttgttggct accttgcccc caccatgcgc gaaggacagg cctaccctgc 21360

taacttcccc tatccgctta taggcaagac cgcagttgac agcattaccc agaaaaagtt 21420

tctttgcgat cgcacccttt ggcgcatccc attctccagt aactttatgt ccatgggcgc 21480

actcacagac ctgggccaaa accttctcta cgccaactcc gcccacgcgc tagacatgac 21540

ttttgaggtg gatcccatgg acgagcccac ccttctttat gttttgtttg aagtctttga 21600

cgtggtccgt gtgcaccggc cgcaccgcgg cgtcatcgaa accgtgtacc tgcgcacgcc 21660

cttctcggcc ggcaacgcca caacataaag aagcaagcaa catcaacaac agctgccgcc 21720

atgggctcca gtgagcagga actgaaagcc attgtcaaag atcttggttg tgggccatat 21780

tttttgggca cctatgacaa gcgctttcca ggctttgttt ctccacacaa gctcgcctgc 21840

gccatagtca atacggccgg tcgcgagact gggggcgtac actggatggc ctttgcctgg 21900

aacccgcact caaaaacatg ctacctcttt gagccctttg gcttttctga ccagcgactc 21960

aagcaggttt accagtttga gtacgagtca ctcctgcgcc gtagcgccat tgcttcttcc 22020

cccgaccgct gtataacgct ggaaaagtcc acccaaagcg tacaggggcc caactcggcc 22080

gcctgtggac tattctgctg catgtttctc cacgcctttg ccaactggcc ccaaactccc 22140

atggatcaca accccaccat gaaccttatt accggggtac ccaactccat gctcaacagt 22200

ccccaggtac agcccaccct gcgtcgcaac caggaacagc tctacagctt cctggagcgc 22260

cactcgccct acttccgcag ccacagtgcg cagattagga gcgccacttc tttttgtcac 22320

ttgaaaaaca tgtaaaaata atgtactaga gacactttca ataaaggcaa atgcttttat 22380

ttgtacactc tcgggtgatt atttaccccc acccttgccg tctgcgccgt ttaaaaatca 22440

aaggggttct gccgcgcatc gctatgcgcc actggcaggg acacgttgcg atactggtgt 22500

ttagtgctcc acttaaactc aggcacaacc atccgcggca gctcggtgaa gttttcactc 22560

cacaggctgc gcaccatcac caacgcgttt agcaggtcgg gcgccgatat cttgaagtcg 22620

cagttggggc ctccgccctg cgcgcgcgag ttgcgataca cagggttgca gcactggaac 22680

actatcagcg ccgggtggtg cacgctggcc agcacgctct tgtcggagat cagatccgcg 22740

tccaggtcct ccgcgttgct cagggcgaac ggagtcaact ttggtagctg ccttcccaaa 22800

aagggcgcgt gcccaggctt tgagttgcac tcgcaccgta gtggcatcaa aaggtgaccg 22860

tgcccggtct gggcgttagg atacagcgcc tgcataaaag ccttgatctg cttaaaagcc 22920

acctgagcct ttgcgccttc agagaagaac atgccgcaag acttgccgga aaactgattg 22980

gccggacagg ccgcgtcgtg cacgcagcac cttgcgtcgg tgttggagat ctgcaccaca 23040

tttcggcccc accggttctt cacgatcttg gccttgctag actgctcctt cagcgcgcgc 23100

tgcccgtttt cgctcgtcac atccatttca atcacgtgct ccttatttat cataatgctt 23160

ccgtgtagac acttaagctc gccttcgatc tcagcgcagc ggtgcagcca caacgcgcag 23220

cccgtgggct cgtgatgctt gtaggtcacc tctgcaaacg actgcaggta cgcctgcagg 23280

aatcgcccca tcatcgtcac aaaggtcttg ttgctggtga aggtcagctg caacccgcgg 23340

tgctcctcgt tcagccaggt cttgcatacg gccgccagag cttccacttg gtcaggcagt 23400

agtttgaagt tcgcctttag atcgttatcc acgtggtact tgtccatcag cgcgcgcgca 23460

gcctccatgc ccttctccca cgcagacacg atcggcacac tcagcgggtt catcaccgta 23520

atttcacttt ccgcttcgct gggctcttcc tcttcctctt gcgtccgcat accacgcgcc 23580

actgggtcgt cttcattcag ccgccgcact gtgcgcttac ctcctttgcc atgcttgatt 23640

