CN114262692A - Oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and construction method and application thereof - Google Patents
Oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, a construction method and application thereof, and belongs to the technical field of medical genetic engineering. The invention deletes 27 basic groups of 920nt-946nt region in E1A conservative sequence 2 region of human 5 type adenovirus gene, inserts gene sequence coding tumor targeting peptide TMVP1 in 19641nt-19655nt region of Hexon hypervariable region 5, simultaneously deletes E3 region in 29477nt-29714nt region of ADP gene to form deletion region, inserts full length coding sequence coding HSV-TK gene in the deletion region and introduces Cla1 enzyme cutting site. The invention also discloses a construction method and application of the oncolytic adenovirus recombinant. The oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has ideal targeting effect and strong killing effect.
Description
Technical Field
The invention relates to an oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, a construction method and application thereof, and belongs to the technical field of medical genetic engineering.
Background
Gene therapy (gene therapy) refers to the introduction of foreign genes into target cells to correct or compensate for diseases caused by gene defects or abnormal expression of genes. As a gene therapy vector, the development prospect of the oncolytic virus for treating malignant tumor is good. Among the numerous vectors for oncolytic viral therapy, recombinant adenoviral vectors are most widely used and have been recognized for their clinical feasibility and safety. The adenovirus gene therapy vector has the following biological advantages and clinical application advantages: (1) the infection spectrum is wide, the cell which is in the division stage or the stationary stage can be effectively attacked by various tissue-derived cells, and the anti-cancer spectrum is wide; (2) after the adenovirus enters cells, the adenovirus is not integrated into host chromosomes, so that the risk of mutation and carcinogenesis is avoided, the clinical application is safe, only cold-like symptoms are generated after the adenovirus is used, and the suppression of hematopoietic function and immunologic function is avoided; (3) the clinical application is convenient, and the oral cavity (such as abdominal cavity, thoracic cavity and cranial cavity) administration, local or tumor in-vivo direct injection (one point or a plurality of points), interventional therapy and other ways are provided; (4) the adenovirus can be continuously expressed in vivo for only two to three weeks, and is particularly suitable for tumor treatment; (5) intravenous or topical application produces only a mild inflammatory response with minor side effects; (6) clinical grade quantities of adenovirus are easy to produce and purify. Based on the advantages, more and more adenovirus vectors are constructed and generated, and the good clinical application prospect is shown.
Although adenovirus has obvious advantages compared with other various gene therapy vectors, the existing adenovirus therapy vectors at home and abroad gradually make some breakthrough progress, but still have some insurmountable defects to limit the wide application of the adenovirus, which are specifically as follows: (1) liver tropism. After the adenovirus enters an organism, no matter intravenous systemic injection or intratumoral injection, local injection in thoracic cavity, abdominal cavity and the like, in the organism, the coagulation factor ten (Fx) can rapidly recognize adenovirus capsid protein Hexon, is combined with the adenovirus capsid protein Hexon and brings the adenovirus to the liver, the other side of the coagulation factor is combined with Heparan Sulfate proteoglycan (HSPGS for short) on the surface of the hepatocyte, namely, the coagulation factor forms a bridge between the adenovirus and the hepatocyte, and the adenovirus captured by the liver is gradually phagocytized by kupffer cells or macrophages in the liver. The unremoved adenovirus is largely replicated in the liver, resulting in acute liver injury, manifested by a rapid rise in transaminase. (2) Adenovirus infects tumor cells, depending on the Coxsackie Adenovirus Receptor (CAR) on the surface of the tumor cell. In most tumor cells, the receptor is in a low expression state, which is not beneficial to the adenovirus entering the tumor cells to play a killing role.
In summary, there are various drawbacks in the current tumor treatment technology and practice, and therefore, there is a need to provide a new therapeutic application approach with both targeting and potent killing, so as to solve the deficiencies in the prior art.
Disclosure of Invention
One of the purposes of the present invention is to provide an oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK.
The technical scheme for solving the problems is as follows: an oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is Ad 5/delta E1A/TMVP1 delta ADP/HSV-TK, the nucleotide sequence of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is shown as SEQ ID No.23, 27 bases shown as SEQ ID No.1 in a 920nt-946nt region are deleted in an E1A conserved sequence 2 region of a human type 5 adenovirus gene, a gene sequence shown as SEQ ID No.15 and used for coding tumor targeting peptide TMVP1 is inserted in a 19641nt-19655nt region of a Hexon hypervariable region 5, the E3 region is deleted in a 29477nt-29714nt region of the ADP gene to form a deletion region, the full-length coding sequence shown as SEQ ID No.22 and used for coding the HSV-TK gene is inserted in the deletion region, and a Cla1 site is introduced.
The inventors of the present application, in order to obtain the above oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, performed the following work:
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 in early stage, being selectively swallowed by the tumor cells, having strong tumor cell toxic effect and the like. The invention inserts the gene sequence of the coding tumor targeting peptide TMVP1 shown as SEQ ID NO.15 into the human type 5 adenovirus, thereby obviously improving the targeting of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK.
HSV-TK gene and herpes simplex virus thymidine kinase gene are common suicide gene, and the thymidine kinase coded by the gene can convert the drug precursor which is nontoxic or has low toxicity to cells into strong cytotoxic substances so as to achieve the aim of killing tumor cells. The invention inserts the full-length coding sequence of the coding HSV-TK gene shown in SEQ ID NO.22 into the human type 5 adenovirus (Ad5), so that the HSV-TK gene is specifically expressed in the tumor, and the tumor cell apoptosis is promoted, thereby remarkably improving the apoptosis promotion effect of an oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK and the killing effect of an oncolytic virus cytolytic cell.
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 HSV-TK can specifically identify tumors and metastasis thereof, has higher tumor targeted replication capacity, utilizes the dual killing effect of the apoptosis promotion effect of HSV-TK gene and the oncolytic virus lytic cell, 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 HSV-TK has the beneficial effects that:
1. the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has strong selectivity of tumor selective replication and therapeutic gene expression;
2. the yield of the progeny oncolytic adenovirus in the tumor cell is high, a high-concentration virus treatment ring can be generated at the local part of the tumor after the cell is cracked, and a strong bystander effect can be formed by combining the advantages of treatment targets;
3. oncolytic adenovirus in tumor cells is efficiently amplified in a large amount, and simultaneously, a large amount of target therapeutic genes are transcribed, so that the tumor cells become a processing plant for synthesizing therapeutic proteins;
4. the immunoregulatory protein of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has complete functions, and the elimination of recombinant adenovirus by an organism is avoided to a certain extent;
5. the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has wide anticancer spectrum. The in vivo model research of the tumor animal model proves that the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has obvious treatment effect on all tested tumor models through the administration way of local tumor injection or intraperitoneal injection, can effectively inhibit tumor metastasis, and has no obvious treatment-related toxicity on treated animals.
The second purpose of the invention is to provide a method for constructing the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK.
The technical scheme for solving the problems is as follows: the construction method of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK comprises the following steps:
step 1: targeted deletion of human adenovirus type 5 genes
Utilizing gene synthesis and homologous recombination to directionally delete 27 bases of 920nt-946nt in the E1A conserved sequence 2 region of the human 5-type adenovirus gene as shown in SEQ ID NO.1 to obtain the directionally deleted human 5-type adenovirus gene Ad 5/delta E1A;
step 2: preparation of Ad 5/. DELTA.E 1A/TMVP1
Inserting a gene sequence which is shown as SEQ ID NO.15 and encodes tumor targeting peptide TMVP1 into the 19641nt-19655nt region of the Hexon hypervariable region 5 of the human adenovirus type 5 gene obtained in the step 1 after targeted deletion to obtain Ad 5/delta E1A/TMVP 1;
and step 3: preparation of oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMVP 1. DELTA.ADP/HSV-TK carrying TMVP1 and HSV-TK
The E3 region of Ad 5/delta E1A/TMVP1 obtained in step 2 is located in the 29477nt-29714nt region of the ADP gene to form a deletion region, the full-length coding sequence of the HSV-TK gene shown in SEQ ID NO.22 is inserted into the deletion region, and the Cla1 enzyme cutting site is introduced, so that the oncolytic adenovirus recombinant Ad 5/delta E1A/TMVP1 delta ADP/HSV-TK carrying the TMVP1 and the HSV-TK shown in SEQ ID NO.23 is obtained.
The construction method of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has the beneficial effects that:
1. the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK constructed by the invention is inserted with a gene sequence coding tumor targeting peptide TMVP1 and a full-length coding sequence coding HSV-TK gene, has the advantages of tumor specific replication, high tumor and metastatic tumor targeting property, specific mass expression of exogenous therapeutic genes, strong tumor specific bystander effect and the like, has high therapeutic index, can solve the key defects in the current tumor therapy technology and practice, and provides an ideal therapeutic application path with targeting and strong killing for tumor therapy.
2. The construction method is simple, easy to operate, low in cost and wide in application prospect.
The third object of the present invention is to provide the use of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK described above.
The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is applied to the preparation of medicines for treating tumors.
The application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has the beneficial effects that:
the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK can be used for preparing medicaments for treating tumors, develops a novel medicament for treating tumors and the application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.
Another object of the present invention is to provide another use of the oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK described above.
The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is applied to the preparation of a gene therapy vector.
The application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has the beneficial effects that:
the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK can be used for preparing gene therapy vectors, not only develops a new gene therapy vector, but also develops the application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.
Another object of the present invention is to provide another use of the oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK described above.
The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is applied to preparation of drugs for improving drug resistance of antitumor chemotherapeutic drugs.
The application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has the beneficial effects that:
the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK can be used for preparing gene therapy vectors, not only develops new drugs for improving drug resistance of antitumor chemotherapeutic drugs, but also develops application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.
Another object of the present invention is to provide another use of the oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK described above.
The technical scheme for solving the problems is as follows: the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is applied to the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.
The application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK has the beneficial effects that:
the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK can be used for preparing gene therapy vectors, develops a new anti-tumor chemotherapeutic drug sensitizer and the application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.
Drawings
FIG. 1 is a structural schematic of oncolytic adenovirus recombinant oncolytic adenovirus Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK carrying TMVP1 and HSV-TK of the present example.
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 graph showing tumor growth in mice in different treatment groups in example 6 of the present invention.
FIG. 4 shows the adenovirus content in each tissue in example 7 of the present invention.
Detailed Description
The principles and features of this invention are described below in conjunction with the following detailed drawings, which are given by way of illustration only and are not intended to limit the scope of the invention.
As shown in figure 1, the invention adopts an adenovirus recombination system AdEasy to construct the target adenovirus by a three-time homologous recombination method. Unless otherwise noted, the enzymes used in the present invention were purchased from gibco, usa, PCR primers were synthesized from biotechnology limited, beijing optimak, and cell lines were purchased from ATCC cell bank, usa, and were cultured using corresponding media, which were purchased from gibco, usa.
Example 1: construction of pAd5/Δ E1A adenovirus packaging plasmid
Step 1.1: construction of shuttle plasmid Blunt-Zero-E1A/Δ E1A
The pXC 1-delta E1A plasmid is 27 bases shown in SEQ ID NO.1 in the 920nt-946nt region deleted in the E1A conserved sequence 2 region of the human adenovirus 5 gene. The construction method comprises the following steps:
the pXC1 plasmid was purchased from 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 E1A with deletion of 121-plus 129AA 920nt-946nt through DNA sequencing, identification and screening.
Taking pXC 1-delta E1A plasmid as a template, and carrying out PCR amplification reaction to obtain an E1A sequence with 27bp deletion. Wherein, the upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQ ID NO.6), downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO. 7). The PCR amplification reaction system is as follows: TransStart FastPfu Fly DNA Polymerase, 1. mu.l; 5 × TransStart FastPfu Fly Buffer, 10 μ l; 10. mu.M of the forward primer, 1. mu.l; 10. mu.M of downstream primer, 1. mu.l; 2.5mM dNTPs, 4. mu.l; adding nuclease-free water to make up the system to 50 mu l; template, 10-30 ng; a total of 50. mu.l was obtained. The procedure of PCR amplification reaction is 95 ℃ for 2 min; at 95 ℃, 20s, -5 ℃, 20s, 72 ℃, 3min, 35 cycles; 72 ℃ for 5 min; and preserving at 4 ℃.
The E1A sequence was ligated into pEASY-Blunt-Zero Blunt-end ligation plasmid (available from Beijing Quanjin Biotechnology Co., Ltd., product No. CB501) by T4 ligase (available from Saimei Feishell technology Co., Ltd., product No. el0011) to obtain shuttle plasmid Blunt-Zero-E1A/delta E1A.
And (3) carrying out PCR amplification reaction by using a shuttle plasmid Blunt-Zero-E1A/delta E1A as a template and adopting a primer carried in the pEASY-Blunt-Zero Blunt end ligation plasmid kit to obtain an E1A shuttle fragment with 27bp deletion, and purifying the shuttle fragment for homologous recombination. The PCR reaction system and the amplification reaction procedure were as described above.
Step 1.2: construction of backbone plasmid pAd-Easy-1
The pAd-Easy-1 vector (available from Agilent technologies, Inc., catalog number 240005) was cleaved with restriction enzyme pme1, and the DNA cleavage product was extracted with phenol chloroform to obtain a backbone fragment for the first homologous recombination, and the DNA concentrations of the shuttle fragment and the backbone fragment were determined.
