CN115572735A - Method for constructing recombinant adenovirus vector, recombinant adenovirus vector constructed by same and application - Google Patents

Abstract

The invention provides a method for constructing a recombinant adenovirus vector, which uses an E4 gene sequence of a C subtype human adenovirus genome to modify an E4 gene sequence of AdC6 so as to improve the yield of recombinant adenovirus. The method can construct a non-replicative adenovirus vector which can be used as a vaccine vector, and experiments prove that exogenous genes can be inserted into the E1/E3/E4/Fiber/Hexon and other regions of the adenovirus vector for vaccine development or gene therapy. The method can also construct a conditionally replicating adenovirus vector, namely an oncolytic virus vector, and various oncolytic-promoting exogenous genes can be inserted into regions such as delta E3, E1 or E4 of the conditionally replicating adenovirus vector so as to enhance the oncolytic effect: experiments prove that the obtained recombinant oncolytic virus can be replicated in various tumor cells and can crack the tumor cells; in animal experiments, the recombinant oncolytic virus can effectively inhibit tumor progression and even eliminate tumor, and shows that the recombinant oncolytic virus has the potential of treating tumor and can be further promoted to clinic.

Description

Method for constructing recombinant adenovirus vector, recombinant adenovirus vector constructed by same and application

Technical Field

The invention relates to a method for constructing a recombinant adenovirus vector, the recombinant adenovirus vector constructed by the method and application of the recombinant adenovirus vector.

Background

Cancer is the leading cause of human death, with 1930 million new cases of cancer and 1000 million cases of cancer death expected to occur globally in 2020. Due to the diversity, drug resistance, infiltration and complex tumor microenvironment of tumors, the traditional treatment method cannot effectively cure the tumors, and the emergence of oncolytic viruses provides a new treatment strategy for the treatment of the tumors. Oncolytic viruses are a class of viruses that selectively infect tumor cells and lyse tumor cells without affecting normal cells.

Commonly used oncolytic viruses are vaccinia virus, adenovirus, herpes virus, newcastle disease virus, poliovirus, and early clinical trials based on these viruses are in progress. Among them, adenovirus has become one of the hot spots of oncolytic virus research due to its unique advantages. The advantages of adenovirus as an oncolytic virus are quite obvious: the research on adenovirus is more thorough, and the structure, function and interaction with host of the gene are studied very deeply; the adenovirus genome is about 36KB, and the gene modification is easy; the adenovirus can not be integrated into a host genome, so that the insertion mutation can not be generated, and the safety is good; adenoviruses can infect a variety of mammalian cells, and thus a number of strategies are used to improve the specificity and selectivity of oncolytic adenoviruses for treating cancer.

Oncolytic virus H101, a human adenovirus type 5 (AdHu 5) vector, is an approved oncolytic adenovirus for use in combination with chemotherapy in the treatment of head and neck squamous cell carcinoma. However, the therapeutic effect of the anti-AdHu 5 pre-existing neutralizing antibody is limited due to the wide existence of the anti-AdHu 5 pre-existing neutralizing antibody in the population; in addition, it has been shown that the direct binding of hypervariable regions in AdHu5 Hexon to coagulation factor X results in AdHu5 having tropism for the liver, thereby affecting its therapeutic efficacy as an oncolytic virus.

Meanwhile, since adenovirus can simultaneously activate T cell immune response and humoral immune response, adenovirus vectors are also widely used in gene therapy and vaccine development, such as: vaccines against diseases such as HIV, influenza, ebola, malaria, rabies, and novel coronaviruses have been developed.

Because the adenovirus genome is relatively large, the construction of the recombinant adenovirus vector is difficult to complete by common molecular biology methods, and therefore, the construction of the adenovirus vector is still a technical bottleneck. Traditional adenovirus vector construction strategies mainly include homologous recombination and direct genome cloning, and are time-consuming and labor-consuming, and take more than 2 months at the fastest speed. A further strategy to improve on this is isothermal assembly, which, although claimed to require only about 1 week, only refers to the time required to join multiple fragments together into one clone by isothermal assembly, not including the time and validation process required to obtain multiple small fragments with complementary sequences at the linker sites. Furthermore, these strategies all rely on first obtaining the wild-type virus and amplifying and purifying the virus to obtain its genomic DNA. If only the adenovirus gene sequence is known, the construction of the adenovirus vector cannot be completed. The rapid development and wide application of synthetic biology bring important innovation for the construction of adenovirus vectors. In contrast to the research direction of traditional biology, synthetic biology is a step-by-step building of parts starting from the most basic elements, in the process of which genetic material is designed and modified to finally obtain ideal new organisms.

Adenovirus type 6 (AdC 6) from chimpanzees is commonly packaged and amplified with HEK293 cells incorporating the E1 gene of a human adenovirus subtype C (e.g., adHu1, adHu2, adHu6, adHu 5). Although the interaction between subtype C human adenovirus E1 and E4 proteins is relatively conserved, the binding of chimpanzee-type adenovirus E4 protein to subtype C human adenovirus E1 may be unstable or less efficient, resulting in reduced chimpanzee adenovirus production.

Disclosure of Invention

Adenovirus type 6 (AdC 6) from chimpanzees, having GenBank accession no: AY530877.1 (Update Date 2004-08-19). The adenovirus genome contains E1-E4 early genes, E1A can activate promoters of E1B, E2A, E2B, E3 and E4, and the adenovirus cannot replicate after deletion. The E3 gene functions primarily to disrupt the host's immune defense mechanisms, independent of replication of the viral genome. The E4 region comprises 7 open reading frames, the products of which are ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 and ORF6/7 respectively, and part of the protein can interact with the E1 region protein to influence the replication of adenovirus. For improved safety, adenoviruses used as vaccine vectors are usually replication-defective, i.e., the E1 gene is deleted; oncolytic adenoviruses are usually conditionally replicating, one of which is the addition of a tumor-specific promoter in front of E1A. The adenovirus is usually packaged and amplified by HEK293 cells integrated with AdHu5 (C subtype human adenoviruses also include AdHu1, adHu2, adHu6 and the like, and the similarity of E1 genes of the AdHu6 and the like) E1 genes of C subtype human adenoviruses. However, the combination of chimpanzee adenovirus E4 protein and subtype C human adenovirus E1 may be unstable or have reduced efficiency, resulting in reduced chimpanzee adenovirus production. The technical problem to be solved by the invention is how to construct adenovirus with higher yield based on chimpanzee adenovirus AdC6 and apply the adenovirus.

In order to solve the above problems, the present invention provides a method for constructing a recombinant adenovirus vector, wherein the method comprises the step of modifying chimpanzee adenovirus AdC6 to obtain the recombinant adenovirus vector, wherein the modification comprises: the E1 gene sequence of AdC6 is completely or partially deleted, the E3 gene sequence of AdC6 is partially deleted, and the E4 gene sequence of AdC6 is modified by the E4 gene sequence of the C subtype human adenovirus genome.

In the above method, the E3 gene sequence of the partially deleted AdC6 is A1 or A2:

a1, reserving ORF1 sequence of AdC6E 3 gene, and partially or completely deleting other sequences of AdC6E 3 gene;

a2, reserving ORF1 sequence and ORF9 sequence of AdC6E 3 gene, and deleting other sequence of AdC6E 3 gene partially or totally.

In the above method, the E4 gene sequence of AdC6 modified by the E4 gene sequence of the C subtype human adenovirus genome is specifically selected from any one of B1-B3:

b1, replacing an ORF6 sequence and/or an ORF6/7 sequence of the AdC6E 4 gene by a corresponding sequence of a C subtype human adenovirus genome;

b2, replacing an ORF4 sequence of the AdC6E 4 gene by a corresponding sequence of a C subtype human adenovirus genome on the basis of the B1;

b3, deleting ORF1 sequence and/or ORF2 sequence of AdC6E 4 gene on the basis of B1 or B2.

The corresponding sequence of the ORF6 sequence of the AdC6E 4 gene is an ORF6 sequence of an E4 gene of a C subtype human adenovirus genome; the corresponding sequence of the ORF6/7 sequence of the AdC6E 4 gene is the ORF6/7 sequence of the E4 gene of the C subtype human adenovirus genome; the corresponding sequence of the ORF4 sequence of the AdC6E 4 gene is the ORF4 sequence of the E4 gene of the subtype C human adenovirus genome.

The subtype C human adenovirus can be selected from human adenovirus type 1 (AdHu 1), human adenovirus type 2 (AdHu 2), human adenovirus type 5 (AdHu 5) or human adenovirus type 6 (AdHu 6), and is preferably human adenovirus type 5 (AdHu 5).

In the above method, the recombinant adenovirus vector is a non-replicative adenovirus vector, and the E1 gene sequence in which the AdC6 is completely or partially deleted is a completely deleted AdC6E1 gene sequence.