agcaccggtg ggttgctgaa acccaccatt tgtagcgcca catcttctct ttcttcctcg 23700

ctgtccacga ttacctctgg tgatggcggg cgctcgggct tgggagaagg gcgcttcttt 23760

ttcttcttgg gcgcaatggc caaatccgcc gccgaggtcg atggccgcgg gctgggtgtg 23820

cgcggcacca gcgcgtcttg tgatgagtct tcctcgtcct cggactcgat acgccgcctc 23880

atccgctttt ttgggggcgc ccggggaggc ggcggcgacg gggacgggga cgacacgtcc 23940

tccatggttg ggggacgtcg cgccgcaccg cgtccgcgct cgggggtggt ttcgcgctgc 24000

tcctcttccc gactggccat ttccttctcc tataggcaga aaaagatcat ggagtcagtc 24060

gagaagaagg acagcctaac cgccccctct gagttcgcca ccaccgcctc caccgatgcc 24120

gccaacgcgc ctaccacctt ccccgtcgag gcacccccgc ttgaggagga ggaagtgatt 24180

atcgagcagg acccaggttt tgtaagcgaa gacgacgagg accgctcagt accaacagag 24240

gataaaaagc aagaccagga caacgcagag gcaaacgagg aacaagtcgg gcggggggac 24300

gaaaggcatg gcgactacct agatgtggga gacgacgtgc tgttgaagca tctgcagcgc 24360

cagtgcgcca ttatctgcga cgcgttgcaa gagcgcagcg atgtgcccct cgccatagcg 24420

gatgtcagcc ttgcctacga acgccaccta ttctcaccgc gcgtaccccc caaacgccaa 24480

gaaaacggca catgcgagcc caacccgcgc ctcaacttct accccgtatt tgccgtgcca 24540

gaggtgcttg ccacctatca catctttttc caaaactgca agatacccct atcctgccgt 24600

gccaaccgca gccgagcgga caagcagctg gccttgcggc agggcgctgt catacctgat 24660

atcgcctcgc tcaacgaagt gccaaaaatc tttgagggtc ttggacgcga cgagaagcgc 24720

gcggcaaacg ctctgcaaca ggaaaacagc gaaaatgaaa gtcactctgg agtgttggtg 24780

gaactcgagg gtgacaacgc gcgcctagcc gtactaaaac gcagcatcga ggtcacccac 24840

tttgcctacc cggcacttaa cctacccccc aaggtcatga gcacagtcat gagtgagctg 24900

atcgtgcgcc gtgcgcagcc cctggagagg gatgcaaatt tgcaagaaca aacagaggag 24960

ggcctacccg cagttggcga cgagcagcta gcgcgctggc ttcaaacgcg cgagcctgcc 25020

gacttggagg agcgacgcaa actaatgatg gccgcagtgc tcgttaccgt ggagcttgag 25080

tgcatgcagc ggttctttgc tgacccggag atgcagcgca agctagagga aacattgcac 25140

tacacctttc gacagggcta cgtacgccag gcctgcaaga tctccaacgt ggagctctgc 25200

aacctggtct cctaccttgg aattttgcac gaaaaccgcc ttgggcaaaa cgtgcttcat 25260

tccacgctca agggcgaggc gcgccgcgac tacgtccgcg actgcgttta cttatttcta 25320

tgctacacct ggcagacggc catgggcgtt tggcagcagt gcttggagga gtgcaacctc 25380

aaggagctgc agaaactgct aaagcaaaac ttgaaggacc tatggacggc cttcaacgag 25440

cgctccgtgg ccgcgcacct ggcggacatc attttccccg aacgcctgct taaaaccctg 25500

caacagggtc tgccagactt caccagtcaa agcatgttgc agaactttag gaactttatc 25560

ctagagcgct caggaatctt gcccgccacc tgctgtgcac ttcctagcga ctttgtgccc 25620

attaagtacc gcgaatgccc tccgccgctt tggggccact gctaccttct gcagctagcc 25680

aactaccttg cctaccactc tgacataatg gaagacgtga gcggtgacgg tctactggag 25740

tgtcactgtc gctgcaacct atgcaccccg caccgctccc tggtttgcaa ttcgcagctg 25800

cttaacgaaa gtcaaattat cggtaccttt gagctgcagg gtccctcgcc tgacgaaaag 25860

tccgcggctc cggggttgaa actcactccg gggctgtgga cgtcggctta ccttcgcaaa 25920