Step 1.3: preparation of Escherichia coli BJ5183 electroconception
Escherichia coli BJ5183 (purchased from Beijing Ke Rui Si Bing Biotechnology Ltd, catalog number st10779) stored at-80 deg.C is thawed, and 1mL of the bacterial liquid is added into 1000mL of LB medium with streptomycin concentration of 30. mu.g/mL. After the bacteria are shaken at the speed of 250rpm/min and the temperature of 37 ℃ for 7h to 8h, the liquid is slightly turbid after observation every 10 min. Subpackaging the bacterial liquid into precooled centrifuge tubes, centrifuging at 4 ℃ and 3000rpm/min for 10min, and discarding the supernatant. And (4) precooling a proper amount of sterile double-distilled water washing bacterial residues twice. Precooling a proper amount of sterile 10% glycerol bacterial washing residues twice. Discarding the supernatant to obtain Escherichia coli BJ5183 electrotransformation competence, and subpackaging in 1.5mL centrifuge tubes for preservation at-80 deg.C.
Step 1.4: homologous recombination construction of pAd 5-E1A/delta E1A adenovirus packaging plasmid
The E1A shuttle fragment obtained in step 1.1 and the backbone fragment obtained in step 1.2 were electroporated into E.coli BJ5183 electroporation competence obtained in step 1.3 using a GenePulser XcellTM electroporation apparatus (available from Bio-Rad) under conditions of 2.5Kv and 25. mu.F. Positive bacteria were screened for kanamycin resistance. Selecting smaller bacterial plaques in bacterial plates, culturing in LB culture medium, extracting plasmids in small quantity, and using an upstream primer: 5'-ttaattaacatcatcaataatataccttatt-3' (SEQ ID NO.8) and the downstream primer: 5'-gatccacataatctaacacaaactc-3' (SEQ ID NO.9), performing PCR amplification reaction, performing the same reaction system and procedure as step 1.1, and sequencing to determine whether the E1A region lacks the region of 920bp-946 bp. The extracted plasmid was transformed into high copy number bacterium DH10B (purchased from Saimer Feishell science Inc., cat # 18290015) to obtain pAd 5-E1A/. DELTA.E 1A adenovirus packaging plasmid, abbreviated pAd 5/. DELTA.E 1A.
Example 2: construction of pAd 5/. DELTA.E 1A/Hexon (HVR/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, plaque development,6-12 plaques were selected. The plaques were transferred to serum-free DMEM medium in 1.5ml EP tubes and incubated at 37 ℃ for 24 h. Seeding in 24-well plates 1X 105293 cells in 10% FBS DMEM, by day two, cells should be 2X 105About 70% of the cells were fused, the liquid was aspirated, and 100. mu.l (about 10) of the above-mentioned incubation liquid was taken3Virus) was added and the liquid was gently shaken 3 times at 37 ℃ with 5% CO2And (5) performing medium incubation for 90 min. Add complete medium to 1ml and place cells at 37 ℃ with 5% CO2And (4) incubating for 5-10 days until complete CPE appears, namely cytotoxic effect, and the cells are rounded and float and mainly comprise nucleolus. If after 10d complete CPE was not present, it was suggested that the virus titer was too low and a second round of amplification was required. Subjecting the plate to three cycles of freezing/thawing to release virus, collecting lysate in a 15ml tube, centrifuging at maximum speed for 10min, collecting supernatant, freezing at-80 deg.C, and collecting the supernatant as second generation virus (about 5 × 10)7Viral/ml. 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 deg.C、5％CO2And (5) performing medium incubation for 90 min. Adding complete medium to 1ml, and placing the cells at 37 deg.C and 5% CO2The cells were scraped and collected in a 1.5ml EP tube, the supernatant was discarded by centrifugation, 300. mu.l PBS was added and three cycles of freeze/thaw were performed to release the virus, the lysate was centrifuged at maximum speed for 10min, the supernatant was collected and frozen at-80 ℃ and DNA was extracted using Qiagen kit mini DNA isolation kit, according to the kit instructions. Performing PCR reaction by using virus DNA as a template, wherein an upstream primer: 5'-cgggatccgggcccccatttcc-3' (SEQ ID NO 10), downstream primer: 5'-tgctctagacacaggtgatgtcg-3' (SEQ ID NO 11), the total reaction system volume of 100. mu.l comprising: containing MgCl 210. mu.l of the buffer solution for PCR of (1); 2mM dNTP, 10. mu.l; 10. mu.M of the forward primer, 1. mu.l; 10. mu.M of downstream primer, 1. mu.l; viral DNA, 10 ng; pfu high fidelity Taq enzyme, 2.5 u; water was added to 100. mu.l. The reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 45 s; 60 ℃ for 1 min; 72 ℃ for 2 min; 28 cycles in total; extension at 72 ℃ for 10 min. The PCR product is 1400bp, 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 fragment was ligated Blunt-ended into pEASY-Blunt-Zero vector (purchased 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 E1A (OMEGA, product number D6692-01) constructed in the step 1.4, utilizing a specific enzyme cutting site AsisI, carrying out enzyme cutting, and then extracting phenol and chloroform to obtain a linear skeleton plasmid.
Step 2.3: the PCR product of pEASY-Hexon (HVR/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 E1A-Hexon (HVR2/TMVP1) plasmid, pAd 5-E1A/delta E1A-Hexon (HVR5/TMVP1) plasmid and pAd 5-E1A/delta E1A-Hexon (HVR7/TMVP1) plasmid are identified.
Example 3: construction of Ad 5/delta E1A/TMVP1/HSV-TK adenovirus vector
Step 3.1 construction of shuttle vector for adenovirus E3 region
PCR was performed using adenovirus Δ 920-946Ad5 as template (described above, including the entire sequence of adenovirus E3 region), with the upstream primer: 5'-tgtcaccactaactgctttactcg-3' (SEQ ID NO 16), downstream primer: 5'-gctgccctgcgtctttcta-3' (SEQ ID NO 17), and a 26342-31140 fragment (i.e., the entire fragment of the E3 region) was obtained and ligated into the pEASY-Blunt-Zero vector to obtain the pEASY-Blunt-Zero-E3 plasmid.
Step 3.2: plasmid pcDNA3.1-E3/Δ ADP is a backbone plasmid, which has a fragment with EcoRI cleavage sites at both ends, into which the complete adenovirus E3 region is inserted but from which a 29477bp-29714bp (the fragment between the two EcoRI cleavage sites of the adenovirus, i.e., the ADP region) fragment is deleted. The construction method comprises the following steps:
the Ad 5E 3 region 29477-29714nt was deleted by 3 PCR methods. Acquisition of fragment 1: primer 1: 5'-atacgcgcccaccgaaac-3' (SEQ ID NO 18), corresponding to 27306nt-27323 nt; primer 2: 5' -aatctatgg atatcgatagggtgggtcgctgtagtt-3' (SEQ ID NO 19), corresponding to 29477-495 nt, the underlined part is the complementary part to primer 3, atcgat is the Cla I cleavage site; carrying out PCR reaction by using Ad5 DNA as a template, wherein the total volume of the reaction system is 100 μ l and comprises: containing MgCl 210. mu.l of the buffer solution for PCR of (1); 2mM dNTP, 10. mu.l; 10 μ M primer 1, 1 μ l; 10 μ M primer 2, 1 μ l; ad5 DNA 200 ng/. mu.l, 1. mu.l; pfu high fidelity Taq enzyme, 2.5 u; water was added to 100. mu.l. The reaction conditions are as follows: 30s at 94 ℃; 30s at 94 ℃; at 46 ℃ for 1 min; 72 ℃ for 1 min; 30 cycles in total; extension at 72 ℃ for 10 min. The PCR product is 2207bp (fragment 1), and after separation and purification by conventional electrophoresis, the concentration is detected and used for subsequent PCR reaction.
Acquisition of fragment 2: primer 3: 5' -cgacccaccctatcgatatccatagattggacggactg-3' (SEQ ID NO 20), corresponding to 29714nt-29734n, the underlined part being the complementary part to primer 2, atcgat being the Cla I cleavage site; primer 4: 5'-atgtctttgaggcttggagg-3' (SEQ ID NO 21), corresponding to 30137nt-30118 nt; the PCR reaction conditions are the same as above, 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 full-length coding sequence of the HSV-TK gene shown in SEQ ID NO.22 is introduced into the Cla1 enzyme cutting site. ClaI enzyme digestion is connected into pcDNA3.1-E3/delta ADP plasmid.
Step 3.3: and (3) carrying out EcoRI digestion on the plasmid constructed in the step (3.2), connecting the plasmid into the pEASY-Blunt-Zero-E3 plasmid constructed in the step (3.1), and carrying out PCR amplification reaction by using a primer carried in the pEASY-Blunt-Zero-E3 plasmid kit to obtain an E3/HSV-TK fragment.
Step 3.4: SpeI digestion of pAd 5-E1A/. DELTA.E 1A-Hexon (HVR2/TMVP1), pAd 5-E1A/. DELTA.E 1A-Hexon (HVR5/TMVP1) and pAd 5-E1A/. DELTA.E 1A-Hexon (HVR7/TMVP1) plasmids constructed in step 2.3, phenol chloroform extraction and recovery of linear backbone plasmids.
The linear backbone plasmid and the E3/HSV-TK fragment obtained in step 3.3 are electrotransferred into Escherichia coli BJ5183 electrotransferred and sensitive peptide obtained in step 1.3, the electrotransferred condition is 2.5Kv and 25 muF, and homologous recombination is carried out to respectively obtain pAd 5/delta 27/HVR2-TMVP 1/delta ADP/HSV-TK plasmid, pAd 5/delta 27/HVR5-TMVP 1/delta ADP/HSV-TK plasmid and pAd 5/delta 27/HVR7-TMVP 1/delta ADP/HSV-TK plasmid.
Example 4: obtaining, amplifying and purifying oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP/HSV-TK carrying TMVP1 and HSV-TK
Step 4.1: obtaining oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP/HSV-TK carrying TMVP1 and HSV-TK
Step 4.1.1: respectively extracting the pAd 5/delta 27/HVR2-TMVP 1/delta ADP/HSV-TK plasmid, pAd 5/delta 27/HVR5-TMVP 1/delta ADP/HSV-TK plasmid and pAd 5/delta 27/HVR7-TMVP 1/delta ADP/HSV-TK plasmid obtained in the step 3.4 in a large quantity, using PacI to enzyme-cut the plasmids, extracting with phenol and chloroform, and recovering enzyme-cut products.
Step 4.1.2: the product recovered in step 4.1.1 was transferred to 293 cells 24-well plates with a confluency of 30% using lipo3000 liposome (purchased from seimer feishell technologies, cat # L3000001) transfection method. Adding fresh DMEM culture medium every 2 days until the fusion degree of 293 cells reaches more than 100%, collecting the cells, repeatedly freezing and thawing to crack and release the virus into the DMEM culture medium, and centrifuging to collect supernatant virus liquid.
Step 4.1.3: transfecting the 293 cell again by adopting a DMEM medium containing the adenovirus, repeating for about 2 weeks until the 293 cell has a CPE effect, collecting the cell, and repeatedly freezing and thawing for lysis to obtain a supernatant virus solution.
Step 4.1.4: three Ad 5/. DELTA.27/TMVP 1/. DELTA.ADP/HSV-TKs obtained in step 4.1.3 were identified, and E1A, Hexon and E3/ADP regions were sequenced after PCR using the corresponding primers, respectively.
Step 4.2: amplification, purification and titer determination of oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP/HSV-TK carrying TMVP1 and HSV-TK
Step 4.2.1: amplification step 4.1.4 sequencing the correct oncolytic adenovirus recombinant Ad 5/delta 27/TMVP 1/delta ADP/HSV-TK carrying TMVP1 and HSV-TK, detecting the titer (MOI) of adenovirus seeds, determining the virus amount infecting 293 cells according to the MOI value, and collecting the cells when CPE effect appears within 48h-72 h. The amplification is repeated until the virus amount reaches the experimental requirement (10)9pfu/ml-1011pfu/ml)。
Step 4.2.2: the adenovirus is purified by the traditional CsCl gradient centrifugation method, and the purified adenovirus is obtained by the dialysis of virus dialysate. Virus particle counts were measured using the UV absorbance method and MOI was measured using the Adeno-XTM Rapid Titer kit (Clonetech, cat # 632250).
Example 5: selection of optimal insertion region for oncolytic adenovirus recombinants carrying TMVP1 and HSV-TK
The purpose of this example was to determine the efficiency of oncolytic adenoviral recombinants carrying TMVP1 and HSV-TK inserted into three different regions of the hypervariable region 2, 5, 7 of Hexon for infection of tumor cells, in order to select the targeted peptide insertion region that has the best effect on 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, oncolytic adenoviral recombinant Ad 5/. DELTA.E 1A/HVR5/TMVP 1. DELTA.ADP/HSV-TK carrying TMVP1 and HSV-TK in tumor cellsThe replication is most active in the cell, and the nucleotide sequence is shown as SEQ ID NO. 23. The hypervariable region 5, the HVR5, which is the optimal adenoviral insert capable of high copy number replication in tumor cells was selected as the final region by in vitro experiments.