In the above method, the recombinant adenovirus vector is any one of the following vectors 1) to 5):

1) Completely deleting an E1 region, an ORF2, an ORF3, an ORF4, an ORF5, an ORF6, an ORF7, an ORF8 and an ORF9 sequence in the E3 region on the basis of AdC6, reserving the ORF1 sequence, and replacing the original E4 region ORF6 sequence of AdC6 with an E4 region ORF6 sequence of a C subtype human adenovirus type 5 to obtain a recombinant adenovirus, which is a corresponding recombinant adenovirus vector named pAdC6XY 2; the nucleotide sequence of pAdC6XY2 is a nucleotide sequence obtained by deleting 563-3413 th sites (the deleted sequence can be replaced by a sequence containing an enzyme cutting site for facilitating subsequent insertion of a foreign gene) in the sequence 1 of the sequence table, deleting 27441-31864 th sites (the deleted sequence can be replaced by a sequence containing an enzyme cutting site for facilitating subsequent insertion of a foreign gene) in the sequence 1 of the sequence table, and replacing 33841-34746 th sites (the sequence containing the enzyme cutting site can be inserted before 33841 and after 34746 th sites for facilitating subsequent insertion of a foreign gene) in the sequence 2 of the sequence table;

2) Completely deleting an E1 region, deleting ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 sequences of the E3 region on the basis of AdC6, reserving the ORF1 sequence, and replacing the original E4 region ORF6/7, ORF6 and ORF4 sequences of the AdC6 by E4 region ORF6/7, ORF6 and ORF4 sequences of a C subtype human adenovirus type 5 to obtain a recombinant adenovirus with a corresponding recombinant adenovirus vector named pAdC6XY 3; the nucleotide sequence of pAdC6XY3 is obtained by deleting 563-3413 th sites (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site), from 27441-31864 th sites (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site) of the sequence 1 in the sequence table, and replacing 33593-35026 th sites (for the convenience of subsequent insertion of a foreign gene, a sequence containing an enzyme cutting site can be inserted before 33593 rd site) of the sequence 1 in the sequence table by the 1 st-1440 th sites of the sequence 2 in the sequence table;

3) Completely deleting an E1 region, an ORF2, an ORF3, an ORF4, an ORF5, an ORF6, an ORF7, an ORF8 and an ORF9 sequence in the E3 region on the basis of AdC6, reserving the ORF1 sequence, replacing the original E4 region ORF6/7, ORF6 and ORF4 sequence of AdC6 by an E4 region ORF6/7, an ORF6 and an ORF4 sequence of a C subtype human adenovirus type 5, and deleting the original E4 region ORF1 and the ORF2 of the AdC6 to obtain a recombinant adenovirus, and a corresponding recombinant adenovirus vector named pAdC6XY 4; the nucleotide sequence of pAdC6XY4 is a nucleotide sequence obtained by deleting 563-3413 th positions (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site), from 27441-31864 th positions (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site), from 1 st position to 1440 th positions (for the convenience of subsequent insertion of a foreign gene, a sequence containing an enzyme cutting site can be inserted before 33593 rd position) of the sequence 1 in the sequence table, from 35381-36190 th positions (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site) of the sequence 1 in the sequence table;

4) Completely deleting the E1 region, deleting the ORF2, ORF3, ORF4, ORF5, ORF6, ORF7 and ORF8 sequences of the E3 region on the basis of AdC6, reserving the ORF1 and ORF9 sequences, and replacing the original E4 region ORF6/7, ORF6 and ORF4 sequences of the AdC6 by the E4 region ORF6/7, ORF6 and ORF4 sequences of the C subtype human adenovirus type 5 to obtain a recombinant adenovirus with a corresponding recombinant adenovirus vector named pAdC6XY 5; the nucleotide sequence of pAdC6XY5 is a nucleotide sequence obtained by deleting 563-3413 th positions (the deleted sequence can be replaced by a sequence containing an enzyme cutting site for facilitating subsequent insertion of a foreign gene) in the sequence 1 of the sequence table, deleting 27441-31456 th positions (the deleted sequence can be replaced by a sequence containing an enzyme cutting site for facilitating subsequent insertion of a foreign gene) in the sequence 1 of the sequence table, and replacing 33593-35026 th positions (the sequence containing an enzyme cutting site for facilitating subsequent insertion of a foreign gene can be inserted before 33593 rd position) in the sequence 1 of the sequence table by using the 1-1440 th positions of the sequence 2 of the sequence table;

5) Based on AdC6, completely deleting the E1 region, deleting the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7 and ORF8 sequences, reserving the ORF1 and ORF9 sequences, replacing the E4 region ORF6/7, ORF6 and ORF4 sequences originally existing in AdC6 by the E4 region ORF6/7, ORF6 and ORF4 sequences of the C subtype human adenovirus 5 type, and deleting the E4 region ORF1 and ORF2 originally existing in AdC6, so as to obtain a recombinant adenovirus, which is a corresponding recombinant adenovirus vector named pAdC6XY 6; the nucleotide sequence of pAdC6XY6 is a nucleotide sequence obtained by deleting 563-3413 th sites in a sequence 1 in the sequence table (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site), deleting 27441-31456 th sites in the sequence 1 in the sequence table (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site), replacing 33593-35026 th sites in the sequence 1 in the sequence table with 1-1440 th sites in the sequence 2 in the sequence table (for the convenience of subsequent insertion of a foreign gene, a sequence containing an enzyme cutting site can be inserted before 33593), and deleting 35381-36190 th sites in the sequence 1 in the sequence table (for the convenience of subsequent insertion of a foreign gene, the deleted sequence can be replaced by a sequence containing an enzyme cutting site).

In the above method, the recombinant adenovirus vector is a conditionally replicating oncolytic adenovirus vector, and the E1 gene sequence of the completely or partially deleted AdC6 is an E1A sequence of the preserved AdC6E1 gene, and the other sequences of the partially or completely deleted AdC6E1 gene;

and the method further comprises the step of placing a tumor specific promoter in front of the E1A gene of said AdC6 to regulate E1A expression.

The tumor specific promoters include, but are not limited to, the human telomerase reverse transcriptase (hTERT) promoter, the survivin promoter, the cyclooxygenase (COX-2) promoter, the alpha-fetoprotein (AFP) promoter, the E2F-1 promoter, the Ki67 promoter, or combinations thereof.

In the above method, the recombinant adenovirus vector is: based on AdC6, deleting E1B 55K in the E1 region, reserving E1A, and placing a human telomerase reverse transcriptase promoter in front of the E1A; the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 sequences are deleted, the ORF1 sequence is reserved, and the original E4 ORF6 sequence of AdC6 is replaced by the E4 ORF6 sequence of human adenovirus type 5, so that the recombinant adenovirus is obtained and is correspondingly named as a recombinant adenovirus vector pAdC6 XYR; the nucleotide sequence of pAdC6XYR is a nucleotide sequence obtained by deleting 1905-3413 th site from a sequence 1 in a sequence table, replacing a sequence from 565-573 th site to 27896-31904 th site from the sequence 1 in the sequence table by using a sequence 4 in the sequence table, replacing 33841-34746 th site from the sequence 1 in the sequence table by using 280-1164 th site from the sequence 2 in the sequence table (for the convenience of subsequent insertion of a foreign gene, a sequence containing an enzyme cutting site can be simultaneously added before 33841 th site and after 34746 th site from the sequence 1 in the sequence table) (a sequence containing an enzyme cutting site can also be simultaneously inserted between 1465 th site and 1466 th site from the sequence 1 in the sequence table).

In the above method, the recombinant adenovirus vector is a conditionally replicating oncolytic adenovirus vector, and the method further comprises the step of inserting an exogenous gene promoting oncolytic expression into the AdC6E1 region or E3 region or E4 region or other regions (such as behind E1A or before a tumor-specific promoter).

The foreign gene promoting oncolytic is a gene enhancing a cancer suppression effect (oncolytic gene), which includes but is not limited to genes encoding the following proteins: human granulocyte macrophage colony stimulating factor (CSF 2), p53, TNF- α, IL-21, IL-12, IL-6, IL-4, CD40L, PD-1 monoclonal antibody, PD-L1 monoclonal antibody, or a combination thereof. Preferably a gene for coding CSF2, the nucleotide sequence of which is shown as 679-1133 of the sequence 2 in the sequence table.

In the above method, the recombinant adenovirus vector is: based on AdC6, deleting E1B 55K in the E1 area, and reserving E1A; the sequences of ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 in the E3 region are deleted, the sequence of ORF1 is reserved to form delta E3, and a CSF2 expression frame (the nucleotide sequence is shown as the sequence 5 in the sequence table) is inserted into the delta E3; and replacing the original E4 ORF6 sequence of AdC6 with the E4 ORF6 sequence of human adenovirus type 5 to obtain the recombinant adenovirus, which is the corresponding recombinant adenovirus vector named AdC6XYR-CSF 2; the nucleotide sequence of pAdC6XYR-CSF2 is obtained by deleting 1905-3413 th bit from the sequence 1 in the sequence table, inserting the sequence 4 in the sequence table between 564 th bit and 574 th bit, deleting 27896-31904 th bit from the sequence 1 in the sequence table to replace the sequence with the sequence 5 in the sequence table, and replacing 33841-34746 th bit from the sequence 1 in the sequence table with 280-1164 th bit from the sequence 2 in the sequence table.

The recombinant adenovirus vector constructed by the method also belongs to the protection scope of the invention.

The recombinant adenovirus obtained by introducing the recombinant adenovirus vector into a packaging cell and packaging also belongs to the protection scope of the invention.

The packaging cell can be a HEK293 cell that incorporates the E1 gene of a human adenovirus subtype C (e.g., adHu1, adHu2, adHu6, adHu5, preferably AdHu 5).

The recombinant adenovirus of the present invention is understood to include not only first-generation to second-generation recombinant viruses but also progeny thereof.