tttgtacctg aggactacca cgcccacgag attaggttct acgaagacca atcccgcccg 25980

ccaaatgcgg agcttaccgc ctgcgtcatt acccagggcc acattcttgg ccaattgcaa 26040

gccatcaaca aagcccgcca agagtttctg ctacgaaagg gacggggggt ttacttggac 26100

ccccagtccg gcgaggagct caacccaatc cccccgccgc cgcagcccta tcagcagcag 26160

ccgcgggccc ttgcttccca ggatggcacc caaaaagaag ctgcagctgc cgccgccacc 26220

cacggacgag gaggaatact gggacagtca ggcagaggag gttttggacg aggaggagga 26280

ggacatgatg gaagactggg agagcctaga cgaggaagct tccgaggtcg aagaggtgtc 26340

agacgaaaca ccgtcaccct cggtcgcatt cccctcgccg gcgccccaga aatcggcaac 26400

cggttccagc atggctacaa cctccgctcc tcaggcgccg ccggcactgc ccgttcgccg 26460

acccaaccgt agatgggaca ccactggaac cagggccggt aagtccaagc agccgccgcc 26520

gttagcccaa gagcaacaac agcgccaagg ctaccgctca tggcgcgggc acaagaacgc 26580

catagttgct tgcttgcaag actgtggggg caacatctcc ttcgcccgcc gctttcttct 26640

ctaccatcac ggcgtggcct tcccccgtaa catcctgcat tactaccgtc atctctacag 26700

cccatactgc accggcggca gcggcagcgg cagcaacagc agcggccaca cagaagcaaa 26760

ggcgaccgga tagcaagact ctgacaaagc ccaagaaatc cacagcggcg gcagcagcag 26820

gaggaggagc gctgcgtctg gcgcccaacg aacccgtatc gacccgcgag cttagaaaca 26880

ggatttttcc cactctgtat gctatatttc aacagagcag gggccaagaa caagagctga 26940

aaataaaaaa caggtctctg cgatccctca cccgcagctg cctgtatcac aaaagcgaag 27000

atcagcttcg gcgcacgctg gaagacgcgg aggctctctt cagtaaatac tgcgcgctga 27060

ctcttaagga ctagtttcgc gccctttctc aaatttaagc gcgaaaacta cgtcatctcc 27120

agcggccaca cccggcgcca gcacctgtcg tcagcgccat tatgagcaag gaaattccca 27180

cgccctacat gtggagttac cagccacaaa tgggacttgc ggctggagct gcccaagact 27240

actcaacccg aataaactac atgagcgcgg gaccccacat gatatcccgg gtcaacggaa 27300

tccgcgccca ccgaaaccga attctcttgg aacaggcggc tattaccacc acacctcgta 27360

ataaccttaa tccccgtagt tggcccgctg ccctggtgta ccaggaaagt cccgctccca 27420

ccactgtggt acttcccaga gacgcccagg ccgaagttca gatgactaac tcaggggcgc 27480

agcttgcggg cggctttcgt cacagggtgc ggtcgcccgg gcagggtata actcacctga 27540

caatcagagg gcgaggtatt cagctcaacg acgagtcggt gagctcctcg cttggtctcc 27600

gtccggacgg gacatttcag atcggcggcg ccggccgtcc ttcattcacg cctcgtcagg 27660

caatcctaac tctgcagacc tcgtcctctg agccgcgctc tggaggcatt ggaactctgc 27720

aatttattga ggagtttgtg ccatcggtct actttaaccc cttctcggga cctcccggcc 27780

actatccgga tcaatttatt cctaactttg acgcggtaaa ggactcggcg gacggctacg 27840

actgaatgtt aagtggagag gcagagcaac tgcgcctgaa acacctggtc cactgtcgcc 27900

gccacaagtg ctttgcccgc gactccggtg agttttgcta ctttgaattg cccgaggatc 27960

atatcgaggg cccggcgcac ggcgtccggc ttaccgccca gggagagctt gcccgtagcc 28020

tgattcggga gtttacccag cgccccctgc tagttgagcg ggacagggga ccctgtgttc 28080

tcactgtgat ttgcaactgt cctaaccttg gattacatca agatctttgt tgccatctct 28140

gtgctgagta taataaatac agaaattaaa atatactggg gctcctatcg ccatcctgta 28200