Example 6: characterization of therapeutic Effect of oncolytic adenovirus Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK
Step 6.1: identification of oncolytic adenovirus recombinant in vitro tumor cell killing Effect carrying TMVP1 and HSV-TK
The purpose of the experiment is to identify the killing effect of the Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK novel oncolytic adenovirus on a series of tumors and normal cells, and the experiment takes wt-Ad5 as a positive control and replication-defective virus Ad5CMV-GFP as a negative control. The cell lines are selected to be a human lung cancer cell line A549, a human high-metastatic prostate cancer cell line PC3M-1E8, a human osteoblastoma cell line U-2OS, a human colon cancer cell line HCT-8, a human breast cancer cell line MCF-7, a human ovarian cancer cell line SKOV-3, a human liver cancer cell line HepG2, a human esophageal cancer cell line Eca-109, a human gastric cancer cell line MKN-45, a human brain glioma cell line SHG-44, a human head and neck squamous cell cancer cell line AGZY-973, a human pancreatic cancer cell line BxPC-3, a human endometrial cancer cell line HEC-1-A, a human rectal cancer cell line Colo320 and a human nasopharyngeal cancer cell line CNE-2. The selected cell lines have different P53, RB gene phenotypes; have different tissue origins and therefore the experimental results may represent different types of tumours. The selected normal cells are primary vascular endothelial cells, primary lung small trachea epithelial cells, prostate epithelial cells and bone marrow mononuclear cells, and the selection principle is as follows: after intravenous administration, a large number of adenovirus normal tissue cells will be contacted.
Seeding in 24-well plates 1X 105Tumor or normal epithelial cells in culture medium of 10% FBS DMEM or RIPM1640, about 70% of the cells fused the next day, aspirated, diluted to 100 μ l with the MOI required to reach 70%, added by gently shaking the liquid three times at 37 deg.C and 5% CO2The culture box of (1) was incubated for 90min, DMEM or RIPM1640 medium containing 10% FBS was added to 2ml while Ganciclovir (GCV) was added, the cells were left at 37 ℃ and 5% CO2Culture ofAnd (5) incubating in the incubator for 3 days, and detecting the apoptosis rate of the cells by using a flow cytometer. The specific experimental results are shown in table 1.
TABLE 1
| wt-Ad5 | Ad5CMV-GFP | Ad5/ΔE1A/HVR5/TMVP1/ΔADP/HSV-TK | |
| A549, day 3 | 37％ | 6％ | 85％ |
| PC3M-1E8, day 3 | 43％ | 4％ | 80％ |
| U-2OS, day 3 | 51％ | 5％ | 75％ |
| HCT-8, day 3 | 50％ | 7％ | 83％ |
| HepG-2, day 3 | 39％ | 6％ | 89％ |
| MCF-7, day 3 | 40％ | 6％ | 74％ |
| SKOV-3, day 3 | 44％ | 5％ | 88％ |
| SHG-44, day 3 | 42％ | 3％ | 81％ |
| BxPC-3, day 3 | 46％ | 5％ | 82％ |
| Colo320, day 3 | 45％ | 3％ | 78％ |
| MKN-45, day 3 | 40％ | 8％ | 83％ |
| HEC-1-A, day 3 | 33％ | 4％ | 85％ |
| CNE-2, day 3 | 32％ | 5％ | 81％ |
| Eca-109, day 3 | 37％ | 7％ | 80％ |
| AGZY-973, day 3 | 45％ | 3％ | 84％ |
| Vascular endothelial cells, day 3 | 44％ | 6％ | 5％ |
| Vascular endothelial cells, day 6 | 100％ | 7％ | 6％ |
| Vascular endothelial cells, day 10 | — | 9％ | 8％ |
| Lung small trachea epithelial cells, day 3 | 57％ | 3％ | 4％ |
| Lung small trachea epithelial cells, day 6 | 100％ | 5％ | 6％ |
| Lung small trachea epithelial cells, day 10 | — | 9％ | 8％ |
The experimental result shows that the positive control wt-Ad5 does not selectively crack tumor cells and normal cells; the killing effect of replication-defective adenovirus Ad5CMV-GFP on cells is very weak, the oncolytic adenovirus Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK + GCV selectively kills tumor cells, the effect is obviously stronger than that of wt-Ad5 on cells, and meanwhile, the recombinant adenovirus construct has no obvious influence on normal control cell lines. The results fully show that the oncolytic adenovirus Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK selectively and efficiently kills tumor cells.
Step 6.2: characterization of in vivo antitumor Effect of oncolytic adenovirus Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK
Tumor cell animal models: selecting BALB/c nude mice of 4-6 weeks of age, taking gastric cancer cell line MKN-45 cells of logarithmic growth phase, and adjusting cell density to be 1 multiplied by 106100 μ l, subcutaneously inoculating 200 μ l to the right dorsal side of each nude mouse, when the average tumor diameter reaches 0.4-0.6cm, dividing into 4 groups according to the tumor size, namely a medium control group, a wt-Ad5 group, an Ad5CMV-GFP group and an Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK group, and injecting PBS, wt-Ad5, Ad5CMV-GFP and Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK into each group of 5 nude mice, respectively, each nude mouse is injected with PBS, wt-Ad5, Ad5CMV-GFP and Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TKOnly 2 x 108pfu/(once), once daily for 5 consecutive days. Intraperitoneal injection of GCV 125 mg/kg on days 2-18-1·d–1. Tumor volumes were observed and measured 2 times per week until the end of the experiment (50 days or tumor volumes greater than 2 cm)3)。
As shown in FIG. 3, at the end of the experiment, the tumors of three nude mice in Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK group completely regressed, the average tumor inhibition rate was 93% +/-6%, and subcutaneous tumors of nude mice in other groups did not shrink significantly. The results are sufficient to show that oncolytic adenovirus Ad 5/. DELTA.E 1A/HVR5/TMVP 1/. DELTA.ADP/HSV-TK has a defined anti-tumor effect in vivo.
Step 6.4: in vivo identification of oncolytic adenoviral recombinants carrying TMVP1 and HSV-TK specifically targeting tumor cells
The specific experimental method comprises the following steps:
4-6 weeks of BALB/c nu nude mice, 2 x 10 abdominal cavity planting6skov3 ovarian cancer cells, 4 weeks later, nude mice were divided into two groups (n ═ 4), and each was intraperitoneally injected with 1 × 10 cells8pfu novel oncolytic adenovirus Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK and its control virus Ad5/Δ E1A. After 48h, the liver, spleen, kidney and tumor tissues of each mouse were taken out, DNA of each tissue was extracted, and the DNA content of adenovirus in 400ng of tissue DNA was detected, with the results shown in FIG. 4: the control virus Ad 5/delta E1A has aggregation in liver, lung and spleen and is distributed less in tumor tissues, the Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK is mainly distributed in tumor tissues, and the liver uptake is obviously reduced; the content in tumor tissues is nearly 100 times that of the control virus Ad 5/delta E1A.
Example 7: identification of oncolytic adenovirus Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK for hepatorenal toxicity
The specific experimental method comprises the following steps: 4-6 weeks of BALB/c nu nude mice, 2 x 10 abdominal cavity planting6skov3 ovarian cancer cells, 4 weeks later, nude mice were divided into two groups (n ═ 4), and each was intraperitoneally injected with 1 × 10 cells8Oncolytic adenovirus Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK with pfu carrying TMVP1 and TK and its control virus Ad5/Δ E1A. 48h later, the liver, spleen, kidney and tumor tissues of each mouse were removed, DNA of each tissue was extracted, and 400ng of DNA was detectedThe DNA content of adenovirus in the tissue DNA is shown in FIG. 4: the control virus Ad5/Δ E1A was aggregated in liver, lung, spleen and distributed in tumor tissue with less TMVP1 and TK carrying, Ad5/Δ E1A/HVR5/TMVP1/Δ ADP/HSV-TK was distributed mainly in tumor tissue, liver uptake was significantly reduced; the content in tumor tissues is nearly 100 times that of the control virus Ad 5/delta E1A.
The results show that the oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK carrying TMVP1 and HSV-TK overcomes hepatotropism, remarkably enhances the targeting property to the tumor, has low toxicity to the liver and the kidney and has higher safety.
Therefore, the oncolytic adenovirus recombinant Ad 5/delta E1A/HVR5/TMVP 1/delta ADP/HSV-TK carrying TMVP1 and HSV-TK can be used for preparing medicaments for treating tumors, not only develops a novel medicament for treating tumors, but also develops the application of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and has positive pharmaceutical value and wide social significance.
Although the present invention has been described with respect to the preferred embodiments, it is not intended to be limited to the embodiments disclosed, and many modifications and variations are possible to those skilled in the art without departing from the spirit of the invention.
Sequence listing
<110> affiliated Tongji hospital of Tongji medical college of Huazhong university of science and technology
<120> oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK, and construction method and application thereof
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gatcttacct gccacgaggc tggcttt 27
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cgggatccgg gcccccattt cc 22
<210> 3
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gtcactgggt ggatcgatca cctccggtac 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gaggtgatcg atccacccag tgacgacgag 30
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tgctctagac acaggtgatg tcg 23
<210> 6
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttaattaaca tcatcaataa tataccttat t 31
<210> 7
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gatccacata atctaacaca aactc 25
<210> 8