The invention also provides any one of the following applications:

p1, the method or the recombinant adenovirus vector are applied to the prepared recombinant adenovirus;

p2, when the recombinant adenovirus vector is a non-replicative adenovirus vector, the method, the constructed recombinant adenovirus vector or the application of the recombinant adenovirus obtained by packaging the constructed recombinant adenovirus vector in the preparation of vaccine vectors;

p3, when the recombinant adenovirus vector is a non-replicative adenovirus vector, the method, the constructed recombinant adenovirus vector or the recombinant adenovirus packaged by the constructed recombinant adenovirus vector are applied to the preparation of vaccines;

p4, when the recombinant adenovirus vector is a non-replicative adenovirus vector, the method, the constructed recombinant adenovirus vector or the application of the recombinant adenovirus obtained by packaging the constructed recombinant adenovirus vector in the preparation of gene therapy medicaments;

p5, when the recombinant adenovirus vector is a conditionally replicating oncolytic adenovirus vector, the method, the constructed recombinant adenovirus vector or the application of the recombinant adenovirus obtained by packaging the constructed recombinant adenovirus vector in the preparation of a tumor cell inhibiting drug;

p6, when the recombinant adenovirus vector is a conditionally replicating oncolytic adenovirus vector, the method, the constructed recombinant adenovirus vector or the application of the recombinant adenovirus packaged by the constructed recombinant adenovirus vector in preparing a medicament for preventing or treating tumors.

The tumor cell can be any one of human embryonic kidney cell (HEK 293), human cervical carcinoma cell (Hela), human malignant embryonal rhabdomyoma cell (RD), human liver cancer cell (Huh 7), human colon cancer cell (SW 620), mouse prostate cancer cell (RM-1), mouse colon cancer cell (MC 38) and the like.

The tumor can be any one of human liver cancer, human malignant embryonal rhabdomyoma, human colon cancer, human cervical cancer, mouse colon cancer, mouse prostate cancer and the like.

The invention provides a method for constructing a recombinant adenovirus vector, which uses an E4 gene sequence of a C subtype human adenovirus genome to modify an E4 gene sequence of AdC6 so as to improve the yield of recombinant adenovirus. The method can construct a non-replicative adenovirus vector which can be used as a vaccine vector, and experiments prove that exogenous genes can be inserted into the E1/E3/E4/Fiber/Hexon and other regions of the adenovirus vector for vaccine development or gene therapy. The method can also construct and obtain a conditionally replication adenovirus vector, namely an oncolytic virus vector, and verifies the application value of the vector for further developing into recombinant oncolytic viruses: various foreign genes promoting oncolytic gene can be inserted into the region such as delta E3, E1 or E4, and the like, so that the oncolytic effect is further enhanced. In vitro experiments show that the recombinant oncolytic viruses can replicate in various tumor cells and lyse the tumor cells. In MC38 and RM-1 tumor-bearing mouse models, the recombinant oncolytic virus can effectively inhibit tumor progression and even eliminate tumors, and the recombinant oncolytic virus based on AdC6 has the potential of treating tumors and can be further promoted to clinic.

Drawings

FIG. 1 shows the formation of plaques of recombinant adenovirus and the restriction enzyme digestion of genome in example 2 of the present invention. Wherein, A picture of figure 1 is a picture of the formation of plaques of three recombinant adenoviruses; FIG. 1B is a diagram showing the results of enzyme digestion and identification of the genomes of three recombinant adenoviruses AdC6XY2, adC6XYR and AdC6XYR-CSF2 in sequence from left to right.

FIG. 2 is a graph showing the measurement of CSF2 expression in various tumor cells according to example 2 of the present invention.

FIG. 3 is a line graph showing the toxicity of 3 recombinant adenoviruses of example 2 of the present invention against 6 tumor cells at different titers.

FIG. 4 is a graph showing the results of different oncolytic viruses inducing apoptosis of tumor cells HeLa in example 2 of the present invention.

FIG. 5 is a graph showing the tumor-suppressing effect of the oncolytic adenovirus of example 2 of the present invention in tumor-bearing mice.

FIG. 6 is a diagram showing the result of plasmid digestion identification of the adenoviral vector in example 1 of the present invention.

FIG. 7 is a diagram showing the results of restriction enzyme digestion of genome after 12 generations of amplification of each recombinant adenovirus in example 1 of the present invention.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The following examples are provided as illustrative of the present technology

Further modifications of the teachings herein will be suggested to those of ordinary skill in the art and are not to be construed as limiting the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Used in the following examples

The materials, reagents and the like in (1) are all conventional biochemical reagents and can be obtained from commercial sources if no special description is provided.

Human embryonic kidney cells (HEK 293), human cervical cancer cells (Hela),Human malignant embryonal rhabdomyoma cells (RD), human liver cancer cells (Huh 7), human colon cancer cells (SW 620), mouse prostate cancer cells (RM-1) and mouse colon cancer cells (MC 38) are all ATCC products. All cells were incubated at 37 ℃ and 5% CO in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin ₂ The incubator of (2) for cultivation.

CCK8 reagent and 0.25% Trypsin-EDTA from Sino Biological, suzhou, N.seimei Biotech, anti-CSF 2 from Sino Biological, anti-Cleaved-PARP (Asp 214) from phosphorylated P53 (Ser 46), bax, anti-phosphorylated BAD (Ser 112) [ Ser75] from Affinity, anti-BIK, anti-P21 Cip1 from Affinity, internal reference beta-actin from protein, HRP coupled goat anti-rabbit secondary antibody from Abcam.

The C57BL/6 mice in the examples described below were standard strains purchased from female, 6-8 weeks old, beijing Beford Biotechnology Ltd.

The experimental data in the following examples are all expressed as mean ± standard deviation, and GraphPadPrism 6 was statistically analyzed, and the mean comparison between the groups was performed by One-way analysis of variance (ANOVA); ANOVA was used if each group of data was normally distributed, and non-parametric tests (Kruskal-Wallis) were used if each group was not normally distributed.

Example 1

In order to avoid the problem of reduced chimpanzee adenovirus production caused by unstable or reduced efficiency of chimpanzee adenovirus E4 protein binding with AdHu 5E 1 and improve the production of recombinant adenovirus AdC6, the inventor takes AdC6 as a base, deletes an E1 region and most of E3, partially replaces the E4 sequence with the E4 sequence of AdHu5, obtains a plurality of recombinant adenovirus AdC6XY2, adC6XY3, adC6XY4, adC6XY5 and AdC6XY6 with different E4 sequences, and designs corresponding recombinant adenovirus vectors pAdC6XY2, pAdC6XY3, pAdC6XY4, pAdC6XY5 and pAdC6XY6. To observe the change in adenovirus production and specific activity before and after the modification of E4 region, adC6XY1 without modifying E4 region was used as a control, which corresponds to pAdC6XY1 as recombinant adenovirus vector.

AdC6: chimpanzee adenovirus type 6, genBank accession No.: AY530877.1, update Date 2004-08-19. The corresponding adenovirus vector is pAdC6, the nucleotide sequence of which is sequence No. 1 (the nucleotide sequence of E1 region is 576-3413 position of sequence No. 1, the nucleotide sequence of E1A is 576-1437 position of the sequence No. 1, the nucleotide sequence of E1B 55K is 1905-3413 position of the sequence No. 1, the nucleotide sequence of E3 region is 27110-31864 position of the sequence No. 1, the nucleotide sequence of E3 ORF1 is 27110-27430 position of the sequence No. 3, the nucleotide sequence of E3 ORF2, ORF3, ORF4, ORF5, ORF6, ORF7 and ORF8 is 27457-31864 position of the sequence No. 3 ORF9, the nucleotide sequence of E4 region is 33593-36181 position of the sequence No. 1, the nucleotide sequence of E4 ORF1, ORF2 is 35377-36181 position of the nucleotide sequence of E4 ORF3, the nucleotide sequence of E33594, the nucleotide sequence of E33746 4-34652 position of the sequence No. 3, the nucleotide sequence of the sequence No. 33746 3/34746 position of the sequence No. 3 and No. 3/34746 sequence No. 3.

pAdC6XY1: based on chimpanzee adenovirus type 6 (AdC 6), the E1 region (i.e. deletion of 563-3413 nucleotides of sequence 1, deletion of 2851bp in size) is completely deleted, the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 sequences are deleted, the ORF1 sequence (i.e. deletion of 27441-31864 nucleotides of sequence 1, deletion of 4424bp in size) is retained, and the obtained adenovirus vector is named pAdC6XY1. The adenovirus thus packaged was designated AdC6XY1.

pAdC6XY2: based on the chimpanzee adenovirus type 6 (AdC 6), the E1 region (ie. The nucleotides 563 to 3413 of AdC6 are deleted and the size is 2851bp; the deleted sequence is replaced with sequence 3, which contains I-CeuI and PI-SceI cleavage sites and may also be replaced with sequences containing other cleavage sites, in order to facilitate subsequent insertion of a foreign gene), the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8, ORF9 sequences are deleted, the ORF1 sequence (ie. The nucleotides 27441 to 31864 of deletion sequence 1, deletion size 4424bp; the foreign gene is inserted for ease of subsequent insertion and may also be replaced with other cleavage sites), and the E4 ORF6 sequence (nucleotide 280 to 1164 of HuAdD 5, genBank accession No.: AC _000008.1, update 2018-08-13) of human adenovirus type 5 (HuAd 5, genBank accession No.: AC000008.1, update 2018-08-13) is used, and the E4 ORF6 sequence (nucleotide 280 to replace the nucleotide 1164 sequence) of AdC6 sequence (nucleotide 12) of AdC 6) (the extra nucleotide sequence, and the extra nucleotide 33746 site is also inserted with the adenovirus vector III, and the extra nucleotide 336 sequence of the adenovirus type 5, the extra adenovirus vector is inserted at the sites of the former site, and the later period of the adenovirus vector is also inserted into the plasmid III site of the adenovirus type III site of the DNA sequence (the DNA sequence). The adenovirus thus packaged was designated AdC6XY2.