aacgccaccg tcttcacccg cccaagcaaa ccaaggcgaa ccttacctgg tacttttaac 28260

atctctccct ctgtgattta caacagtttc aacccagacg gagtgagtct acgagagaac 28320

ctctccgagc tcagctactc catcagaaaa aacaccaccc tccttacctg ccgggaacgt 28380

acgagtgcgt caccggccgc tgcaccacac ctaccgcctg accgtaaacc agactttttc 28440

cggacagacc tcaataactc tgtttaccag aacaggaggt gagcttagaa aacccttagg 28500

gtattaggcc aaaggcgcag ctactgtggg gtttatgaac aattcaagca actctacggg 28560

ctattctaat tcaggtttct ctagaatcgg ggttggggtt attctctgtc ttgtgattct 28620

ctttattctt atactaacgc ttctctgcct aaggctcgcc gcctgctgtg tgcacatttg 28680

catttattgt cagcttttta aacgctgggg tcgccaccca agatgattag gtacataatc 28740

ctaggtttac tcacccttgc gtcagcccac ggtaccaccc aaaaggtgga ttttaaggag 28800

ccagcctgta atgttacatt cgcagctgaa gctaatgagt gcaccactct tataaaatgc 28860

accacagaac atgaaaagct gcttattcgc cacaaaaaca aaattggcaa gtatgctgtt 28920

tatgctattt ggcagccagg tgacactaca gagtataatg ttacagtttt ccagggtaaa 28980

agtcataaaa cttttatgta tacttttcca ttttatgaaa tgtgcgacat taccatgtac 29040

atgagcaaac agtataagtt gtggccccca caaaattgtg tggaaaacac tggcactttc 29100

tgctgcactg ctatgctaat tacagtgctc gctttggtct gtaccctact ctatattaaa 29160

tacaaaagca gacgcagctt tattgaggaa aagaaaatgc cttaatttac taagttacaa 29220

agctaatgtc accactaact gctttactcg ctgcttgcaa aacaaattca aaaagttagc 29280

attataatta gaataggatt taaacccccc ggtcatttcc tgctcaatac cattcccctg 29340

aacaattgac tctatgtggg atatgctcca gcgctacaac cttgaagtca ggcttcctgg 29400

atgtcagcat ctgactttgg ccagcacctg tcccgcggat ttgttccagt ccaactacag 29460

cgacccaccc taacggcaac cgcagcagcc actcccgctt gggaagaata gaggcagatt 29520

ctggaagtca agaagacatc atccggaata ttgccaggca cctcgcccag gtcggggaca 29580

gcatggaccg tagcatccct ccgggcctgg tgaacggcct ggccctgcag ctcaggaaca 29640

ccagccggtc ggaggaggac cggaacaggg acctggccac tgccctggag cagctgctgc 29700

aggcctaccc tagagacatg gagaaggaga agaccatgct ggtgctggcc ctgctgctgg 29760

ccaagaaggt ggccagtcac acgccgtcct tgctccgtga tgtctttcac acaacagtga 29820

attttattaa ccagaaccta cgcacctacg tgaggagctt agccagaaat gggatggatg 29880

aaatcgattg atggaatcca tagattggac ggactgaaac acatgttctt ttctcttaca 29940

gtatgattaa atgagacatg attcctcgag tttttatatt actgaccctt gttgcgcttt 30000

tttgtgcgtg ctccacattg gctgcggttt ctcacatcga agtagactgc attccagcct 30060

tcacagtcta tttgctttac ggatttgtca ccctcacgct catctgcagc ctcatcactg 30120

tggtcatcgc ctttatccag tgcattgact gggtctgtgt gcgctttgca tatctcagac 30180

accatcccca gtacagggac aggactatag ctgagcttct tagaattctt taattatgaa 30240

atttactgtg acttttctgc tgattatttg caccctatct gcgttttgtt ccccgacctc 30300

caagcctcaa agacatatat catgcagatt cactcgtata tggaatattc caagttgcta 30360

caatgaaaaa agcgatcttt ccgaagcctg gttatatgca atcatctctg ttatggtgtt 30420

ctgcagtacc atcttagccc tagctatata tccctacctt gacattggct ggaaacgaat 30480

agatgccatg aaccacccaa ctttccccgc gcccgctatg cttccactgc aacaagttgt 30540