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ttaattaaca tcatcaataa tataccttat t 31
<210> 9
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gatccacata atctaacaca aactc 25
<210> 10
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgggatccgg gcccccattt cc 22
<210> 11
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tgctctagac acaggtgatg tcg 23
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
<210> 14
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
<210> 15
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
<210> 16
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
tgtcaccact aactgcttta ctcg 24
<210> 17
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gctgccctgc gtctttcta 19
<210> 18
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
atacgcgccc accgaaac 18
<210> 19
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aatctatgga tatcgatagg gtgggtcgct gtagtt 36
<210> 20
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cgacccaccc tatcgatatc catagattgg acggactg 38
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
<210> 22
<211> 1131
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
atggcttcgt acccctgcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60
ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120
cgcccggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc ccacgggatg 180
gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240
gtacccgagc cgatgactta ctggcgggtg ctgggggctt ccgagacaat cgcgaacatc 300
tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360
atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420
cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480
ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcggta ccttatgggc 540
agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600
acaaacatcg tgttgggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660
cagcgccccg gtgagcggct tgacctggct atgctggccg cgattcgccg cgtttacggg 720
ctacttgcca atacggtgcg gtatctgcag tgcggcgggt cgtggcggga ggattgggga 780
cagctttcgg ggacggcctt gacgccccag ggtgccgagc cccagagcaa cgcgggccca 840
cgaccccata tcggggaaac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900
aacggcgacc tgtacaacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960
cccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020
ctgcaactta cctccgggat ggtccagacc catgtcacca ccccaggctc cataccgacg 1080
atctgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctcactg a 1131
<210> 23
<211> 36830
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60
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 gaggtgatcn tccacccagt gacgacgagg atgaagaggg tgaggagttt 960
gtgttagatt atgtggagca ccccgggcac ggttgcaggt cttgtcatta tcaccggagg 1020
aatacggggg acccagatat tatgtgttcg ctttgctata tgaggacctg tggcatgttt 1080
gtctacagta agtgaaaatt atgggcagtg ggtgatagag tggtgggttt ggtgtggtaa 1140
tttttttttt aatttttaca gttttgtggt ttaaagaatt ttgtattgtg atttttttaa 1200
aaggtcctgt gtctgaacct gagcctgagc ccgagccaga accggagcct gcaagaccta 1260
cccgccgtcc taaaatggcg cctgctatcc tgagacgccc gacatcacct gtgtctagag 1320
aatgcaatag tagtacggat agctgtgact ccggtccttc taacacacct cctgagatac 1380
acccggtggt cccgctgtgc cccattaaac cagttgccgt gagagttggt gggcgtcgcc 1440
aggctgtgga atgtatcgag gacttgctta acgagcctgg gcaacctttg gacttgagct 1500
gtaaacgccc caggccataa ggtgtaaacc tgtgattgcg tgtgtggtta acgcctttgt 1560
ttgctgaatg agttgatgta agtttaataa agggtgagat aatgtttaac ttgcatggcg 1620
tgttaaatgg ggcggggctt aaagggtata taatgcgccg tgggctaatc ttggttacat 1680
ctgacctcat ggaggcttgg gagtgtttgg aagatttttc tgctgtgcgt aacttgctgg 1740
aacagagctc taacagtacc tcttggtttt ggaggtttct gtggggctca tcccaggcaa 1800
agttagtctg cagaattaag gaggattaca agtgggaatt tgaagagctt ttgaaatcct 1860
gtggtgagct gtttgattct ttgaatctgg gtcaccaggc gcttttccaa gagaaggtca 1920
tcaagacttt ggatttttcc acaccggggc gcgctgcggc tgctgttgct tttttgagtt 1980
ttataaagga taaatggagc gaagaaaccc atctgagcgg ggggtacctg ctggattttc 2040
tggccatgca tctgtggaga gcggttgtga gacacaagaa tcgcctgcta ctgttgtctt 2100
ccgtccgccc ggcgataata ccgacggagg agcagcagca gcagcaggag gaagccaggc 2160
ggcggcggca ggagcagagc ccatggaacc cgagagccgg cctggaccct cgggaatgaa 2220
tgttgtacag gtggctgaac tgtatccaga actgagacgc attttgacaa ttacagagga 2280
tgggcagggg ctaaaggggg taaagaggga gcggggggct tgtgaggcta cagaggaggc 2340
taggaatcta gcttttagct taatgaccag acaccgtcct gagtgtatta cttttcaaca 2400
gatcaaggat aattgcgcta atgagcttga tctgctggcg cagaagtatt ccatagagca 2460
gctgaccact tactggctgc agccagggga tgattttgag gaggctatta gggtatatgc 2520
aaaggtggca cttaggccag attgcaagta caagatcagc aaacttgtaa atatcaggaa 2580
ttgttgctac atttctggga acggggccga ggtggagata gatacggagg atagggtggc 2640
ctttagatgt agcatgataa atatgtggcc gggggtgctt ggcatggacg gggtggttat 2700
tatgaatgta aggtttactg gccccaattt tagcggtacg gttttcctgg ccaataccaa 2760
ccttatccta cacggtgtaa gcttctatgg gtttaacaat acctgtgtgg aagcctggac 2820
cgatgtaagg gttcggggct gtgcctttta ctgctgctgg aagggggtgg tgtgtcgccc 2880
caaaagcagg gcttcaatta agaaatgcct ctttgaaagg tgtaccttgg gtatcctgtc 2940
tgagggtaac tccagggtgc gccacaatgt ggcctccgac tgtggttgct tcatgctagt 3000
gaaaagcgtg gctgtgatta agcataacat ggtatgtggc aactgcgagg acagggcctc 3060
tcagatgctg acctgctcgg acggcaactg tcacctgctg aagaccattc acgtagccag 3120
ccactctcgc aaggcctggc cagtgtttga gcataacata ctgacccgct gttccttgca 3180
tttgggtaac aggagggggg tgttcctacc ttaccaatgc aatttgagtc acactaagat 3240
attgcttgag cccgagagca tgtccaaggt gaacctgaac ggggtgtttg acatgaccat 3300
gaagatctgg aaggtgctga ggtacgatga gacccgcacc aggtgcagac cctgcgagtg 3360
tggcggtaaa catattagga accagcctgt gatgctggat gtgaccgagg agctgaggcc 3420
cgatcacttg gtgctggcct gcacccgcgc tgagtttggc tctagcgatg aagatacaga 3480
ttgaggtact gaaatgtgtg ggcgtggctt aagggtggga aagaatatat aaggtggggg 3540
tcttatgtag ttttgtatct gttttgcagc agccgccgcc gccatgagca ccaactcgtt 3600
tgatggaagc attgtgagct catatttgac aacgcgcatg cccccatggg ccggggtgcg 3660
tcagaatgtg atgggctcca gcattgatgg tcgccccgtc ctgcccgcaa actctactac 3720
cttgacctac gagaccgtgt ctggaacgcc gttggagact gcagcctccg ccgccgcttc 3780
agccgctgca gccaccgccc gcgggattgt gactgacttt gctttcctga gcccgcttgc 3840
aagcagtgca gcttcccgtt catccgcccg cgatgacaag ttgacggctc ttttggcaca 3900
attggattct ttgacccggg aacttaatgt cgtttctcag cagctgttgg atctgcgcca 3960
gcaggtttct gccctgaagg cttcctcccc tcccaatgcg gtttaaaaca taaataaaaa 4020
accagactct gtttggattt ggatcaagca agtgtcttgc tgtctttatt taggggtttt 4080
gcgcgcgcgg taggcccggg accagcggtc tcggtcgttg agggtcctgt gtattttttc 4140
caggacgtgg taaaggtgac tctggatgtt cagatacatg ggcataagcc cgtctctggg 4200
gtggaggtag caccactgca gagcttcatg ctgcggggtg gtgttgtaga tgatccagtc 4260
gtagcaggag cgctgggcgt ggtgcctaaa aatgtctttc agtagcaagc tgattgccag 4320
gggcaggccc ttggtgtaag tgtttacaaa gcggttaagc tgggatgggt gcatacgtgg 4380
ggatatgaga tgcatcttgg actgtatttt taggttggct atgttcccag ccatatccct 4440
ccggggattc atgttgtgca gaaccaccag cacagtgtat ccggtgcact tgggaaattt 4500
gtcatgtagc ttagaaggaa atgcgtggaa gaacttggag acgcccttgt gacctccaag 4560
attttccatg cattcgtcca taatgatggc aatgggccca cgggcggcgg cctgggcgaa 4620
gatatttctg ggatcactaa cgtcatagtt gtgttccagg atgagatcgt cataggccat 4680
ttttacaaag cgcgggcgga gggtgccaga ctgcggtata atggttccat ccggcccagg 4740
ggcgtagtta ccctcacaga tttgcatttc ccacgctttg agttcagatg gggggatcat 4800
gtctacctgc ggggcgatga agaaaacggt ttccggggta ggggagatca gctgggaaga 4860
aagcaggttc ctgagcagct gcgacttacc gcagccggtg ggcccgtaaa tcacacctat 4920
taccgggtgc aactggtagt taagagagct gcagctgccg tcatccctga gcaggggggc 4980
cacttcgtta agcatgtccc tgactcgcat gttttccctg accaaatccg ccagaaggcg 5040
ctcgccgccc agcgatagca gttcttgcaa ggaagcaaag tttttcaacg gtttgagacc 5100
gtccgccgta ggcatgcttt tgagcgtttg accaagcagt tccaggcggt cccacagctc 5160
ggtcacctgc tctacggcat ctcgatccag catatctcct cgtttcgcgg gttggggcgg 5220
ctttcgctgt acggcagtag tcggtgctcg tccagacggg ccagggtcat gtctttccac 5280
gggcgcaggg tcctcgtcag cgtagtctgg gtcacggtga aggggtgcgc tccgggctgc 5340
gcgctggcca gggtgcgctt gaggctggtc ctgctggtgc tgaagcgctg ccggtcttcg 5400
ccctgcgcgt cggccaggta gcatttgacc atggtgtcat agtccagccc ctccgcggcg 5460
tggcccttgg cgcgcagctt gcccttggag gaggcgccgc acgaggggca gtgcagactt 5520
ttgagggcgt agagcttggg cgcgagaaat accgattccg gggagtaggc atccgcgccg 5580
caggccccgc agacggtctc gcattccacg agccaggtga gctctggccg ttcggggtca 5640
aaaaccaggt ttcccccatg ctttttgatg cgtttcttac ctctggtttc catgagccgg 5700
tgtccacgct cggtgacgaa aaggctgtcc gtgtccccgt atacagactt gagaggcctg 5760
tcctcgagcg gtgttccgcg gtcctcctcg tatagaaact cggaccactc tgagacaaag 5820
gctcgcgtcc aggccagcac gaaggaggct aagtgggagg ggtagcggtc gttgtccact 5880
agggggtcca ctcgctccag ggtgtgaaga cacatgtcgc cctcttcggc atcaaggaag 5940
gtgattggtt tgtaggtgta ggccacgtga ccgggtgttc ctgaaggggg gctataaaag 6000
ggggtggggg cgcgttcgtc ctcactctct tccgcatcgc tgtctgcgag ggccagctgt 6060
tggggtgagt actccctctg aaaagcgggc atgacttctg cgctaagatt gtcagtttcc 6120
aaaaacgagg aggatttgat attcacctgg cccgcggtga tgcctttgag ggtggccgca 6180
tccatctggt cagaaaagac aatctttttg ttgtcaagct tggtggcaaa cgacccgtag 6240
agggcgttgg acagcaactt ggcgatggag cgcagggttt ggtttttgtc gcgatcggcg 6300
cgctccttgg ccgcgatgtt tagctgcacg tattcgcgcg caacgcaccg ccattcggga 6360
aagacggtgg tgcgctcgtc gggcaccagg tgcacgcgcc aaccgcggtt gtgcagggtg 6420
acaaggtcaa cgctggtggc tacctctccg cgtaggcgct cgttggtcca gcagaggcgg 6480
ccgcccttgc gcgagcagaa tggcggtagg gggtctagct gcgtctcgtc cggggggtct 6540
gcgtccacgg taaagacccc gggcagcagg cgcgcgtcga agtagtctat cttgcatcct 6600
tgcaagtcta gcgcctgctg ccatgcgcgg gcggcaagcg cgcgctcgta tgggttgagt 6660
gggggacccc atggcatggg gtgggtgagc gcggaggcgt acatgccgca aatgtcgtaa 6720
acgtagaggg gctctctgag tattccaaga tatgtagggt agcatcttcc accgcggatg 6780
ctggcgcgca cgtaatcgta tagttcgtgc gagggagcga ggaggtcggg accgaggttg 6840
ctacgggcgg gctgctctgc tcggaagact atctgcctga agatggcatg tgagttggat 6900