pAdC6XY3: based on the chimpanzee adenovirus type 6 (AdC 6), the E1 region is completely deleted (i.e., nucleotides 563 to 3413 of AdC6 are deleted, the size is 2851bp; the deleted sequence is replaced with sequence 3 containing I-CeuI and PI-SceI cleavage sites and may be replaced with sequences containing other cleavage sites for the purpose of subsequent insertion of a foreign gene), the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8, ORF9 sequences are deleted, the ORF1 sequence is retained (i.e., nucleotides 27441 to 31864 of deletion sequence 1, nucleotide 4424bp in size is deleted), the foreign gene is inserted for the purpose of subsequent insertion of HpaI cleavage sites and may be replaced with other cleavage sites), and the E4 region ORF6/7, ORF6, ORF4 sequence (HuAd 5, genBank accession No.: AC _000008.1, update 2018-08-13) of human adenovirus type 5 (HuAd 5, nucleotide 3501-3508-08-13) is used, the ORF6/7 sequence (nucleotide 3506 of sequence 2) of the E4 region, ORF6, the sequence is replaced with nucleotide 3506, the foreign gene, the sequence of the foreign gene, the former nucleotide 336, the later nucleotide 336, and the sequence of the vector is inserted in the DNA 3 region, and the ORF3 region ORF3, nucleotide 593, nucleotide I site is also inserted for the later nucleotide I site. The adenovirus thus packaged was named AdC6XY3.

pAdC6XY4: based on chimpanzee adenovirus type 6 (AdC 6), completely deleting the E1 region (namely deleting 563-3413 nucleotides of AdC6, and deleting 2851bp in size; in order to facilitate the subsequent insertion of a foreign gene, the deleted sequence is replaced by a sequence 3 which comprises I-CeuI and PI-SceI enzyme cutting sites, and sequences comprising other enzyme cutting sites can also be used for replacing the deleted sequence), ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 sequences of the E3 region are deleted, the ORF1 sequence is reserved (namely the 27441-31864 nucleotides of the sequence 1 are deleted, and the 4424bp size is deleted; for the purpose of subsequent insertion of a foreign gene, the deleted sequence is replaced with HpaI cleavage site and may be replaced with another cleavage site), and the E4 region ORF6/7, ORF6, ORF4 sequence (nucleotides 1 to 1440 of sequence 2) originally existing in AdC6 is replaced with human adenovirus type 5 (AdHu 5, genBank accession No.: AC-000008.1, update Date 2018-08-13) E4 region ORF6/7, ORF6, ORF4 sequence (nucleotides 33593 to 35026 of sequence 1) to facilitate the later insertion of a foreign gene, asiSI cleavage site is added before 33593 and may be replaced with another cleavage site), and the E4 region ORF1, ORF2 originally existing in AdC6 is deleted (i.e., nucleotides 381 to 36190 of sequence 1 are deleted to facilitate the subsequent insertion of a foreign gene, the deleted sequence deleted is replaced with AsiSI site and may be replaced with another cleavage site), and the resulting vector pAdCYX4 is named AdC6, thus packaged as XY4 adenovirus 6.

pAdC6XY5: based on chimpanzee adenovirus type 6 (AdC 6), completely deleting the E1 region (namely deleting 563-3413 nucleotides of AdC6, and deleting 2851bp in size; for the purpose of subsequent insertion of the foreign gene, the deleted sequence was replaced with sequence 3, which contains I-CeuI and PI-SceI cleavage sites and may be replaced with sequences containing other cleavage sites, the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 sequences were deleted, the ORF1 and ORF9 sequences were retained (i.e., nucleotides 27441-31456 of deletion sequence 1, deletion size 4016bp; for the purpose of subsequent insertion of the foreign gene, the deleted sequence was replaced with an HpaI cleavage site and may be replaced with other cleavage sites), and the E4 region ORF6/7, ORF6, ORF4 sequences (nucleotides 1-35008 of sequence 2) originally owned by AdC6 were replaced with adenovirus type 5 (AdHu 5, genBank accession No.: AC-000008.1, update Date 2018-08-13), the E4 region ORF6/7, ORF4 sequences (nucleotides 33593-35026 of sequence 1; nucleotides 1-3503 of sequence 2), and the adenovirus sites were also replaced with the plasmid pA 595, which was obtained for the purpose of adding the adenovirus vector before the enzyme cleavage site, pA 595 and the adenovirus sites. The adenovirus thus packaged was named AdC6XY5.

pAdC6XY6: based on chimpanzee adenovirus type 6 (AdC 6), completely deleting the E1 region (namely deleting 563-3413 nucleotides of AdC6, and deleting 2851bp in size; in order to facilitate the subsequent insertion of the foreign gene, the deleted sequence is replaced by a sequence 3 which comprises I-CeuI and PI-SceI enzyme cutting sites, and a sequence comprising other enzyme cutting sites can be used for replacing the deleted sequence), the ORF2, ORF3, ORF4, ORF5, ORF6, ORF7 and ORF8 sequences of the E3 region are deleted, the ORF1 and ORF9 sequences are reserved (namely, the 27441-31456 nucleotides of the sequence 1 are deleted, and the deletion size is 4016bp; for the purpose of subsequent insertion of a foreign gene, the deleted sequence is replaced with HpaI cleavage site and may be replaced with another cleavage site), and the E4 region ORF6/7, ORF6, ORF4 sequence (nucleotides 1 to 1440 of SEQ ID NO: 2) originally existing in AdC6 is replaced with human adenovirus type 5 (AdHu 5, genBank accession No.: AC-000008.1, update Date 2018-08-13) E4 region ORF6/7, ORF6, ORF4 sequence (nucleotides 33593 to 35026 of SEQ ID NO: 1) for the purpose of later insertion of the foreign gene, asiSI cleavage site is added before 33593 and may be replaced with another cleavage site), and the E4 region ORF1, ORF2 originally existing in AdC6 is deleted (i.e., nucleotides 381 to 36190 of SEQ ID NO: 1 is deleted for the purpose of subsequent insertion of the foreign gene, the deleted sequence is replaced with AsiSI site and may be replaced with another cleavage site), and the resulting vector is named pAdCYX6. AdC6 thus packaged into an XY6 adenovirus type 6.

Specific modifications to obtain pAdC6XY1, pAdC6XY2, pAdC6XY3, pAdC6XY4, pAdC6XY5, pAdC6XY6 starting from pAdC6 (SEQ ID NO: 1 of the sequence listing) are shown in Table 1:

TABLE 1

After a series of adenovirus vector plasmids (pAdC 6XY1, pAdC6XY2, pAdC6XY3, pAdC6XY4, pAdC6XY5 and pAdC6XY 6) obtained in the above-mentioned manner are cut by using various enzymes (ApaI, ageI and XhoI), 1% agarose gel electrophoresis is carried out, the obtained plasmid cut identification electrophoresis picture is shown in figure 6, and the obtained DNA band proves that the series of adenovirus plasmid molecular cloning is complete and correct.

HEK293 cells were transfected after digestion of pAdC6XY1, pAdC6XY2, pAdC6XY3, pAdC6XY4, pAdC6XY5, pAdC6XY6 with PacI, respectively, and were significantly diseased 6 or 11 days after rescue: and (3) collecting cells after rounding, floating and comet-shaped plaques are changed, repeatedly freezing and thawing for three times between 80 ℃ below zero and 37 ℃, continuously infecting HEK293 cells to amplify viruses, and purifying the viruses by a cesium chloride density gradient centrifugation mode after a certain amount of viruses are collected to obtain the recombinant adenovirus. The recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY1 is AdC6XY1, the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY2 is AdC6XY2, the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY3 is AdC6XY3, the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY4 is AdC6XY4, the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY5 is AdC6XY5, and the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY6 is AdC6XY6.

The yields and specific activities (specific activity = number of viral particles/viral infectious titer) of adenoviruses AdC6XY1, adC6XY2, adC6XY3, adC6XY4, adC6XY5, adC6XY6 were compared and the results are shown in table 2.

Table 2:

name of adenovirus

Saving time

Number of particles (vp/ml)

Volume of

Viral yield

Specific activity

Load capacity

AdC6XY1

11 days

6.32×10 12

1.6ml

1.01×10 13 vp

487

6.5kb

AdC6XY2

6 days

1.63×10 13

3.5ml

5.70×10 13 vp

138

7.0kb

AdC6XY3

6 days

1.32×10 13

3.3ml

4.36×10 13 vp

126

7.0kb

AdC6XY4

6 days

1.04×10 13

2.5ml

2.60×10 13 vp

235

8.0kb

AdC6XY5

6 days

2.00×10 13

3.5ml

7.00×10 13 vp

105

7.0kb

AdC6XY6

6 days

1.72×10 13

3.0ml

5.16×10 13 vp

215

7.5kb

Analysis results show that compared with AdC6XY1, the E4 region can obviously improve the yield of adenovirus by about 2.5-7 times after being replaced by an AdHu5 source (AdC 6XY2, adC6XY3, adC6XY4, adC6XY5 and AdC6XY 6); meanwhile, the specific activity of the adenovirus can be improved, the adenovirus with better quality can be obtained, and the virus-releasing time of the adenovirus is obviously shortened (from 11 days to 6 days). The reservation of ORF1, ORF9 of the E3 region (AdC 6XY5, adC6XY 6) contributes to a further increase in adenovirus production. Furthermore, partial deletion of the E4 region (ORF 1-2) still rescues adenovirus and further increases the loading of foreign genes, but one of ORF6 or ORF6/7 must be retained.