tgccggcggc tttgtcccag ccaatcagcc tcgccccact tctcccaccc ccactgaaat 30600

cagctacttt aatctaacag gaggagatga ctgacaccct agatctagaa atggacggaa 30660

ttattacaga gcagcgcctg ctagaaagac gcagggcagc ggccgagcaa cagcgcatga 30720

atcaagagct ccaagacatg gttaacttgc accagtgcaa aaggggtatc ttttgtctgg 30780

taaagcaggc caaagtcacc tacgacagta ataccaccgg acaccgcctt agctacaagt 30840

tgccaaccaa gcgtcagaaa ttggtggtca tggtgggaga aaagcccatt accataactc 30900

agcactcggt agaaaccgaa ggctgcattc actcaccttg tcaaggacct gaggatctct 30960

gcacccttat taagaccctg tgcggtctca aagatcttat tccctttaac taataaaaaa 31020

aaataataaa gcatcactta cttaaaatca gttagcaaat ttctgtccag tttattcagc 31080

agcacctcct tgccctcctc ccagctctgg tattgcagct tcctcctggc tgcaaacttt 31140

ctccacaatc taaatggaat gtcagtttcc tcctgttcct gtccatccgc acccactatc 31200

ttcatgttgt tgcagatgaa gcgcgcaaga ccgtctgaag ataccttcaa ccccgtgtat 31260

ccatatgaca cggaaaccgg tcctccaact gtgccttttc ttactcctcc ctttgtatcc 31320

cccaatgggt ttcaagagag tccccctggg gtactctctt tgcgcctatc cgaacctcta 31380

gttacctcca atggcatgct tgcgctcaaa atgggcaacg gcctctctct ggacgaggcc 31440

ggcaacctta cctcccaaaa tgtaaccact gtgagcccac ctctcaaaaa aaccaagtca 31500

aacataaacc tggaaatatc tgcacccctc acagttacct cagaagccct aactgtggct 31560

gccgccgcac ctctaatggt cgcgggcaac acactcacca tgcaatcaca ggccccgcta 31620

accgtgcacg actccaaact tagcattgcc acccaaggac ccctcacagt gtcagaagga 31680

aagctagccc tgcaaacatc aggccccctc accaccaccg atagcagtac ccttactatc 31740

actgcctcac cccctctaac tactgccact ggtagcttgg gcattgactt gaaagagccc 31800

atttatacac aaaatggaaa actaggacta aagtacgggg ctcctttgca tgtaacagac 31860

gacctaaaca ctttgaccgt agcaactggt ccaggtgtga ctattaataa tacttccttg 31920

caaactaaag ttactggagc cttgggtttt gattcacaag gcaatatgca acttaatgta 31980

gcaggaggac taaggattga ttctcaaaac agacgcctta tacttgatgt tagttatccg 32040

tttgatgctc aaaaccaact aaatctaaga ctaggacagg gccctctttt tataaactca 32100

gcccacaact tggatattaa ctacaacaaa ggcctttact tgtttacagc ttcaaacaat 32160

tccaaaaagc ttgaggttaa cctaagcact gccaaggggt tgatgtttga cgctacagcc 32220

atagccatta atgcaggaga tgggcttgaa tttggttcac ctaatgcacc aaacacaaat 32280

cccctcaaaa caaaaattgg ccatggccta gaatttgatt caaacaaggc tatggttcct 32340

aaactaggaa ctggccttag ttttgacagc acaggtgcca ttacagtagg aaacaaaaat 32400

aatgataagc taactttgtg gaccacacca gctccatctc ctaactgtag actaaatgca 32460

gagaaagatg ctaaactcac tttggtctta acaaaatgtg gcagtcaaat acttgctaca 32520

gtttcagttt tggctgttaa aggcagtttg gctccaatat ctggaacagt tcaaagtgct 32580

catcttatta taagatttga cgaaaatgga gtgctactaa acaattcctt cctggaccca 32640

gaatattgga actttagaaa tggagatctt actgaaggca cagcctatac aaacgctgtt 32700

ggatttatgc ctaacctatc agcttatcca aaatctcacg gtaaaactgc caaaagtaac 32760

attgtcagtc aagtttactt aaacggagac aaaactaaac ctgtaacact aaccattaca 32820