gatatggttg gacgctggaa gacgttgaag ctggcgtctg tgagacctac cgcgtcacgc 6960
acgaaggagg cgtaggagtc gcgcagcttg ttgaccagct cggcggtgac ctgcacgtct 7020
agggcgcagt agtccagggt ttccttgatg atgtcatact tatcctgtcc cttttttttc 7080
cacagctcgc ggttgaggac aaactcttcg cggtctttcc agtactcttg gatcggaaac 7140
ccgtcggcct ccgaacggta agagcctagc atgtagaact ggttgacggc ctggtaggcg 7200
cagcatccct tttctacggg tagcgcgtat gcctgcgcgg ccttccggag cgaggtgtgg 7260
gtgagcgcaa aggtgtccct gaccatgact ttgaggtact ggtatttgaa gtcagtgtcg 7320
tcgcatccgc cctgctccca gagcaaaaag tccgtgcgct ttttggaacg cggatttggc 7380
agggcgaagg tgacatcgtt gaagagtatc tttcccgcgc gaggcataaa gttgcgtgtg 7440
atgcggaagg gtcccggcac ctcggaacgg ttgttaatta cctgggcggc gagcacgatc 7500
tcgtcaaagc cgttgatgtt gtggcccaca atgtaaagtt ccaagaagcg cgggatgccc 7560
ttgatggaag gcaatttttt aagttcctcg taggtgagct cttcagggga gctgagcccg 7620
tgctctgaaa gggcccagtc tgcaagatga gggttggaag cgacgaatga gctccacagg 7680
tcacgggcca ttagcatttg caggtggtcg cgaaaggtcc taaactggcg acctatggcc 7740
attttttctg gggtgatgca gtagaaggta agcgggtctt gttcccagcg gtcccatcca 7800
aggttcgcgg ctaggtctcg cgcggcagtc actagaggct catctccgcc gaacttcatg 7860
accagcatga agggcacgag ctgcttccca aaggccccca tccaagtata ggtctctaca 7920
tcgtaggtga caaagagacg ctcggtgcga ggatgcgagc cgatcgggaa gaactggatc 7980
tcccgccacc aattggagga gtggctattg atgtggtgaa agtagaagtc cctgcgacgg 8040
gccgaacact cgtgctggct tttgtaaaaa cgtgcgcagt actggcagcg gtgcacgggc 8100
tgtacatcct gcacgaggtt gacctgacga ccgcgcacaa ggaagcagag tgggaatttg 8160
agcccctcgc ctggcgggtt tggctggtgg tcttctactt cggctgcttg tccttgaccg 8220
tctggctgct cgaggggagt tacggtggat cggaccacca cgccgcgcga gcccaaagtc 8280
cagatgtccg cgcgcggcgg tcggagcttg atgacaacat cgcgcagatg ggagctgtcc 8340
atggtctgga gctcccgcgg cgtcaggtca ggcgggagct cctgcaggtt tacctcgcat 8400
agacgggtca gggcgcgggc tagatccagg tgatacctaa tttccagggg ctggttggtg 8460
gcggcgtcga tggcttgcaa gaggccgcat ccccgcggcg cgactacggt accgcgcggc 8520
gggcggtggg ccgcgggggt gtccttggat gatgcatcta aaagcggtga cgcgggcgag 8580
cccccggagg tagggggggc tccggacccg ccgggagagg gggcaggggc acgtcggcgc 8640
cgcgcgcggg caggagctgg tgctgcgcgc gtaggttgct ggcgaacgcg acgacgcggc 8700
ggttgatctc ctgaatctgg cgcctctgcg tgaagacgac gggcccggtg agcttgagcc 8760
tgaaagagag ttcgacagaa tcaatttcgg tgtcgttgac ggcggcctgg cgcaaaatct 8820
cctgcacgtc tcctgagttg tcttgatagg cgatctcggc catgaactgc tcgatctctt 8880
cctcctggag atctccgcgt ccggctcgct ccacggtggc ggcgaggtcg ttggaaatgc 8940
gggccatgag ctgcgagaag gcgttgaggc ctccctcgtt ccagacgcgg ctgtagacca 9000
cgcccccttc ggcatcgcgg gcgcgcatga ccacctgcgc gagattgagc tccacgtgcc 9060
gggcgaagac ggcgtagttt cgcaggcgct gaaagaggta gttgagggtg gtggcggtgt 9120
gttctgccac gaagaagtac ataacccagc gtcgcaacgt ggattcgttg atatccccca 9180
aggcctcaag gcgctccatg gcctcgtaga agtccacggc gaagttgaaa aactgggagt 9240
tgcgcgccga cacggttaac tcctcctcca gaagacggat gagctcggcg acagtgtcgc 9300
gcacctcgcg ctcaaaggct acaggggcct cttcttcttc ttcaatctcc tcttccataa 9360
gggcctcccc ttcttcttct tctggcggcg gtgggggagg ggggacacgg cggcgacgac 9420
ggcgcaccgg gaggcggtcg acaaagcgct cgatcatctc cccgcggcga cggcgcatgg 9480
tctcggtgac ggcgcggccg ttctcgcggg ggcgcagttg gaagacgccg cccgtcatgt 9540
cccggttatg ggttggcggg gggctgccat gcggcaggga tacggcgcta acgatgcatc 9600
tcaacaattg ttgtgtaggt actccgccgc cgagggacct gagcgagtcc gcatcgaccg 9660
gatcggaaaa cctctcgaga aaggcgtcta accagtcaca gtcgcaaggt aggctgagca 9720
ccgtggcggg cggcagcggg cggcggtcgg ggttgtttct ggcggaggtg ctgctgatga 9780
tgtaattaaa gtaggcggtc ttgagacggc ggatggtcga cagaagcacc atgtccttgg 9840
gtccggcctg ctgaatgcgc aggcggtcgg ccatgcccca ggcttcgttt tgacatcggc 9900
gcaggtcttt gtagtagtct tgcatgagcc tttctaccgg cacttcttct tctccttcct 9960
cttgtcctgc atctcttgca tctatcgctg cggcggcggc ggagtttggc cgtaggtggc 10020
gccctcttcc tcccatgcgt gtgaccccga agcccctcat cggctgaagc agggctaggt 10080
cggcgacaac gcgctcggct aatatggcct gctgcacctg cgtgagggta gactggaagt 10140
catccatgtc cacaaagcgg tggtatgcgc ccgtgttgat ggtgtaagtg cagttggcca 10200
taacggacca gttaacggtc tggtgacccg gctgcgagag ctcggtgtac ctgagacgcg 10260
agtaagccct cgagtcaaat acgtagtcgt tgcaagtccg caccaggtac tggtatccca 10320
ccaaaaagtg cggcggcggc tggcggtaga ggggccagcg tagggtggcc ggggctccgg 10380
gggcgagatc ttccaacata aggcgatgat atccgtagat gtacctggac atccaggtga 10440
tgccggcggc ggtggtggag gcgcgcggaa agtcgcggac gcggttccag atgttgcgca 10500
gcggcaaaaa gtgctccatg gtcgggacgc tctggccggt caggcgcgcg caatcgttga 10560
cgctctagac cgtgcaaaag gagagcctgt aagcgggcac tcttccgtgg tctggtggat 10620
aaattcgcaa gggtatcatg gcggacgacc ggggttcgag ccccgtatcc ggccgtccgc 10680
cgtgatccat gcggttaccg cccgcgtgtc gaacccaggt gtgcgacgtc agacaacggg 10740
ggagtgctcc ttttggcttc cttccaggcg cggcggctgc tgcgctagct tttttggcca 10800
ctggccgcgc gcagcgtaag cggttaggct ggaaagcgaa agcattaagt ggctcgctcc 10860
ctgtagccgg agggttattt tccaagggtt gagtcgcggg acccccggtt cgagtctcgg 10920
accggccgga ctgcggcgaa cgggggtttg cctccccgtc atgcaagacc ccgcttgcaa 10980
attcctccgg aaacagggac gagccccttt tttgcttttc ccagatgcat ccggtgctgc 11040
ggcagatgcg cccccctcct cagcagcggc aagagcaaga gcagcggcag acatgcaggg 11100
caccctcccc tcctcctacc gcgtcaggag gggcgacatc cgcggttgac gcggcagcag 11160
atggtgatta cgaacccccg cggcgccggg cccggcacta cctggacttg gaggagggcg 11220
agggcctggc gcggctagga gcgccctctc ctgagcggta cccaagggtg cagctgaagc 11280
gtgatacgcg tgaggcgtac gtgccgcggc agaacctgtt tcgcgaccgc gagggagagg 11340
agcccgagga gatgcgggat cgaaagttcc acgcagggcg cgagctgcgg catggcctga 11400
atcgcgagcg gttgctgcgc gaggaggact ttgagcccga cgcgcgaacc gggattagtc 11460
ccgcgcgcgc acacgtggcg gccgccgacc tggtaaccgc atacgagcag acggtgaacc 11520
aggagattaa ctttcaaaaa agctttaaca accacgtgcg tacgcttgtg gcgcgcgagg 11580
aggtggctat aggactgatg catctgtggg actttgtaag cgcgctggag caaaacccaa 11640
atagcaagcc gctcatggcg cagctgttcc ttatagtgca gcacagcagg gacaacgagg 11700
cattcaggga tgcgctgcta aacatagtag agcccgaggg ccgctggctg ctcgatttga 11760
taaacatcct gcagagcata gtggtgcagg agcgcagctt gagcctggct gacaaggtgg 11820
ccgccatcaa ctattccatg cttagcctgg gcaagtttta cgcccgcaag atataccata 11880
ccccttacgt tcccatagac aaggaggtaa agatcgaggg gttctacatg cgcatggcgc 11940
tgaaggtgct taccttgagc gacgacctgg gcgtttatcg caacgagcgc atccacaagg 12000
ccgtgagcgt gagccggcgg cgcgagctca gcgaccgcga gctgatgcac agcctgcaaa 12060
gggccctggc tggcacgggc agcggcgata gagaggccga gtcctacttt gacgcgggcg 12120
ctgacctgcg ctgggcccca agccgacgcg ccctggaggc agctggggcc ggacctgggc 12180
tggcggtggc acccgcgcgc gctggcaacg tcggcggcgt ggaggaatat gacgaggacg 12240
atgagtacga gccagaggac ggcgagtact aagcggtgat gtttctgatc agatgatgca 12300
agacgcaacg gacccggcgg tgcgggcggc gctgcagagc cagccgtccg gccttaactc 12360
cacggacgac tggcgccagg tcatggaccg catcatgtcg ctgactgcgc gcaatcctga 12420
cgcgttccgg cagcagccgc aggccaaccg gctctccgca attctggaag cggtggtccc 12480
ggcgcgcgca aaccccacgc acgagaaggt gctggcgatc gtaaacgcgc tggccgaaaa 12540
cagggccatc cggcccgacg aggccggcct ggtctacgac gcgctgcttc agcgcgtggc 12600
tcgttacaac agcggcaacg tgcagaccaa cctggaccgg ctggtggggg atgtgcgcga 12660
ggccgtggcg cagcgtgagc gcgcgcagca gcagggcaac ctgggctcca tggttgcact 12720
aaacgccttc ctgagtacac agcccgccaa cgtgccgcgg ggacaggagg actacaccaa 12780
ctttgtgagc gcactgcggc taatggtgac tgagacaccg caaagtgagg tgtaccagtc 12840
tgggccagac tattttttcc agaccagtag acaaggcctg cagaccgtaa acctgagcca 12900
ggctttcaaa aacttgcagg ggctgtgggg ggtgcgggct cccacaggcg accgcgcgac 12960
cgtgtctagc ttgctgacgc ccaactcgcg cctgttgctg ctgctaatag cgcccttcac 13020
ggacagtggc agcgtgtccc gggacacata cctaggtcac ttgctgacac tgtaccgcga 13080
ggccataggt caggcgcatg tggacgagca tactttccag gagattacaa gtgtcagccg 13140
cgcgctgggg caggaggaca cgggcagcct ggaggcaacc ctaaactacc tgctgaccaa 13200
ccggcggcag aagatcccct cgttgcacag tttaaacagc gaggaggagc gcattttgcg 13260
ctacgtgcag cagagcgtga gccttaacct gatgcgcgac ggggtaacgc ccagcgtggc 13320
gctggacatg accgcgcgca acatggaacc gggcatgtat gcctcaaacc ggccgtttat 13380
caaccgccta atggactact tgcatcgcgc ggccgccgtg aaccccgagt atttcaccaa 13440
tgccatcttg aacccgcact ggctaccgcc ccctggtttc tacaccgggg gattcgaggt 13500
gcccgagggt aacgatggat tcctctggga cgacatagac gacagcgtgt tttccccgca 13560
accgcagacc ctgctagagt tgcaacagcg cgagcaggca gaggcggcgc tgcgaaagga 13620
aagcttccgc aggccaagca gcttgtccga tctaggcgct gcggccccgc ggtcagatgc 13680
tagtagccca tttccaagct tgatagggtc tcttaccagc actcgcacca cccgcccgcg 13740
cctgctgggc gaggaggagt acctaaacaa ctcgctgctg cagccgcagc gcgaaaaaaa 13800
cctgcctccg gcatttccca acaacgggat agagagccta gtggacaaga tgagtagatg 13860
gaagacgtac gcgcaggagc acagggacgt gccaggcccg cgcccgccca cccgtcgtca 13920
aaggcacgac cgtcagcggg gtctggtgtg ggaggacgat gactcggcag acgacagcag 13980
cgtcctggat ttgggaggga gtggcaaccc gtttgcgcac cttcgcccca ggctggggag 14040
aatgttttaa aaaaaaaaaa gcatgatgca aaataaaaaa ctcaccaagg ccatggcacc 14100
gagcgttggt tttcttgtat tccccttagt atgcggcgcg cggcgatgta tgaggaaggt 14160
cctcctccct cctacgagag tgtggtgagc gcggcgccag tggcggcggc gctgggttct 14220
cccttcgatg ctcccctgga cccgccgttt gtgcctccgc ggtacctgcg gcctaccggg 14280
gggagaaaca gcatccgtta ctctgagttg gcacccctat tcgacaccac ccgtgtgtac 14340
ctggtggaca acaagtcaac ggatgtggca tccctgaact accagaacga ccacagcaac 14400
tttctgacca cggtcattca aaacaatgac tacagcccgg gggaggcaag cacacagacc 14460
atcaatcttg acgaccggtc gcactggggc ggcgacctga aaaccatcct gcataccaac 14520
atgccaaatg tgaacgagtt catgtttacc aataagttta aggcgcgggt gatggtgtcg 14580
cgcttgccta ctaaggacaa tcaggtggag ctgaaatacg agtgggtgga gttcacgctg 14640
cccgagggca actactccga gaccatgacc atagacctta tgaacaacgc gatcgtggag 14700
cactacttga aagtgggcag acagaacggg gttctggaaa gcgacatcgg ggtaaagttt 14760
gacacccgca acttcagact ggggtttgac cccgtcactg gtcttgtcat gcctggggta 14820
tatacaaacg aagccttcca tccagacatc attttgctgc caggatgcgg ggtggacttc 14880
acccacagcc gcctgagcaa cttgttgggc atccgcaagc ggcaaccctt ccaggagggc 14940
tttaggatca cctacgatga tctggagggt ggtaacattc ccgcactgtt ggatgtggac 15000