Thereafter, the inventors continued to infect HEK293 cells with the resulting series of adenoviruses (AdC 6XY1, adC6XY2, adC6XY3, adC6XY4, adC6XY5, adC6XY 6) and passaged until the 12 th passage P12 (the rescued adenovirus was designated as the first generation P1). The genome of each adenovirus of generation P12 was extracted, digested with various enzymes (ApaI, ageI, xhoI), electrophoresed on 1% agarose gel, and the stability of each adenovirus was confirmed by the restriction band, and the results are shown in FIG. 7. Analysis results show that each adenovirus still maintains the correctness and integrity of the genome and has good stability after continuous passage amplification for 12 times.

To test the possibility of developing the AdC6XY series of vectors as vaccines or gene therapy products, the inventors also performed the expression of a wide variety of types of genes, some representative foreign genes and their expression profiles are shown in Table 3.

TABLE 3

The inventor modifies a series of replication-defective recombinant adenovirus vectors based on AdC6, and proves that the modified adenovirus vectors have improved yield, better quality and larger foreign gene loading capacity, and can be further developed into vaccines or gene therapy products.

Example 2

It has been found in the prior art that adenoviruses of rare human serotypes or derived from other species can be used as vectors for oncolytic viruses. The tumor specific promoter is used to drive the expression of adenovirus E1A, so that adenovirus can selectively replicate in tumor cells, and the specificity and selectivity of oncolytic virus for treating tumor can be improved. Common tumor specific promoters include human telomerase promoter, prostate specific antigen promoter, tyrosinase promoter, etc. The inventor also modified E1 based on AdC6 on example 1: the E1 gene is reinserted into each adenovirus delta E1 region in the embodiment 1, only E1B 55K of the E1 region is deleted, E1A is reserved, and meanwhile, a human telomerase reverse transcriptase (hTERT) promoter (sequence 4 in a sequence table) is selected to be placed in front of the E1A to regulate and control the expression of the adenovirus E1A, so that the conditionally replicating oncolytic adenovirus is obtained. For example, on the basis of AdC6XY2, the E1 gene is reinserted into the Delta E1 region of AdC6XY2, only E1B 55K of the E1 region is deleted, E1A is reserved, and an hTERT promoter is inserted before E1A to obtain the conditionally replicative adenovirus which is named as AdC68XYR.

In order to enhance the killing ability of conditionally replication competent oncolytic adenoviruses against tumor cells, foreign genes that contribute to oncolytic can be inserted into oncolytic viral vectors, and are generally classified into the following groups: the anti-tumor immune response-enhancing gene silencing agent comprises (1) molecules for directly inducing tumor cell death, (2) anti-angiogenesis molecules, (3) immune regulating molecules, cytokines (IL-2, IL-18, 20, IL-12, 21, CSF2, interferon), chemokines (CCL 5, CCL20, CCL 21) and other molecules for enhancing anti-tumor immune response, and (4) small RNA molecules such as miRNA, siRNA, shRNA and lncRNA and the like for silencing tumor-related genes. In the invention, CSF2 (the nucleotide sequence of the coding gene of the CSF2 is shown as 683-1113 th site of a sequence 5 in a sequence table, and the sequence 5 in the sequence table is a CSF2 expression frame) is selected as a representative, and the CSF2 is cloned to delta E3 of pAdC6XYR to obtain a recombinant oncolytic adenovirus vector pAdC6XYR-CSF2.

Specific modifications to pAdC6XYR and pAdC6XYR-CSF2 starting from pAdC6 (SEQ ID NO: 1) were as follows (see Table 4):

pAdC6XYR: based on chimpanzee adenovirus type 6 (AdC 6), E1B 55K in the E1 region is deleted, E1A is reserved (namely nucleotides 1905 to 3413 in a sequence 1 are deleted, and the deletion size is 1509 bp), and a human telomerase reverse transcriptase (hTERT) promoter is placed in front of the E1A (the sequence 4 in a sequence table is used for replacing the bits 565 to 573 in the sequence 1 in the sequence table); the E3 region ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 sequences were deleted, the ORF1 sequence (i.e., nucleotides 27896-31904 of the deleted sequence 1, deletion size 4009 bp) was retained, and the E4 ORF6 sequence (nucleotides 280-1164 of the sequence 2) of human adenovirus type 5 (AdHu 5, genBank accession No.: AC-000008.1, update Date 8-08-13) was used in place of the E4 ORF6 sequence (nucleotides 33841-34746 of the sequence 1; for subsequent insertion of a foreign gene, asiSI cleavage site was added before and after 33201841 and 34746 at the same time, and may be replaced with another cleavage site) (for subsequent insertion of a foreign gene after E1A, PI-SceI cleavage site may be inserted between 1465 and 1466 of the sequence 1 of the sequence list at the same time, and pAdC6 may be replaced with another cleavage site), and the resulting adenovirus vector pAdC6 was named pAdC. The adenovirus thus packaged was designated AdC6XYR.

AdC6XYR-CSF2: based on chimpanzee adenovirus type 6 (AdC 6), E1B 55K in an E1 region is deleted, E1A is reserved (namely nucleotides 1905 to 3413 in a sequence 1 are deleted, and the deletion size is 1509 bp), and a human telomerase reverse transcriptase (hTERT) promoter (sequence 4) is placed in front of the E1A (the sequence 4 in a sequence table is used for replacing the bits 565 to 573 in the sequence 1 in the sequence table); deleting ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8 and ORF9 sequences in the E3 region, reserving an ORF1 sequence (namely, the 27896-31904 th nucleotide of the sequence 1 is deleted, the deletion size is 4009 bp) to form delta E3, and inserting a CSF2 expression frame at the delta E3 (the nucleotide sequence is shown as a sequence 5 in a sequence table); and the E4 ORF6 sequence (nucleotides 280-1164 of SEQ ID NO: 2) of human adenovirus type 5 (AdHu 5, genBank accession No.: AC-000008.1, update Date 2018-08-13) was used in place of the original E4 ORF6 sequence (nucleotides 33841-34746 of SEQ ID NO: 1) of AdC6, and the resulting adenovirus vector was named pAdC6XYR-CSF2. The adenovirus thus packaged was named AdC6XYR-CSF2.

TABLE 4

1. Construction of adenovirus

The oncolytic adenoviral vector pAdC6XYR, which drives adenoviral replication by the telomerase promoter, was synthesized by general biosystems (Anhui) Inc.

Granulocyte-macrophage colony stimulating factor (GM-CSF/CSF 2) was cloned into the Δ E3 region of pAdC6XYR using conventional molecular cloning homologous recombination to give pAdC6XYR-CSF2.

Replication-defective adenovirus vector pAdC6XY2 (see example 1) was used as a negative control.

HEK293 cells were transfected after digestion of pAdC6XYR-CSF2, pAdC6XYR and pAdC6XY2 with PacI, respectively, until the cells exhibited overt lesion characteristics: after rounding, floating and comet-shaped plaques (A picture in figure 1), collecting cells, repeatedly freezing and thawing for three times between-80 ℃ and 37 ℃, continuously infecting HEK293 cells to amplify viruses, after collecting a certain amount of viruses, purifying the viruses by a cesium chloride density gradient centrifugation mode to obtain the recombinant adenovirus. The recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XYR-CSF2 is AdC6XYR-CSF2, the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XYR is AdC6XYR, and the recombinant adenovirus obtained by transfecting HEK293 cells with pAdC6XY2 is AdC6XY2. After amplification and purification, the genomes of three recombinant adenoviruses AdC6XYR-CSF2, adC6XYR and AdC6XY2 are extracted. The purified viral genomes were digested with ApaI, bglII, xho I, and the digested fragments of the three recombinant adenoviruses were completely correct when compared to the standard map (FIG. 1, panel B). This demonstrates that the recombinant adenovirus was successfully constructed without fragment deletion.

2. Western Blot to detect CSF2 expression

To detect that recombinant adenovirus can infect different tumor cells and express CSF2, the dose was 10 ¹⁰ Different tumor cells (human cervical cancer cells (Hela), human malignant embryonal rhabdomyoma cells (RD), human liver cancer cells (Huh 7), human colon cancer cells (SW 620), mouse prostate cancer cells (RM-1) and mouse colon cancer cells (MC 38)) are infected by the AdC6XYR-CSF2 of vp, the tumor cells infected by the AdC6XY2 and the AdC6XYR of the same dosage are used as negative controls, cell supernatants are collected after infection for 24 hours, blot is carried out by using an anti-Western CSF2 antibody as a primary antibody to detect the expression of the CSF2, and the internal reference is a beta-actin antibody.

The result is shown in fig. 2, the recombinant adenovirus AdC6XYR-CSF2 can replicate in different tumor cells and normally express CSF2, which indicates that the recombinant adenovirus AdC6XYR-CSF2 can effectively infect and replicate in different tumor cells, and successfully package out viral particles capable of normally expressing CSF2, demonstrating that the recombinant oncolytic virus using AdC6 as a vector can infect various tumor cells and has a broad spectrum of infection. Two recombinant adenoviruses, adC6XYR and AdC6XY2, do not express CSF2.

3. Survival rate of tumor cells detected by CCK8 experiment

6 tumor cells, namely human cervical cancer cells (Hela), human malignant embryonal rhabdomyoma cells (RD), human liver cancer cells (Huh 7), human colon cancer cells (SW 620), mouse prostate cancer cells (RM-1) and mouse colon cancer cells (MC 38), are respectively inoculated to a 96-well plate in advance one day, each well is 100 mu l, and the volume of each well is 1 multiplied by 10 ⁴ And (4) placing the individual cells in a cell culture box for overnight culture. After 24h, 6 tumor cells are infected by 3 recombinant adenoviruses AdC6XYR-CSF2, adC6XYR and AdC6XY2 respectively with 3 titers (1 MOI, 10MOI and 100 MOI), each titer is subjected to 5 duplicate wells, a cell well without virus is used as a control, the cells are placed in an incubator and then are continuously cultured, 10 mu l of CCK8 reagent is added to each well after 72 hours, the cells are placed in the incubator and incubated for 1-2h, and then the absorbance is measured at 450nm and 620nm, and the cell survival rate is calculated. Each experiment was repeated 3 times.