ctaaacggta cacaggaaac aggagacaca actccaagtg catactctat gtcattttca 32880

tgggactggt ctggccacaa ctacattaat gaaatatttg ccacatcctc ttacactttt 32940

tcatacattg cccaagaata aagaatcgtt tgtgttatgt ttcaacgtgt ttatttttca 33000

attgcagaaa atttcaagtc atttttcatt cagtagtata gccccaccac cacatagctt 33060

atacagatca ccgtacctta atcaaactca cagaacccta gtattcaacc tgccacctcc 33120

ctcccaacac acagagtaca cagtcctttc tccccggctg gccttaaaaa gcatcatatc 33180

atgggtaaca gacatattct taggtgttat attccacacg gtttcctgtc gagccaaacg 33240

ctcatcagtg atattaataa actccccggg cagctcactt aagttcatgt cgctgtccag 33300

ctgctgagcc acaggctgct gtccaacttg cggttgctta acgggcggcg aaggagaagt 33360

ccacgcctac atgggggtag agtcataatc gtgcatcagg atagggcggt ggtgctgcag 33420

cagcgcgcga ataaactgct gccgccgccg ctccgtcctg caggaataca acatggcagt 33480

ggtctcctca gcgatgattc gcaccgcccg cagcataagg cgccttgtcc tccgggcaca 33540

gcagcgcacc ctgatctcac ttaaatcagc acagtaactg cagcacagca ccacaatatt 33600

gttcaaaatc ccacagtgca aggcgctgta tccaaagctc atggcgggga ccacagaacc 33660

cacgtggcca tcataccaca agcgcaggta gattaagtgg cgacccctca taaacacgct 33720

ggacataaac attacctctt ttggcatgtt gtaattcacc acctcccggt accatataaa 33780

cctctgatta aacatggcgc catccaccac catcctaaac cagctggcca aaacctgccc 33840

gccggctata cactgcaggg aaccgggact ggaacaatga cagtggagag cccaggactc 33900

gtaaccatgg atcatcatgc tcgtcatgat atcaatgttg gcacaacaca ggcacacgtg 33960

catacacttc ctcaggatta caagctcctc ccgcgttaga accatatccc agggaacaac 34020

ccattcctga atcagcgtaa atcccacact gcagggaaga cctcgcacgt aactcacgtt 34080

gtgcattgtc aaagtgttac attcgggcag cagcggatga tcctccagta tggtagcgcg 34140

ggtttctgtc tcaaaaggag gtagacgatc cctactgtac ggagtgcgcc gagacaaccg 34200

agatcgtgtt ggtcgtagtg tcatgccaaa tggaacgccg gacgtagtca tatttcctga 34260

agcaaaacca ggtgcgggcg tgacaaacag atctgcgtct ccggtctcgc cgcttagatc 34320

gctctgtgta gtagttgtag tatatccact ctctcaaagc atccaggcgc cccctggctt 34380

cgggttctat gtaaactcct tcatgcgccg ctgccctgat aacatccacc accgcagaat 34440

aagccacacc cagccaacct acacattcgt tctgcgagtc acacacggga ggagcgggaa 34500

gagctggaag aaccatgttt ttttttttat tccaaaagat tatccaaaac ctcaaaatga 34560

agatctatta agtgaacgcg ctcccctccg gtggcgtggt caaactctac agccaaagaa 34620

cagataatgg catttgtaag atgttgcaca atggcttcca aaaggcaaac ggccctcacg 34680

tccaagtgga cgtaaaggct aaacccttca gggtgaatct cctctataaa cattccagca 34740

ccttcaacca tgcccaaata attctcatct cgccaccttc tcaatatatc tctaagcaaa 34800

tcccgaatat taagtccggc cattgtaaaa atctgctcca gagcgccctc caccttcagc 34860

ctcaagcagc gaatcatgat tgcaaaaatt caggttcctc acagacctgt ataagattca 34920

aaagcggaac attaacaaaa ataccgcgat cccgtaggtc ccttcgcagg gccagctgaa 34980

cataatcgtg caggtctgca cggaccagcg cggccacttc cccgccagga accatgacaa 35040

aagaacccac actgattatg acacgcatac tcggagctat gctaaccagc gtagccccga 35100

tgtaagcttg ttgcatgggc ggcgatataa aatgcaaggt gctgctcaaa aaatcaggca 35160