gcctaccagg cgagcttgaa agatgacacc gaacagggcg ggggtggcgc aggcggcagc 15060
aacagcagtg gcagcggcgc ggaagagaac tccaacgcgg cagccgcggc aatgcagccg 15120
gtggaggaca tgaacgatca tgccattcgc ggcgacacct ttgccacacg ggctgaggag 15180
aagcgcgctg aggccgaagc agcggccgaa gctgccgccc ccgctgcgca acccgaggtc 15240
gagaagcctc agaagaaacc ggtgatcaaa cccctgacag aggacagcaa gaaacgcagt 15300
tacaacctaa taagcaatga cagcaccttc acccagtacc gcagctggta ccttgcatac 15360
aactacggcg accctcagac cggaatccgc tcatggaccc tgctttgcac tcctgacgta 15420
acctgcggct cggagcaggt ctactggtcg ttgccagaca tgatgcaaga ccccgtgacc 15480
ttccgctcca cgcgccagat cagcaacttt ccggtggtgg gcgccgagct gttgcccgtg 15540
cactccaaga gcttctacaa cgaccaggcc gtctactccc aactcatccg ccagtttacc 15600
tctctgaccc acgtgttcaa tcgctttccc gagaaccaga ttttggcgcg cccgccagcc 15660
cccaccatca ccaccgtcag tgaaaacgtt cctgctctca cagatcacgg gacgctaccg 15720
ctgcgcaaca gcatcggagg agtccagcga gtgaccatta ctgacgccag acgccgcacc 15780
tgcccctacg tttacaaggc cctgggcata gtctcgccgc gcgtcctatc gagccgcact 15840
ttttgagcaa gcatgtccat ccttatatcg cccagcaata acacaggctg gggcctgcgc 15900
ttcccaagca agatgtttgg cggggccaag aagcgctccg accaacaccc agtgcgcgtg 15960
cgcgggcact accgcgcgcc ctggggcgcg cacaaacgcg gccgcactgg gcgcaccacc 16020
gtcgatgacg ccatcgacgc ggtggtggag gaggcgcgca actacacgcc cacgccgcca 16080
ccagtgtcca cagtggacgc ggccattcag accgtggtgc gcggagcccg gcgctatgct 16140
aaaatgaaga gacggcggag gcgcgtagca cgtcgccacc gccgccgacc cggcactgcc 16200
gcccaacgcg cggcggcggc cctgcttaac cgcgcacgtc gcaccggccg acgggcggcc 16260
atgcgggccg ctcgaaggct ggccgcgggt attgtcactg tgccccccag gtccaggcga 16320
cgagcggccg ccgcagcagc cgcggccatt agtgctatga ctcagggtcg caggggcaac 16380
gtgtattggg tgcgcgactc ggttagcggc ctgcgcgtgc ccgtgcgcac ccgccccccg 16440
cgcaactaga ttgcaagaaa aaactactta gactcgtact gttgtatgta tccagcggcg 16500
gcggcgcgca acgaagctat gtccaagcgc aaaatcaaag aagagatgct ccaggtcatc 16560
gcgccggaga tctatggccc cccgaagaag gaagagcagg attacaagcc ccgaaagcta 16620
aagcgggtca aaaagaaaaa gaaagatgat gatgatgaac ttgacgacga ggtggaactg 16680
ctgcacgcta ccgcgcccag gcgacgggta cagtggaaag gtcgacgcgt aaaacgtgtt 16740
ttgcgacccg gcaccaccgt agtctttacg cccggtgagc gctccacccg cacctacaag 16800
cgcgtgtatg atgaggtgta cggcgacgag gacctgcttg agcaggccaa cgagcgcctc 16860
ggggagtttg cctacggaaa gcggcataag gacatgctgg cgttgccgct ggacgagggc 16920
aacccaacac ctagcctaaa gcccgtaaca ctgcagcagg tgctgcccgc gcttgcaccg 16980
tccgaagaaa agcgcggcct aaagcgcgag tctggtgact tggcacccac cgtgcagctg 17040
atggtaccca agcgccagcg actggaagat gtcttggaaa aaatgaccgt ggaacctggg 17100
ctggagcccg aggtccgcgt gcggccaatc aagcaggtgg cgccgggact gggcgtgcag 17160
accgtggacg ttcagatacc cactaccagt agcaccagta ttgccaccgc cacagagggc 17220
atggagacac aaacgtcccc ggttgcctca gcggtggcgg atgccgcggt gcaggcggtc 17280
gctgcggccg cgtccaagac ctctacggag gtgcaaacgg acccgtggat gtttcgcgtt 17340
tcagcccccc ggcgcccgcg cggttcgagg aagtacggcg ccgccagcgc gctactgccc 17400
gaatatgccc tacatccttc cattgcgcct acccccggct atcgtggcta cacctaccgc 17460
cccagaagac gagcaactac ccgacgccga accaccactg gaacccgccg ccgccgtcgc 17520
cgtcgccagc ccgtgctggc cccgatttcc gtgcgcaggg tggctcgcga aggaggcagg 17580
accctggtgc tgccaacagc gcgctaccac cccagcatcg tttaaaagcc ggtctttgtg 17640
gttcttgcag atatggccct cacctgccgc ctccgtttcc cggtgccggg attccgagga 17700
agaatgcacc gtaggagggg catggccggc cacggcctga cgggcggcat gcgtcgtgcg 17760
caccaccggc ggcggcgcgc gtcgcaccgt cgcatgcgcg gcggtatcct gcccctcctt 17820
attccactga tcgccgcggc gattggcgcc gtgcccggaa ttgcatccgt ggccttgcag 17880
gcgcagagac actgattaaa aacaagttgc atgtggaaaa atcaaaataa aaagtctgga 17940
ctctcacgct cgcttggtcc tgtaactatt ttgtagaatg gaagacatca actttgcgtc 18000
tctggccccg cgacacggct cgcgcccgtt catgggaaac tggcaagata tcggcaccag 18060
caatatgagc ggtggcgcct tcagctgggg ctcgctgtgg agcggcatta aaaatttcgg 18120
ttccaccgtt aagaactatg gcagcaaggc ctggaacagc agcacaggcc agatgctgag 18180
ggataagttg aaagagcaaa atttccaaca aaaggtggta gatggcctgg cctctggcat 18240
tagcggggtg gtggacctgg ccaaccaggc agtgcaaaat aagattaaca gtaagcttga 18300
tccccgccct cccgtagagg agcctccacc ggccgtggag acagtgtctc cagaggggcg 18360
tggcgaaaag cgtccgcgcc ccgacaggga agaaactctg gtgacgcaaa tagacgagcc 18420
tccctcgtac gaggaggcac taaagcaagg cctgcccacc acccgtccca tcgcgcccat 18480
ggctaccgga gtgctgggcc agcacacacc cgtaacgctg gacctgcctc cccccgccga 18540
cacccagcag aaacctgtgc tgccaggccc gaccgccgtt gttgtaaccc gtcctagccg 18600
cgcgtccctg cgccgcgccg ccagcggtcc gcgatcgttg cggcccgtag ccagtggcaa 18660
ctggcaaagc acactgaaca gcatcgtggg tctgggggtg caatccctga agcgccgacg 18720
atgcttctga atagctaacg tgtcgtatgt gtgtcatgta tgcgtccatg tcgccgccag 18780
aggagctgct gagccgccgc gcgcccgctt tccaagatgg ctaccccttc gatgatgccg 18840
cagtggtctt acatgcacat ctcgggccag gacgcctcgg agtacctgag ccccgggctg 18900
gtgcagtttg cccgcgccac cgagacgtac ttcagcctga ataacaagtt tagaaacccc 18960
acggtggcgc ctacgcacga cgtgaccaca gaccggtccc agcgtttgac gctgcggttc 19020
atccctgtgg accgtgagga tactgcgtac tcgtacaagg cgcggttcac cctagctgtg 19080
ggtgataacc gtgtgctgga catggcttcc acgtactttg acatccgcgg cgtgctggac 19140
aggggcccta cttttaagcc ctactctggc actgcctaca acgccctggc tcccaagggt 19200
gccccaaatc cttgcgaatg ggatgaagct gctactgctc ttgaaataaa cctagaagaa 19260
gaggacgatg acaacgaaga cgaagtagac gagcaagctg agcagcaaaa aactcacgta 19320
tttgggcagg cgccttattc tggtataaat attacaaagg agggtattca aataggtgtc 19380
gaaggtcaaa cacctaaata tgccgataaa acatttcaac ctgaacctca aataggagaa 19440
tctcagtggt acgaaactga aattaatcat gcagctggga gagtccttaa aaagactacc 19500
ccaatgaaac catgttacgg ttcatatgca aaacccacaa atgaaaatgg agggcaaggc 19560
attcttgtaa agcaacaaaa tggaaagcta gaaagtcaag tggaaatgca attttcttgc 19620
tcgtggacgt tctcaactac tgaggcagcc gcaggcaatg gtgataactt gactcctaaa 19680
gtggtattgt acagtgaaga tgtagatata gaaaccccag acactcatat ttcttacatg 19740
cccactatta aggaaggtaa ctcacgagaa ctaatgggcc aacaatctat gcccaacagg 19800
cctaattaca ttgcttttag ggacaatttt attggtctaa tgtattacaa cagcacgggt 19860
aatatgggtg ttctggcggg ccaagcatcg cagttgaatg ctgttgtaga tttgcaagac 19920
agaaacacag agctttcata ccagcttttg cttgattcca ttggtgatag aaccaggtac 19980
ttttctatgt ggaatcaggc tgttgacagc tatgatccag atgttagaat tattgaaaat 20040
catggaactg aagatgaact tccaaattac tgctttccac tgggaggtgt gattaataca 20100
gagactctta ccaaggtaaa acctaaaaca ggtcaggaaa atggatggga aaaagatgct 20160
acagaatttt cagataaaaa tgaaataaga gttggaaata attttgccat ggaaatcaat 20220
ctaaatgcca acctgtggag aaatttcctg tactccaaca tagcgctgta tttgcccgac 20280
aagctaaagt acagtccttc caacgtaaaa atttctgata acccaaacac ctacgactac 20340
atgaacaagc gagtggtggc tcccgggtta gtggactgct acattaacct tggagcacgc 20400
tggtcccttg actatatgga caacgtcaac ccatttaacc accaccgcaa tgctggcctg 20460
cgctaccgct caatgttgct gggcaatggt cgctatgtgc ccttccacat ccaggtgcct 20520
cagaagttct ttgccattaa aaacctcctt ctcctgccgg gctcatacac ctacgagtgg 20580
aacttcagga aggatgttaa catggttctg cagagctccc taggaaatga cctaagggtt 20640
gacggagcca gcattaagtt tgatagcatt tgcctttacg ccaccttctt ccccatggcc 20700
cacaacaccg cctccacgct tgaggccatg cttagaaacg acaccaacga ccagtccttt 20760
aacgactatc tctccgccgc caacatgctc taccctatac ccgccaacgc taccaacgtg 20820
cccatatcca tcccctcccg caactgggcg gctttccgcg gctgggcctt cacgcgcctt 20880
aagactaagg aaaccccatc actgggctcg ggctacgacc cttattacac ctactctggc 20940
tctataccct acctagatgg aaccttttac ctcaaccaca cctttaagaa ggtggccatt 21000
acctttgact cttctgtcag ctggcctggc aatgaccgcc tgcttacccc caacgagttt 21060
gaaattaagc gctcagttga cggggagggt tacaacgttg cccagtgtaa catgaccaaa 21120
gactggttcc tggtacaaat gctagctaac tacaacattg gctaccaggg cttctatatc 21180
ccagagagct acaaggaccg catgtactcc ttctttagaa acttccagcc catgagccgt 21240
caggtggtgg atgatactaa atacaaggac taccaacagg tgggcatcct acaccaacac 21300
aacaactctg gatttgttgg ctaccttgcc cccaccatgc gcgaaggaca ggcctaccct 21360
gctaacttcc cctatccgct tataggcaag accgcagttg acagcattac ccagaaaaag 21420
tttctttgcg atcgcaccct ttggcgcatc ccattctcca gtaactttat gtccatgggc 21480
gcactcacag acctgggcca aaaccttctc tacgccaact ccgcccacgc gctagacatg 21540
acttttgagg tggatcccat ggacgagccc acccttcttt atgttttgtt tgaagtcttt 21600
gacgtggtcc gtgtgcaccg gccgcaccgc ggcgtcatcg aaaccgtgta cctgcgcacg 21660
cccttctcgg ccggcaacgc cacaacataa agaagcaagc aacatcaaca acagctgccg 21720
ccatgggctc cagtgagcag gaactgaaag ccattgtcaa agatcttggt tgtgggccat 21780
attttttggg cacctatgac aagcgctttc caggctttgt ttctccacac aagctcgcct 21840
gcgccatagt caatacggcc ggtcgcgaga ctgggggcgt acactggatg gcctttgcct 21900
ggaacccgca ctcaaaaaca tgctacctct ttgagccctt tggcttttct gaccagcgac 21960
tcaagcaggt ttaccagttt gagtacgagt cactcctgcg ccgtagcgcc attgcttctt 22020
cccccgaccg ctgtataacg ctggaaaagt ccacccaaag cgtacagggg cccaactcgg 22080
ccgcctgtgg actattctgc tgcatgtttc tccacgcctt tgccaactgg ccccaaactc 22140
ccatggatca caaccccacc atgaacctta ttaccggggt acccaactcc atgctcaaca 22200
gtccccaggt acagcccacc ctgcgtcgca accaggaaca gctctacagc ttcctggagc 22260
gccactcgcc ctacttccgc agccacagtg cgcagattag gagcgccact tctttttgtc 22320
acttgaaaaa catgtaaaaa taatgtacta gagacacttt caataaaggc aaatgctttt 22380
atttgtacac tctcgggtga ttatttaccc ccacccttgc cgtctgcgcc gtttaaaaat 22440
caaaggggtt ctgccgcgca tcgctatgcg ccactggcag ggacacgttg cgatactggt 22500
gtttagtgct ccacttaaac tcaggcacaa ccatccgcgg cagctcggtg aagttttcac 22560
tccacaggct gcgcaccatc accaacgcgt ttagcaggtc gggcgccgat atcttgaagt 22620
cgcagttggg gcctccgccc tgcgcgcgcg agttgcgata cacagggttg cagcactgga 22680
acactatcag cgccgggtgg tgcacgctgg ccagcacgct cttgtcggag atcagatccg 22740
cgtccaggtc ctccgcgttg ctcagggcga acggagtcaa ctttggtagc tgccttccca 22800
aaaagggcgc gtgcccaggc tttgagttgc actcgcaccg tagtggcatc aaaaggtgac 22860
cgtgcccggt ctgggcgtta ggatacagcg cctgcataaa agccttgatc tgcttaaaag 22920
ccacctgagc ctttgcgcct tcagagaaga acatgccgca agacttgccg gaaaactgat 22980
tggccggaca ggccgcgtcg tgcacgcagc accttgcgtc ggtgttggag atctgcacca 23040