Cell viability = [ OD450 (experimental) -OD620 (experimental) ]/[ OD450 (control) -OD620 (control) ].

The results are shown in FIG. 3: experimental results prove that the oncolytic viruses AdC6XYR-CSF2 and AdC6XYR can generate obvious killing effects on various tumor cells, when 10MOI is infected, the survival rate of the tumor cells of the AdC6XYR and AdC6XYR-CSF2 is reduced compared with that of a replication-defective adenovirus AdC6XY2 infected group, and when the multiplicity of infection is 100MOI, the survival rate of the tumor cells of replication-type oncolytic adenoviruses AdC6XYR and AdC6XYR-CSF2 is obviously reduced compared with that of the replication-defective adenovirus AdC6XY2 infected group, namely the killing effect on the tumor cells is extremely obvious. The recombinant oncolytic viruses AdC6XYR and AdC6XYR-CSF2 are further proved to be capable of effectively killing tumor cells and have broad spectrum.

4. Study of the oncolytic mechanism

To investigate whether recombinant oncolytic viruses exert oncolytic effects by inducing apoptosis in tumor cells, the inventors infected HeLa cells with AdC6XYR-CSF2, adC6XYR and AdC6XY2 at 10MOI, respectively, collected proteins at 12h, 24h and 48h time points, and examined apoptotic proteins PARP, P53 (Ser 46), bax, bad (Ser 112) [ Ser75] antibody, BIK antibody and P21 Cip1 by WesternBlot method using anti-cleared-PARP (Asp 214) antibody, anti-phosphorylated P53 (Ser 46), bax, bad (Ser 75), bik, P21, β -actin as internal references, respectively, anti-phosphorylated BAD (Ser 112) [ Ser75] antibody, anti-BIK antibody and anti-P21 Cip1 antibody.

As a result, as shown in fig. 4, when HeLa cells were infected with oncolytic viruses AdC6XYR-CSF2 and AdC6XYR, the expression level of phosphorylated P53 protein was significantly increased and the expression level of downstream gene P21 was relatively increased, compared to the control group (AdC 6XY 2). The protein encoded by the P21 gene is a cell cycle-dependent kinase inhibitor, and can bind to the corresponding protein kinase to cause G1 phase arrest, and in addition, the expression of Bad, bik, and Bax phosphorylated by proapoptotic proteins is also significantly increased. The oncolytic virus AdC6XYR-CSF2 and AdC6XYR are shown to kill tumor cells, and the tumor cells are mainly killed by inducing the tumor cells to die.

5. Construction and treatment of MC38 and RM-1 tumor-bearing mice

6-8 weeks old C57BL/6 mice, female, were injected subcutaneously into the back of each mouse with mouse colon cancer MC38 cell suspension or mouse prostate RM-1 cell suspension, mouse colon cancer MC38 cells andthe injection dose of mouse prostate RM-1 cells is 1X 10 ⁶ One cell/one, 50ul in volume. Tumor size was measured at daily observation, and tumor volume (V) was calculated as: v (mm) ³ ) = length × width/2. When the tumor grows to 50-100mm ³ Randomly divided into 4 groups of 5 pieces each. The specific grouping is as follows:

PBS group: intratumorally, 50 μ l of PBS was injected every 3 days for 3 total injections.

AdC6XY2 group: 50 μ l of AdC6XY2 suspension (liquid obtained by diluting AdC6XY2 with PBS) was injected intratumorally, once every 3 days, for a total of 3 injections. AdC6XY2 was injected at a dose of 1X 10 ⁸ PFU)/one.

AdC6XYR group: 50 μ l of AdC6XYR suspension (liquid obtained by diluting AdC6XYR with PBS) was injected intratumorally, once every 3 days, for 3 injections. AdC6XYR was injected at a dose of 1X 10 ⁸ PFU)/only.

AdC6XYR-CSF2 group: 50 μ l of AdC6XYR-CSF2 suspension (liquid obtained by diluting AdC6XYR-CSF2 with PBS) was injected intratumorally, once every 3 days, for 3 injections. AdC6XYR-CSF2 was injected at a dose of 1X 10 ⁸ PFU)/only.

Tumor sizes were then measured every third day and recorded. When the tumor grows to 2500-3000mm ³ Mice were euthanized.

The results are shown in FIG. 5: in RM-1 tumor-bearing mice, the tumor growth rate of the AdC6XYR-CSF2 and AdC6XYR oncolytic virus treatment groups is obviously reduced compared with that of the PBS group and the replication-defective adenovirus AdC6XY2 treatment group, wherein the PBS group and the replication-defective adenovirus AdC6XY2 treatment group have no significant difference (P & gt 0.05), the oncolytic virus AdC6XYR-CSF2 and AdC6XYR treatment groups have significant difference (P & lt 0.05) compared with that of the PBS group and the replication-defective adenovirus AdC6XY2 treatment group, and the oncolytic virus AdC6XYR-CSF2 treatment group and the AdC6XYR treatment group have no significant difference (P & gt 0.05).

The treatment effect is more obvious in MC38 tumor-bearing mice. The oncolytic adenovirus AdC6XYR-CSF2 and AdC6XYR can obviously inhibit the growth of tumors, the tumor volume is obviously reduced after treatment, and the therapeutic effect on the tumors is very good. Wherein the PBS group and the replication-defective adenovirus AdC6XY2 treatment group have no significant difference (P > 0.05), the oncolytic virus AdC6XYR-CSF2 and AdC6XYR treatment groups have significant difference (P < 0.01) with the PBS group and the replication-defective adenovirus AdC6XY2 treatment group, and the oncolytic virus AdC6XYR-CSF2 treatment group and the AdC6XYR treatment group have no significant difference (P > 0.05).

The above results show that: the novel oncolytic viruses AdC6XYR-CSF2 and AdC6XYR taking AdC6 as a carrier can obviously inhibit the growth of tumors in MC38 and RM-1 tumor-bearing mice and induce the reduction of the tumor volume, and a novel treatment method is expected to be provided for patients with prostate cancer and colorectal cancer.

The inventor researches the tumor killing effect of AdC 6-based oncolytic adenoviruses AdC6XYR-CSF2 and AdC6XYR and discusses an anti-tumor mechanism, and in vitro and in vivo experiments show that the adenovirus AdC6XYR has a good anti-tumor effect, and particularly in MC38 and RM-1 tumor-bearing mice, the adenovirus AdC6XYR can remarkably inhibit the growth of tumors and generate a good anti-tumor effect. The research of related mechanisms shows that AdC6XYR-CSF2 and AdC6XYR kill tumor cells, and mainly regulate the expression of apoptosis-promoting proteins by activating a P53 pathway to induce the apoptosis of the tumor cells so as to kill the tumor cells, wherein the mechanism is different from that of inducing the apoptosis of the tumor cells by a mitochondrion pathway independent of P53 by virtue of an oncolytic virus derived from chimpanzee 7 and inducing the autophagy of the tumor cells by the oncolytic virus derived from AdHu 5. In conclusion, the study proves that the replicative chimpanzee adenovirus AdC6 can be transformed into oncolytic virus, has huge potential for treating tumors, and provides basic theory and experimental basis for the biological treatment research of tumors.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> Suzhou photo game biotechnology, inc

<120> method for constructing recombinant adenovirus vector, recombinant adenovirus vector constructed by same and application