aagcctcgcg caaaaaagaa agcacatcgt agtcatgctc atgcagataa aggcaggtaa 35220

gctccggaac caccacagaa aaagacacca tttttctctc aaacatgtct gcgggtttct 35280

gcataaacac aaaataaaat aacaaaaaaa catttaaaca ttagaagcct gtcttacaac 35340

aggaaaaaca acccttataa gcataagacg gactacggcc atgccggcgt gaccgtaaaa 35400

aaactggtca ccgtgattaa aaagcaccac cgacagctcc tcggtcatgt ccggagtcat 35460

aatgtaagac tcggtaaaca catcaggttg attcacatcg gtcagtgcta aaaagcgacc 35520

gaaatagccc gggggaatac atacccgcag gcgtagagac aacattacag cccccatagg 35580

aggtataaca aaattaatag gagagaaaaa cacataaaca cctgaaaaac cctcctgcct 35640

aggcaaaata gcaccctccc gctccagaac aacatacagc gcttccacag cggcagccat 35700

aacagtcagc cttaccagta aaaaagaaaa cctattaaaa aaacaccact cgacacggca 35760

ccagctcaat cagtcacagt gtaaaaaagg gccaagtgca gagcgagtat atataggact 35820

aaaaaatgac gtaacggtta aagtccacaa aaaacaccca gaaaaccgca cgcgaaccta 35880

cgcccagaaa cgaaagccaa aaaacccaca acttcctcaa atcgtcactt ccgttttccc 35940

acgttacgta acttcccatt ttaagaaaac tacaattccc aacacataca agttactccg 36000

ccctaaaacc tacgtcaccc gccccgttcc cacgccccgc gccacgtcac aaactccacc 36060

ccctcattat catattggct tcaatccaaa ataaggtata ttattgatga tg 36112

Claims

1. An oncolytic adenovirus recombinant carrying TMVP1 and tBId is Ad 5/delta 27/TMVP 1/delta ADP-tBId, the nucleotide sequence of which is shown in SEQ ID NO.23, the oncolytic adenovirus recombinant carrying TMVP1 and tBId is characterized in that 27 bases shown in SEQ ID NO.1 in the 920nt-946nt region are deleted in the E1A conserved sequence 2 region of the human type 5 adenovirus gene, the gene sequence coding tumor targeting peptide TMVP1 shown in SEQ ID NO.15 is inserted in the 19641nt-19655nt region of the Hexon hypervariable region 5, the E3 region is deleted in the 29477nt-29714nt region of the ADP gene to form a deletion region, the gene sequence of mitochondrial apoptosis peptide tBId shown in SEQ ID NO.22 is inserted in the deletion region, and the enzyme cutting site of Cla1 is introduced.

2. The method for constructing an oncolytic adenoviral recombinant carrying TMVP1 and tBId according to claim 1, comprising the steps of:

step 1: targeted deletion of human adenovirus type 5 genes

step 2: preparation of Ad 5/. DELTA.27/TMVP 1

3. Use of the oncolytic adenoviral recombinant carrying TMVP1 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 TMVP1 and tBid according to claim 1 for the preparation of a gene therapy vector.

5. Use of the oncolytic adenoviral recombinant carrying TMVP1 and tBid according to claim 1 for the preparation of a medicament for improving resistance to anti-tumor chemotherapeutic drugs.

6. The use of the oncolytic adenoviral recombinant carrying TMVP1 and tBid according to claim 1 for the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.