catttcggcc ccaccggttc ttcacgatct tggccttgct agactgctcc ttcagcgcgc 23100
gctgcccgtt ttcgctcgtc acatccattt caatcacgtg ctccttattt atcataatgc 23160
ttccgtgtag acacttaagc tcgccttcga tctcagcgca gcggtgcagc cacaacgcgc 23220
agcccgtggg ctcgtgatgc ttgtaggtca cctctgcaaa cgactgcagg tacgcctgca 23280
ggaatcgccc catcatcgtc acaaaggtct tgttgctggt gaaggtcagc tgcaacccgc 23340
ggtgctcctc gttcagccag gtcttgcata cggccgccag agcttccact tggtcaggca 23400
gtagtttgaa gttcgccttt agatcgttat ccacgtggta cttgtccatc agcgcgcgcg 23460
cagcctccat gcccttctcc cacgcagaca cgatcggcac actcagcggg ttcatcaccg 23520
taatttcact ttccgcttcg ctgggctctt cctcttcctc ttgcgtccgc ataccacgcg 23580
ccactgggtc gtcttcattc agccgccgca ctgtgcgctt acctcctttg ccatgcttga 23640
ttagcaccgg tgggttgctg aaacccacca tttgtagcgc cacatcttct ctttcttcct 23700
cgctgtccac gattacctct ggtgatggcg ggcgctcggg cttgggagaa gggcgcttct 23760
ttttcttctt gggcgcaatg gccaaatccg ccgccgaggt cgatggccgc gggctgggtg 23820
tgcgcggcac cagcgcgtct tgtgatgagt cttcctcgtc ctcggactcg atacgccgcc 23880
tcatccgctt ttttgggggc gcccggggag gcggcggcga cggggacggg gacgacacgt 23940
cctccatggt tgggggacgt cgcgccgcac cgcgtccgcg ctcgggggtg gtttcgcgct 24000
gctcctcttc ccgactggcc atttccttct cctataggca gaaaaagatc atggagtcag 24060
tcgagaagaa ggacagccta accgccccct ctgagttcgc caccaccgcc tccaccgatg 24120
ccgccaacgc gcctaccacc ttccccgtcg aggcaccccc gcttgaggag gaggaagtga 24180
ttatcgagca ggacccaggt tttgtaagcg aagacgacga ggaccgctca gtaccaacag 24240
aggataaaaa gcaagaccag gacaacgcag aggcaaacga ggaacaagtc gggcgggggg 24300
acgaaaggca tggcgactac ctagatgtgg gagacgacgt gctgttgaag catctgcagc 24360
gccagtgcgc cattatctgc gacgcgttgc aagagcgcag cgatgtgccc ctcgccatag 24420
cggatgtcag ccttgcctac gaacgccacc tattctcacc gcgcgtaccc cccaaacgcc 24480
aagaaaacgg cacatgcgag cccaacccgc gcctcaactt ctaccccgta tttgccgtgc 24540
cagaggtgct tgccacctat cacatctttt tccaaaactg caagataccc ctatcctgcc 24600
gtgccaaccg cagccgagcg gacaagcagc tggccttgcg gcagggcgct gtcatacctg 24660
atatcgcctc gctcaacgaa gtgccaaaaa tctttgaggg tcttggacgc gacgagaagc 24720
gcgcggcaaa cgctctgcaa caggaaaaca gcgaaaatga aagtcactct ggagtgttgg 24780
tggaactcga gggtgacaac gcgcgcctag ccgtactaaa acgcagcatc gaggtcaccc 24840
actttgccta cccggcactt aacctacccc ccaaggtcat gagcacagtc atgagtgagc 24900
tgatcgtgcg ccgtgcgcag cccctggaga gggatgcaaa tttgcaagaa caaacagagg 24960
agggcctacc cgcagttggc gacgagcagc tagcgcgctg gcttcaaacg cgcgagcctg 25020
ccgacttgga ggagcgacgc aaactaatga tggccgcagt gctcgttacc gtggagcttg 25080
agtgcatgca gcggttcttt gctgacccgg agatgcagcg caagctagag gaaacattgc 25140
actacacctt tcgacagggc tacgtacgcc aggcctgcaa gatctccaac gtggagctct 25200
gcaacctggt ctcctacctt ggaattttgc acgaaaaccg ccttgggcaa aacgtgcttc 25260
attccacgct caagggcgag gcgcgccgcg actacgtccg cgactgcgtt tacttatttc 25320
tatgctacac ctggcagacg gccatgggcg tttggcagca gtgcttggag gagtgcaacc 25380
tcaaggagct gcagaaactg ctaaagcaaa acttgaagga cctatggacg gccttcaacg 25440
agcgctccgt ggccgcgcac ctggcggaca tcattttccc cgaacgcctg cttaaaaccc 25500
tgcaacaggg tctgccagac ttcaccagtc aaagcatgtt gcagaacttt aggaacttta 25560
tcctagagcg ctcaggaatc ttgcccgcca cctgctgtgc acttcctagc gactttgtgc 25620
ccattaagta ccgcgaatgc cctccgccgc tttggggcca ctgctacctt ctgcagctag 25680
ccaactacct tgcctaccac tctgacataa tggaagacgt gagcggtgac ggtctactgg 25740
agtgtcactg tcgctgcaac ctatgcaccc cgcaccgctc cctggtttgc aattcgcagc 25800
tgcttaacga aagtcaaatt atcggtacct ttgagctgca gggtccctcg cctgacgaaa 25860
agtccgcggc tccggggttg aaactcactc cggggctgtg gacgtcggct taccttcgca 25920
aatttgtacc tgaggactac cacgcccacg agattaggtt ctacgaagac caatcccgcc 25980
cgccaaatgc ggagcttacc gcctgcgtca ttacccaggg ccacattctt ggccaattgc 26040
aagccatcaa caaagcccgc caagagtttc tgctacgaaa gggacggggg gtttacttgg 26100
acccccagtc cggcgaggag ctcaacccaa tccccccgcc gccgcagccc tatcagcagc 26160
agccgcgggc ccttgcttcc caggatggca cccaaaaaga agctgcagct gccgccgcca 26220
cccacggacg aggaggaata ctgggacagt caggcagagg aggttttgga cgaggaggag 26280
gaggacatga tggaagactg ggagagccta gacgaggaag cttccgaggt cgaagaggtg 26340
tcagacgaaa caccgtcacc ctcggtcgca ttcccctcgc cggcgcccca gaaatcggca 26400
accggttcca gcatggctac aacctccgct cctcaggcgc cgccggcact gcccgttcgc 26460
cgacccaacc gtagatggga caccactgga accagggccg gtaagtccaa gcagccgccg 26520
ccgttagccc aagagcaaca acagcgccaa ggctaccgct catggcgcgg gcacaagaac 26580
gccatagttg cttgcttgca agactgtggg ggcaacatct ccttcgcccg ccgctttctt 26640
ctctaccatc acggcgtggc cttcccccgt aacatcctgc attactaccg tcatctctac 26700
agcccatact gcaccggcgg cagcggcagc ggcagcaaca gcagcggcca cacagaagca 26760
aaggcgaccg gatagcaaga ctctgacaaa gcccaagaaa tccacagcgg cggcagcagc 26820
aggaggagga gcgctgcgtc tggcgcccaa cgaacccgta tcgacccgcg agcttagaaa 26880
caggattttt cccactctgt atgctatatt tcaacagagc aggggccaag aacaagagct 26940
gaaaataaaa aacaggtctc tgcgatccct cacccgcagc tgcctgtatc acaaaagcga 27000
agatcagctt cggcgcacgc tggaagacgc ggaggctctc ttcagtaaat actgcgcgct 27060
gactcttaag gactagtttc gcgccctttc tcaaatttaa gcgcgaaaac tacgtcatct 27120
ccagcggcca cacccggcgc cagcacctgt cgtcagcgcc attatgagca aggaaattcc 27180
cacgccctac atgtggagtt accagccaca aatgggactt gcggctggag ctgcccaaga 27240
ctactcaacc cgaataaact acatgagcgc gggaccccac atgatatccc gggtcaacgg 27300
aatccgcgcc caccgaaacc gaattctctt ggaacaggcg gctattacca ccacacctcg 27360
taataacctt aatccccgta gttggcccgc tgccctggtg taccaggaaa gtcccgctcc 27420
caccactgtg gtacttccca gagacgccca ggccgaagtt cagatgacta actcaggggc 27480
gcagcttgcg ggcggctttc gtcacagggt gcggtcgccc gggcagggta taactcacct 27540
gacaatcaga gggcgaggta ttcagctcaa cgacgagtcg gtgagctcct cgcttggtct 27600
ccgtccggac gggacatttc agatcggcgg cgccggccgt ccttcattca cgcctcgtca 27660
ggcaatccta actctgcaga cctcgtcctc tgagccgcgc tctggaggca ttggaactct 27720
gcaatttatt gaggagtttg tgccatcggt ctactttaac cccttctcgg gacctcccgg 27780
ccactatccg gatcaattta ttcctaactt tgacgcggta aaggactcgg cggacggcta 27840
cgactgaatg ttaagtggag aggcagagca actgcgcctg aaacacctgg tccactgtcg 27900
ccgccacaag tgctttgccc gcgactccgg tgagttttgc tactttgaat tgcccgagga 27960
tcatatcgag ggcccggcgc acggcgtccg gcttaccgcc cagggagagc ttgcccgtag 28020
cctgattcgg gagtttaccc agcgccccct gctagttgag cgggacaggg gaccctgtgt 28080
tctcactgtg atttgcaact gtcctaacct tggattacat caagatcttt gttgccatct 28140
ctgtgctgag tataataaat acagaaatta aaatatactg gggctcctat cgccatcctg 28200
taaacgccac cgtcttcacc cgcccaagca aaccaaggcg aaccttacct ggtactttta 28260
acatctctcc ctctgtgatt tacaacagtt tcaacccaga cggagtgagt ctacgagaga 28320
acctctccga gctcagctac tccatcagaa aaaacaccac cctccttacc tgccgggaac 28380
gtacgagtgc gtcaccggcc gctgcaccac acctaccgcc tgaccgtaaa ccagactttt 28440
tccggacaga cctcaataac tctgtttacc agaacaggag gtgagcttag aaaaccctta 28500
gggtattagg ccaaaggcgc agctactgtg gggtttatga acaattcaag caactctacg 28560
ggctattcta attcaggttt ctctagaatc ggggttgggg ttattctctg tcttgtgatt 28620
ctctttattc ttatactaac gcttctctgc ctaaggctcg ccgcctgctg tgtgcacatt 28680
tgcatttatt gtcagctttt taaacgctgg ggtcgccacc caagatgatt aggtacataa 28740
tcctaggttt actcaccctt gcgtcagccc acggtaccac ccaaaaggtg gattttaagg 28800
agccagcctg taatgttaca ttcgcagctg aagctaatga gtgcaccact cttataaaat 28860
gcaccacaga acatgaaaag ctgcttattc gccacaaaaa caaaattggc aagtatgctg 28920
tttatgctat ttggcagcca ggtgacacta cagagtataa tgttacagtt ttccagggta 28980
aaagtcataa aacttttatg tatacttttc cattttatga aatgtgcgac attaccatgt 29040
acatgagcaa acagtataag ttgtggcccc cacaaaattg tgtggaaaac actggcactt 29100
tctgctgcac tgctatgcta attacagtgc tcgctttggt ctgtacccta ctctatatta 29160
aatacaaaag cagacgcagc tttattgagg aaaagaaaat gccttaattt actaagttac 29220
aaagctaatg tcaccactaa ctgctttact cgctgcttgc aaaacaaatt caaaaagtta 29280
gcattataat tagaatagga tttaaacccc ccggtcattt cctgctcaat accattcccc 29340
tgaacaattg actctatgtg ggatatgctc cagcgctaca accttgaagt caggcttcct 29400
ggatgtcagc atctgacttt ggccagcacc tgtcccgcgg atttgttcca gtccaactac 29460
agcgacatcg atatggcttc gtatcccggc catcagcacg cgtctgcgtt cgaccaggct 29520
gcgcgttctc gcggccatag caaccgacgt acggcgttgc gccctcgccg gcagcaagaa 29580
gccacggaag tccgcccgga gcagaaaatg cccacgctac tgcgggttta tatagacggt 29640
ccccacggga tggggaaaac caccaccacg caactgctgg tggccctggg ttcgcgcgac 29700
gatatcgtct acgtacccga gccgatgact tactggcggg tgctgggggc ttccgagaca 29760
atcgcgaaca tctacaccac acaacaccgc ctcgaccagg gtgagatatc ggccggggac 29820
gcggcggtgg taatgacaag cgcccagata acaatgggca tgccttatgc cgtgaccgac 29880
gccgttctgg ctcctcatat cgggggggag gctgggagct cacatgcccc gcccccggcc 29940
ctcaccctca tcttcgaccg ccatcccatc gccgccctcc tgtgctaccc gcccgcgcga 30000
taccttatgg gcagcatgac cccccaggcc gtgccggcgt tcgtggccct catcccgccg 30060
accttgcccg gcacaaacat cgtgttgggg gcccttccgg aggacagaca catcgaccgc 30120
ctggccaaac gccagcgccc cggcgagcgg cttgacctgg ctatgctggc cgcgattcgc 30180
cgcgtttacg ggctgcttgc caatacggtg cggtatctgc agggcggcgg gtcgtggcgg 30240
gaggattggg gacagctttc ggggacggcc gtgccgcccc agggtgccga gccccagagc 30300
aacgcgggcc cacgacccca tatcggggac acgttattta ccctgtttcg ggcccccgag 30360
ttgctggccc ccaacggcga cctgtacaac gtgtttgcct gggccttgga cgtcttggcc 30420
aaacgcctcc gtcccatgca cgtccttatc ctggattacg accaatcgcc cgccggctgc 30480
cgggacgccc tgctgcaact tacctccggg atgatccaga cccacgtcac caccccaggc 30540
tccataccga cgatctgcga cctggcgcgc acgtttgccc gggagatggg ggcggctcac 30600
tgaatcgatt gatggaatcc atagattgga cggactgaaa cacatgttct tttctcttac 30660
agtatgatta aatgagacat gattcctcga gtttttatat tactgaccct tgttgcgctt 30720
ttttgtgcgt gctccacatt ggctgcggtt tctcacatcg aagtagactg cattccagcc 30780
ttcacagtct atttgcttta cggatttgtc accctcacgc tcatctgcag cctcatcact 30840
gtggtcatcg cctttatcca gtgcattgac tgggtctgtg tgcgctttgc atatctcaga 30900
caccatcccc agtacaggga caggactata gctgagcttc ttagaattct ttaattatga 30960
aatttactgt gacttttctg ctgattattt gcaccctatc tgcgttttgt tccccgacct 31020
ccaagcctca aagacatata tcatgcagat tcactcgtat atggaatatt ccaagttgct 31080