<130> GNCSY211420

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 39294

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

catcatcaat aatatacctc aaacttttgg tgcgcgttaa tatgcaaatg agctgtttga 60

atttggggag ggaggaaggt gattggctgc gggagcggcg accgttaggg gcggggcggg 120

tgacgttttg atgacgtggc tatgaggcgg agccggtttg caagttctcg tgggaaaagt 180

gacgtcaaac gaggtgtggt ttgaacacgg aaatactcaa ttttcccgcg ctctctgaca 240

ggaaatgagg tgtttctggg cggatgcaag tgaaaacggg ccattttcgc gcgaaaactg 300

aatgaggaag tgaaaatctg agtaatttcg cgtttatggc agggaggagt atttgccgag 360

ggccgagtag actttgaccg attacgtggg ggtttcgatt accgtatttt tcacctaaat 420

ttccgcgtac ggtgtcaaag tccggtgttt ttacgtaggc gtcagctgat cgccagggta 480

tttaaacctg cgctctctag tcaagaggcc actcttgagt gccagcgagt agagttttct 540

cctccgcgcc gcgagtcaga tctacacttt gaaagatgag gcacctgaga gacctgcccg 600

gtaatgtttt cctggctact gggaacgaga ttctggaatt ggtggtggac gccatgatgg 660

gtgacgaccc tccagagccc cctaccccat ttgaggcgcc ttcgctgtac gatttgtatg 720

atctggaggt ggatgtgccc gagagcgacc ctaacgagga ggcggtgaat gatttgttta 780

gcgatgccgc gctgctggct gccgagcagg ctaatacgga ctctggctca gacagcgatt 840

cctctctcca taccccgaga cccggcagag gtgagaaaaa gatccccgag cttaaagggg 900

aagagctcga cctgcgctgc tatgaggaat gcttgcctcc gagcgatgat gaggaggacg 960

aggaggcgat tcgagctgcg gtgaaccagg gagtgaaaac tgcgggcgag agctttagcc 1020

tggactgtcc tactctgccc ggacacggct gtaagtcttg tgaatttcat cgcatgaata 1080

ctggagataa gaatgtgatg tgtgccctgt gctatatgag agcttacaac cattgtgttt 1140

acagtaagtg tgattaactt tagttgggaa ggcagagggt gactgggtgc tgactggttt 1200

atttatgtat atgttttttt atgtgtaggt cccgtctctg acgtagatga gacccccact 1260

tcagagtgca tttcatcacc cccagaaatt ggcgaggaac cgcccgaaga tattattcat 1320

agaccagttg cagtgagagt caccgggcgg agagcagctg tggagagttt ggatgacttg 1380

ctacagggtg gggatgaacc tttggacttg tgtacccgga aacgccccag gcactaagtg 1440

ccacacatgt gtgtttactt aaggtgatgt cagtatttat agggtgtgga gtgcaataaa 1500

atccgtgttg actttaagtg cgtgttttat gactcagggg tggggactgt gggtatataa 1560

gcaggtgcag acctgtgtgg tcagttcaga gcaggactca tggagatctg gactgtcttg 1620

gaagactttc accagactag acagttgcta gagaactcat cggagggagt ctcttacctg 1680

tggagattct gcttcggtgg gcctctagct aagctagtct atagggccaa acaggattat 1740

aaggaacaat ttgaggatat tttgagagag tgtcctggta tttttgactc tctcaacttg 1800

ggccatcagt ctcactttaa ccagagtatt ctgagagccc ttgacttttc tactcctggc 1860

agaactaccg ccgcggtagc cttttttgcc tttattcttg acaaatggag tcaagaaacc 1920

catttcagca gggattaccg tctggactgc ttagcagtag ctttgtggag aacatggagg 1980

tgccagcgcc tgaatgcaat ctccggctac ttgccagtac agccggtaga cacgctgagg 2040

atcctgagtc tccagtcacc ccaggaacac caacgccgcc agcagccgca gcaggagcag 2100

cagcaagagg aggaccgaga agagaacccg agagccggtc tggaccctcc ggtggcggag 2160

gaggaggagt agctgacttg tttcccgagc tgcgccgggt gctgactagg tcttccagtg 2220

gacgggagag ggggattaag cgggagaggc atgaggagac tagccacaga actgaactga 2280

ctgtcagtct gatgagccgc aggcgcccag aatcggtgtg gtggcatgag gtgcagtcgc 2340

aggggataga tgaggtctcg gtgatgcatg agaaatattc cctagaacaa gtcaagactt 2400

gttggttgga gcccgaggat gattgggagg tagccatcag gaattatgcc aagctggctc 2460

tgaagccaga caagaagtac aagattacca aactgattaa tatcagaaat tcctgctaca 2520

tttcagggaa tggggccgag gtggagatca gtacccagga gagggtggcc ttcagatgtt 2580

gtatgatgaa tatgtacccg ggggtggtgg gcatggaggg agtcaccttt atgaacacga 2640

ggttcagggg tgatgggtat aatggggtgg tctttatggc caacaccaag ctgacagtgc 2700

acggatgctc cttctttggc ttcaataaca tgtgcatcga ggcctggggc agtgtttcag 2760

tgaggggatg cagcttttca gccaactgga tgggggtcgt gggcagaacc aagagcaagg 2820

tgtcagtgaa gaaatgcctg ttcgagaggt gccacctggg ggtgatgagc gagggcgaag 2880

ccaaagtcaa acactgcgcc tctaccgaga cgggctgctt tgtgctgatc aagggcaatg 2940

cccaagtcaa gcataacatg atctgtgggg cctcggatga gcgcggctac cagatgctga 3000

cctgcgccgg tgggaacagc catatgctgg ccaccgtgca tgtggcctcg cacccccgca 3060

agacatggcc cgagttcgag cacaacgtca tgacccgctg caatgtgcac ctgggctccc 3120

gccgaggcat gttcatgccc taccagtgca acatgcaatt tgtgaaggtg ctgctggagc 3180

ccgatgccat gtccagagtg agcctgacgg gggtgtttga catgaatgtg gagctgtgga 3240

aaattctgag atatgatgaa tccaagacca ggtgccgggc ctgcgaatgc ggaggcaagc 3300

acgccaggct tcagcccgtg tgtgtggagg tgacggagga cctgcgaccc gatcatttgg 3360

tgttgtcctg caacgggacg gagttcggct ccagcgggga agaatctgac tagagtgagt 3420

agtgtttggg gctgggtgtg agcctgcatg aggggcagaa tgactaaaat ctgtggtttt 3480

ctgtgtgttg cagcagcatg agcggaagcg cctcctttga gggaggggta ttcagccctt 3540

atctgacggg gcgtctcccc tcctgggcgg gagtgcgtca gaatgtgatg ggatccacgg 3600

tggacggccg gcccgtgcag cccgcgaact cttcaaccct gacctacgcg accctgagct 3660

cctcgtccgt ggacgcagct gccgccgcag ctgctgcttc cgccgccagc gccgtgcgcg 3720

gaatggccct gggcgccggc tactacagct ctctggtggc caactcgagt tccaccaata 3780

atcccgccag cctgaacgag gagaagctgc tgctgctgat ggcccagctc gaggccctga 3840

cccagcgcct gggcgagctg acccagcagg tggctcagct gcaggcggag acgcgggccg 3900

cggttgccac ggtgaaaacc aaataaaaaa tgaatcaata aataaacgga gacggttgtt 3960

gattttaaca cagagtcttg aatctttatt tgatttttcg cgcgcggtag gccctggacc 4020

accggtctcg atcattgagc acccggtgga tcttttccag gacccggtag aggtgggctt 4080

ggatgttgag gtacatgggc atgagcccgt cccgggggtg gaggtagctc cattgcaggg 4140

cctcgtgctc ggggatggtg ttgtaaatca cccagtcata gcaggggcgc agggcgtggt 4200

gctgcacgat gtccttgagg aggagactga tggccacggg cagccccttg gtgtaggtgt 4260

tgacgaacct gttgagctgg gagggatgca tgcgggggga gatgagatgc atcttggcct 4320

ggatcttgag attggcgatg ttcccgccca gatcccgccg ggggttcatg ttgtgcagga 4380

ccaccagcac ggtgtatccg gtgcacttgg ggaatttgtc atgcaacttg gaagggaagg 4440

cgtgaaagaa tttggagacg cccttgtgac cgcccaggtt ttccatgcac tcatccatga 4500

tgatggcgat gggcccgtgg gcggcggcct gggcaaagac gtttcggggg tcggacacat 4560

cgtagttgtg gtcctgggtg agctcgtcat aggccatttt aatgaatttg gggcggaggg 4620

tgcccgactg ggggacgaag gtgccctcga tcccgggggc gtagttgccc tcgcagatct 4680

gcatctccca ggccttgagc tcggaggggg ggatcatgtc cacctgcggg gcgatgaaaa 4740

aaacggtttc cggggcgggg gagatgagct gggccgaaag caggttccgg agcagctggg 4800

acttgccgca accggtgggg ccgtagatga ccccgatgac cggctgcagg tggtagttga 4860

gggagagaca gctgccgtcc tcgcggagga ggggggccac ctcgttcatc atctcgcgca 4920

catgcatgtt ctcgcgcacg agttccgcca ggaggcgctc gccccccagc gagaggagct 4980

cttgcagcga ggcgaagttt ttcagcggct tgagtccgtc ggccatgggc attttggaga 5040

gggtctgttg caagagttcc agacggtccc agagctcggt gatgtgctct agggcatctc 5100

gatccagcag acctcctcgt ttcgcgggtt ggggcgactg cgggagtagg gcaccaggcg 5160

atgggcgtcc agcgaggcca gggtccggtc cttccagggc cgcagggtcc gcgtcagcgt 5220

ggtctccgtc acggtgaagg ggtgcgcgcc gggctgggcg cttgcgaggg tgcgcttcag 5280

gctcatccgg ctggtcgaga accgctcccg gtcggcgccc tgcgcgtcgg ccaggtagca 5340

attgagcatg agttcgtagt tgagcgcctc ggccgcgtgg cccttggcgc ggagcttacc 5400

tttggaagtg tgtccgcaga cgggacagag gagggacttg agggcgtaga gcttgggggc 5460

gaggaagacg gactcggggg cgtaggcgtc cgcgccgcag ctggcgcaga cggtctcgca 5520