acaatgaaaa aagcgatctt tccgaagcct ggttatatgc aatcatctct gttatggtgt 31140
tctgcagtac catcttagcc ctagctatat atccctacct tgacattggc tggaaacgaa 31200
tagatgccat gaaccaccca actttccccg cgcccgctat gcttccactg caacaagttg 31260
ttgccggcgg ctttgtccca gccaatcagc ctcgccccac ttctcccacc cccactgaaa 31320
tcagctactt taatctaaca ggaggagatg actgacaccc tagatctaga aatggacgga 31380
attattacag agcagcgcct gctagaaaga cgcagggcag cggccgagca acagcgcatg 31440
aatcaagagc tccaagacat ggttaacttg caccagtgca aaaggggtat cttttgtctg 31500
gtaaagcagg ccaaagtcac ctacgacagt aataccaccg gacaccgcct tagctacaag 31560
ttgccaacca agcgtcagaa attggtggtc atggtgggag aaaagcccat taccataact 31620
cagcactcgg tagaaaccga aggctgcatt cactcacctt gtcaaggacc tgaggatctc 31680
tgcaccctta ttaagaccct gtgcggtctc aaagatctta ttccctttaa ctaataaaaa 31740
aaaataataa agcatcactt acttaaaatc agttagcaaa tttctgtcca gtttattcag 31800
cagcacctcc ttgccctcct cccagctctg gtattgcagc ttcctcctgg ctgcaaactt 31860
tctccacaat ctaaatggaa tgtcagtttc ctcctgttcc tgtccatccg cacccactat 31920
cttcatgttg ttgcagatga agcgcgcaag accgtctgaa gataccttca accccgtgta 31980
tccatatgac acggaaaccg gtcctccaac tgtgcctttt cttactcctc cctttgtatc 32040
ccccaatggg tttcaagaga gtccccctgg ggtactctct ttgcgcctat ccgaacctct 32100
agttacctcc aatggcatgc ttgcgctcaa aatgggcaac ggcctctctc tggacgaggc 32160
cggcaacctt acctcccaaa atgtaaccac tgtgagccca cctctcaaaa aaaccaagtc 32220
aaacataaac ctggaaatat ctgcacccct cacagttacc tcagaagccc taactgtggc 32280
tgccgccgca cctctaatgg tcgcgggcaa cacactcacc atgcaatcac aggccccgct 32340
aaccgtgcac gactccaaac ttagcattgc cacccaagga cccctcacag tgtcagaagg 32400
aaagctagcc ctgcaaacat caggccccct caccaccacc gatagcagta cccttactat 32460
cactgcctca ccccctctaa ctactgccac tggtagcttg ggcattgact tgaaagagcc 32520
catttataca caaaatggaa aactaggact aaagtacggg gctcctttgc atgtaacaga 32580
cgacctaaac actttgaccg tagcaactgg tccaggtgtg actattaata atacttcctt 32640
gcaaactaaa gttactggag ccttgggttt tgattcacaa ggcaatatgc aacttaatgt 32700
agcaggagga ctaaggattg attctcaaaa cagacgcctt atacttgatg ttagttatcc 32760
gtttgatgct caaaaccaac taaatctaag actaggacag ggccctcttt ttataaactc 32820
agcccacaac ttggatatta actacaacaa aggcctttac ttgtttacag cttcaaacaa 32880
ttccaaaaag cttgaggtta acctaagcac tgccaagggg ttgatgtttg acgctacagc 32940
catagccatt aatgcaggag atgggcttga atttggttca cctaatgcac caaacacaaa 33000
tcccctcaaa acaaaaattg gccatggcct agaatttgat tcaaacaagg ctatggttcc 33060
taaactagga actggcctta gttttgacag cacaggtgcc attacagtag gaaacaaaaa 33120
taatgataag ctaactttgt ggaccacacc agctccatct cctaactgta gactaaatgc 33180
agagaaagat gctaaactca ctttggtctt aacaaaatgt ggcagtcaaa tacttgctac 33240
agtttcagtt ttggctgtta aaggcagttt ggctccaata tctggaacag ttcaaagtgc 33300
tcatcttatt ataagatttg acgaaaatgg agtgctacta aacaattcct tcctggaccc 33360
agaatattgg aactttagaa atggagatct tactgaaggc acagcctata caaacgctgt 33420
tggatttatg cctaacctat cagcttatcc aaaatctcac ggtaaaactg ccaaaagtaa 33480
cattgtcagt caagtttact taaacggaga caaaactaaa cctgtaacac taaccattac 33540
actaaacggt acacaggaaa caggagacac aactccaagt gcatactcta tgtcattttc 33600
atgggactgg tctggccaca actacattaa tgaaatattt gccacatcct cttacacttt 33660
ttcatacatt gcccaagaat aaagaatcgt ttgtgttatg tttcaacgtg tttatttttc 33720
aattgcagaa aatttcaagt catttttcat tcagtagtat agccccacca ccacatagct 33780
tatacagatc accgtacctt aatcaaactc acagaaccct agtattcaac ctgccacctc 33840
cctcccaaca cacagagtac acagtccttt ctccccggct ggccttaaaa agcatcatat 33900
catgggtaac agacatattc ttaggtgtta tattccacac ggtttcctgt cgagccaaac 33960
gctcatcagt gatattaata aactccccgg gcagctcact taagttcatg tcgctgtcca 34020
gctgctgagc cacaggctgc tgtccaactt gcggttgctt aacgggcggc gaaggagaag 34080
tccacgccta catgggggta gagtcataat cgtgcatcag gatagggcgg tggtgctgca 34140
gcagcgcgcg aataaactgc tgccgccgcc gctccgtcct gcaggaatac aacatggcag 34200
tggtctcctc agcgatgatt cgcaccgccc gcagcataag gcgccttgtc ctccgggcac 34260
agcagcgcac cctgatctca cttaaatcag cacagtaact gcagcacagc accacaatat 34320
tgttcaaaat cccacagtgc aaggcgctgt atccaaagct catggcgggg accacagaac 34380
ccacgtggcc atcataccac aagcgcaggt agattaagtg gcgacccctc ataaacacgc 34440
tggacataaa cattacctct tttggcatgt tgtaattcac cacctcccgg taccatataa 34500
acctctgatt aaacatggcg ccatccacca ccatcctaaa ccagctggcc aaaacctgcc 34560
cgccggctat acactgcagg gaaccgggac tggaacaatg acagtggaga gcccaggact 34620
cgtaaccatg gatcatcatg ctcgtcatga tatcaatgtt ggcacaacac aggcacacgt 34680
gcatacactt cctcaggatt acaagctcct cccgcgttag aaccatatcc cagggaacaa 34740
cccattcctg aatcagcgta aatcccacac tgcagggaag acctcgcacg taactcacgt 34800
tgtgcattgt caaagtgtta cattcgggca gcagcggatg atcctccagt atggtagcgc 34860
gggtttctgt ctcaaaagga ggtagacgat ccctactgta cggagtgcgc cgagacaacc 34920
gagatcgtgt tggtcgtagt gtcatgccaa atggaacgcc ggacgtagtc atatttcctg 34980
aagcaaaacc aggtgcgggc gtgacaaaca gatctgcgtc tccggtctcg ccgcttagat 35040
cgctctgtgt agtagttgta gtatatccac tctctcaaag catccaggcg ccccctggct 35100
tcgggttcta tgtaaactcc ttcatgcgcc gctgccctga taacatccac caccgcagaa 35160
taagccacac ccagccaacc tacacattcg ttctgcgagt cacacacggg aggagcggga 35220
agagctggaa gaaccatgtt ttttttttta ttccaaaaga ttatccaaaa cctcaaaatg 35280
aagatctatt aagtgaacgc gctcccctcc ggtggcgtgg tcaaactcta cagccaaaga 35340
acagataatg gcatttgtaa gatgttgcac aatggcttcc aaaaggcaaa cggccctcac 35400
gtccaagtgg acgtaaaggc taaacccttc agggtgaatc tcctctataa acattccagc 35460
accttcaacc atgcccaaat aattctcatc tcgccacctt ctcaatatat ctctaagcaa 35520
atcccgaata ttaagtccgg ccattgtaaa aatctgctcc agagcgccct ccaccttcag 35580
cctcaagcag cgaatcatga ttgcaaaaat tcaggttcct cacagacctg tataagattc 35640
aaaagcggaa cattaacaaa aataccgcga tcccgtaggt cccttcgcag ggccagctga 35700
acataatcgt gcaggtctgc acggaccagc gcggccactt ccccgccagg aaccttgaca 35760
aaagaaccca cactgattat gacacgcata ctcggagcta tgctaaccag cgtagccccg 35820
atgtaagctt tgttgcatgg gcggcgatat aaaatgcaag gtgctgctca aaaaatcagg 35880
caaagcctcg cgcaaaaaag aaagcacatc gtagtcatgc tcatgcagat aaaggcaggt 35940
aagctccgga accaccacag aaaaagacac catttttctc tcaaacatgt ctgcgggttt 36000
ctgcataaac acaaaataaa ataacaaaaa aacatttaaa cattagaagc ctgtcttaca 36060
acaggaaaaa caacccttat aagcataaga cggactacgg ccatgccggc gtgaccgtaa 36120
aaaaactggt caccgtgatt aaaaagcacc accgacagct cctcggtcat gtccggagtc 36180
ataatgtaag actcggtaaa cacatcaggt tgattcatcg gtcagtgcta aaaagcgacc 36240
gaaatagccc gggggaatac atacccgcag gcgtagagac aacattacag cccccatagg 36300
aggtataaca aaattaatag gagagaaaaa cacataaaca cctgaaaaac cctcctgcct 36360
aggcaaaata gcaccctccc gctccagaac aacatacagc gcttcacagc ggcagcctaa 36420
cagtcagcct taccagtaaa aaagaaaacc tattaaaaaa acaccactcg acacggcacc 36480
agctcaatca gtcacagtgt aaaaaagggc caagtgcaga gcgagtatat ataggactaa 36540
aaaatgacgt aacggttaaa gtccacaaaa aacacccaga aaaccgcacg cgaacctacg 36600
cccagaaacg aaagccaaaa aacccacaac ttcctcaaat cgtcacttcc gttttcccac 36660
gttacgtaac ttcccatttt aagaaaacta caattcccaa cacatacaag ttactccgcc 36720
ctaaaaccta cgtcacccgc cccgttccca cgccccgcgc cacgtcacaa actccacccc 36780
ctcattatca tattggcttc aatccaaaat aaggtatatt attgatgatg 36830
Claims (6)
1. An oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is Ad 5/delta E1A/TMVP1 delta ADP/HSV-TK, the nucleotide sequence of the oncolytic adenovirus recombinant carrying TMVP1 and HSV-TK is shown as SEQ ID No.23, 27 basic groups shown as SEQ ID No.1 in the 920nt-946nt region are deleted in the E1A conserved sequence 2 region of a human 5-type adenovirus gene, the 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-29714 region of the ADP gene to form a deletion region, and the deletion region is inserted in the full-length enzyme digestion coding sequence shown as SEQ ID No.22 and used for coding HSV-TK gene and is introduced into Cla1 site.
2. The method of constructing an oncolytic adenoviral recombinant harboring TMVP1 and HSV-TK according to claim 1, comprising the steps of:
step 1: targeted deletion of human adenovirus type 5 genes
Utilizing gene synthesis and homologous recombination to directionally delete 27 bases of 920nt-946nt in the E1A conserved sequence 2 region of the human 5-type adenovirus gene as shown in SEQ ID NO.1 to obtain the directionally deleted human 5-type adenovirus gene Ad 5/delta E1A;
step 2: preparation of Ad 5/. DELTA.E 1A/TMVP1
Inserting a gene sequence which is shown as SEQ ID NO.15 and encodes tumor targeting peptide TMVP1 into the 19641nt-19655nt region of the Hexon hypervariable region 5 of the human adenovirus type 5 gene obtained in the step 1 after targeted deletion to obtain Ad 5/delta E1A/TMVP 1;
and step 3: preparation of oncolytic adenovirus recombinant Ad 5/. DELTA.E 1A/TMVP 1. DELTA.ADP/HSV-TK carrying TMVP1 and HSV-TK
The E3 region of Ad 5/delta E1A/TMVP1 obtained in step 2 is located in the 29477nt-29714nt region of the ADP gene to form a deletion region, the full-length coding sequence of the HSV-TK gene shown in SEQ ID NO.22 is inserted into the deletion region, and the Cla1 enzyme cutting site is introduced, so that the oncolytic adenovirus recombinant Ad 5/delta E1A/TMVP1 delta ADP/HSV-TK carrying the TMVP1 and the HSV-TK shown in SEQ ID NO.23 is obtained.
3. Use of an oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK according to claim 1 for the preparation of a medicament for the treatment of a tumour.
4. Use of an oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK according to claim 1 for the preparation of a gene therapy vector.
5. Use of an oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK according to claim 1 for the preparation of a medicament for the amelioration of resistance to anti-tumour chemotherapeutic agents.
6. Use of the oncolytic adenoviral recombinant carrying TMVP1 and HSV-TK of claim 1 for the preparation of a sensitizer for anti-tumor chemotherapeutic drugs.
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