ctccacgagc caggtgaggt cggggcggtt ggggtcaaaa acgaggtttc ctccgtgctt 5580

tttgatgcgt ttcttacctc tggtctccat gagctcgtgt ccccgctggg tgacaaagag 5640

gctgtccgtg tccccgtaga ccgactttat gggccggtcc tcgagcgggg tgccgcggtc 5700

ctcgtcgtag aggaaccccg cccactccga gacgaaggcc cgggtccagg ccagcacgaa 5760

ggaggccacg tgggaggggt agcggtcgtt gtccaccagc gggtccacct tctccagggt 5820

atgcaagcac atgtccccct cgtccacatc caggaaggtg attggcttgt aagtgtaggc 5880

cacgtgaccg ggggtcccgg ccgggggggt ataaaagggg gcgggcccct gctcgtcctc 5940

actgtcttcc ggatcgctgt ccaggagcgc cagctgttgg ggtaggtatt ccctctcgaa 6000

ggcgggcatg acctcggcac tcaggttgtc agtttctaga aacgaggagg atttgatatt 6060

gacggtgccg ttggagacgc ctttcatgag cccctcgtcc atttggtcag aaaagacgat 6120

ctttttgttg tcgagcttgg tggcgaagga gccgtagagg gcgttggaga gcagcttggc 6180

gatggagcgc atggtctggt tcttttcctt gtcggcgcgc tccttggcgg cgatgttgag 6240

ctgcacgtac tcgcgcgcca cgcacttcca ttcggggaag acggtggtga gctcgtcggg 6300

cacgattctg acccgccagc cgcggttgtg cagggtgatg aggtccacgc tggtggccac 6360

ctcgccgcgc aggggctcgt tggtccagca gaggcgcccg cccttgcgcg agcagaaggg 6420

gggcagcggg tccagcatga gctcgtcggg ggggtcggcg tccacggtga agatgccggg 6480

caggagctcg gggtcgaagt agctgatgca ggtgcccaga ttgtccagcg ccgcttgcca 6540

gtcgcgcacg gccagcgcgc gctcgtaggg gctgaggggc gtgccccagg gcatggggtg 6600

cgtgagcgcg gaggcgtaca tgccgcagat gtcgtagacg tagaggggct cctcgaggac 6660

gccgatgtag gtggggtagc agcgcccccc gcggatgctg gcgcgcacgt agtcgtacag 6720

ctcgtgcgag ggcgcgagga gccccgtgcc gaggttggag cgttgcggct tttcggcgcg 6780

gtagacgatc tggcggaaga tggcgtggga gttggaggag atggtgggcc tttggaagat 6840

gttgaagtgg gcgtggggca ggccgaccga gtccctgatg aagtgggcgt aggagtcctg 6900

cagcttggcg acgagctcgg cggtgacgag gacgtccagg gcgcagtagt cgagggtctc 6960

ttggatgatg tcatacttga gctggccctt ctgcttccac agctcgcggt tgagaaggaa 7020

ctcttcgcgg tccttccagt actcttcgag ggggaacccg tcctgatcgg cacggtaaga 7080

gcccaccatg tagaactggt tgacggcctt gtaggcgcag cagcccttct ccacggggag 7140

ggcgtaagct tgcgcggcct tgcgcaggga ggtgtgggtg agggcgaagg tgtcgcgcac 7200

catgaccttg aggaactggt gcttgaagtc gaggtcgtcg cagccgccct gctcccagag 7260

ttggaagtcc gtgcgcttct tgtaggcggg gttaggcaaa gcgaaagtaa catcgttgaa 7320

gaggatcttg cccgcgcggg gcatgaagtt gcgagtgatg cggaaaggct ggggcacctc 7380

ggcccggttg ttgatgacct gggcggcgag gacgatctcg tcgaagccgt tgatgttgtg 7440

cccgacgatg tagagttcca cgaatcgcgg gcggcccttg acgtggggca gcttcttgag 7500

ctcgtcgtag gtgagctcgg cggggtcgct gagcccgtgc tgctcgaggg cccagtcggc 7560

gacgtggggg ttggcgctga ggaaggaagt ccagagatcc acggccaggg cggtctgcaa 7620

gcggtcccgg tactgacgga actgttggcc cacggccatt ttttcggggg tgacgcagta 7680

gaaggtgcgg gggtcgccgt gccagcggtc ccacttgagc tggagggcga ggtcgtgggc 7740

gagctcgacg agcggcgggt ccccggagag tttcatgacc agcatgaagg ggacgagctg 7800

cttgccgaag gaccccatcc aggtgtaggt ttccacatcg taggtgagga agagcctttc 7860

ggtgcgagga tgcgagccga tggggaagaa ctggatctcc tgccaccagt tggaggaatg 7920

gctgttgatg tgatggaagt agaaatgccg acggcgcgcc gagcactcgt gcttgtgttt 7980

atacaagcgt ccgcagtgct cgcaacgctg cacgggatgc acgtgctgca cgagctgtac 8040

ctgggttcct ttggcgagga atttcagtgg gcagtggagc gctggcggct gcatctcgtg 8100

ctgtactacg tcttggccat cggcgtggcc atcgtctgcc tcgatggtgg tcatgctgac 8160

gagcccgcgc gggaggcagg tccagacctc ggctcggacg ggtcggagag cgaggacgag 8220

ggcgcgcagg ccggagctgt ccagggtcct gagacgctgc ggagtcaggt cagtgggcag 8280

cggcggcgcg cggttgactt gcaggagctt ttccagggcg cgcgggaggt ccagatggta 8340

cttgatctcc acggcgccgt tggtggctac gtccacggct tgcagggtgc cgtgcccctg 8400

gggcgccacc accgtgcccc gtttcttctt gggcgctgct tccatgtcgg tcagaagcgg 8460

cggcgaggac gcgcgccggg cggcaggggc ggctcggggc ccggaggcag gggcggcagg 8520

ggcacgtcgg cgccgcgcgc gggcaggttc tggtactgcg cccggagaag actggcgtga 8580

gcgacgacgc gacggttgac gtcctggatc tgacgcctct gggtgaaggc cacgggaccc 8640

gtgagtttga acctgaaaga gagttcgaca gaatcaatct cggtatcgtt gacggcggcc 8700

tgccgcagga tctcttgcac gtcgcccgag ttgtcctggt aggcgatctc ggtcatgaac 8760

tgctcgatct cctcctcctg aaggtctccg cggccggcgc gctcgacggt ggccgcgagg 8820

tcgttggaga tgcggcccat gagctgcgag aaggcgttca tgccggcctc gttccagacg 8880

cggctgtaga ccacggctcc gtcggggtcg cgcgcgcgca tgaccacctg ggcgaggttg 8940

agctcgacgt ggcgcgtgaa gaccgcgtag ttgcagaggc gctggtagag gtagttgagc 9000

gtggtggcga tgtgctcggt gacgaagaag tacatgatcc agcggcggag cggcatctcg 9060

ctgacgtcgc ccagggcttc caagcgttcc atggcctcgt agaagtccac ggcgaagttg 9120

aaaaactggg agttgcgcgc cgagacggtc aactcctcct ccagaagacg gatgagctcg 9180

gcgatggtgg cgcgcacctc gcgctcgaag gccccggggg gctcctcttc catctcctcc 9240

tcttcctcct ccactaacat ctcttctact tcctcctcag gaggcggtgg cgggggaggg 9300

gccctgcgtc gccggcggcg cacgggcaga cggtcgatga agcgctcgat ggtctccccg 9360

cgccggcgac gcatggtctc ggtgacggcg cgcccgtcct cgcggggccg cagcatgaag 9420

acgccgccgc gcatctccag gtggccgccg ggggggtctc cgttgggcag ggagagggcg 9480

ctgacgatgc atcttatcaa ttgacccgta gggactccgc gcaaggacct gagcgtctcg 9540

agatccacgg gatccgaaaa ccgctgaacg aaggcttcga gccagtcgca gtcgcaaggt 9600

aggctgagcc cggtttcttg ttcttcgggt atttggtcgg gaggcgggcg ggcgatgctg 9660

ctggtgatga agttgaagta ggcggtcctg agacggcgga tggtggcgag gagcaccagg 9720

tccttgggcc cggcttgctg gatgcgcaga cggtcggcca tgccccaggc gtggtcctga 9780

cacctggcga ggtccttgta gtagtcctgc atgagccgct ccacgggcac ctcctcctcg 9840

cccgcgcggc cgtgcatgcg cgtgagcccg aacccgcgct gcggctggac gagcgccagg 9900

tcggcgacga cgcgctcggt gaggatggcc tgctggatct gggtgagggt ggtctggaag 9960

tcgtcgaagt cgacgaagcg gtggtaggct ccggtgttga tggtgtagga gcagttggcc 10020

atgacggacc agttgacggt ctggtggccg ggtcgcacga gctcgtggta cttgaggcgc 10080

gagtaggcgc gcgtgtcgaa gatgtagtcg ttgcaggcgc gcacgaggta ctggtatccg 10140

acgaggaagt gcggcggcgg ctggcggtag agcggccatc gctcggtggc gggggcgccg 10200

ggcgcgaggt cctcgagcat gaggcggtgg tagccgtaga tgtacctgga catccaggtg 10260

atgccggcgg cggtggtgga ggcgcgcggg aactcgcgga cgcggttcca gatgttgcgc 10320

agcggcagga agtagttcat ggtggccgcg gtctggcccg tgaggcgcgc gcagtcgtgg 10380

atgctctaga catacgggca aaaacgaaag cggtcagcgg ctcgactccg tggcctggag 10440

gctaagcgaa cgggttgggc tgcgcgtgta ccccggttcg aatctcgaat caggctggag 10500

ccgcagctaa cgtggtactg gcactcccgt ctcgacccaa gcctgctaac gaaacctcca 10560

ggatacggag gcgggtcgtt ttttggcctt ggtcgctggt catgaaaaac tagtaagcgc 10620

ggaaagcggc cgcccgcgat ggctcgctgc cgtagtctgg agaaagaatc gccagggttg 10680

cgttgcggtg tgccccggtt cgagcctcag cgctcggcgc cggccggatt ccgcggctaa 10740

cgtgggcgtg gctgccccgt cgtttccaag accccttagc cagccgactt ctccagttac 10800

ggagcgagcc cctctttttt tttcttgtgt ttttgccaga tgcatcccgt actgcggcag 10860

atgcgccccc accctccacc acaaccgccc ctaccgcagc agcagcaaca gccggcgctt 10920

ctgcccccgc cccagcagca gccagccact accgcggcgg ccgccgtgag cggagccggc 10980

gttcagtatg acctggcctt ggaagagggc gaggggctgg cgcggctggg ggcgtcgtcg 11040

ccggagcggc acccgcgcgt gcagatgaaa agggacgctc gcgaggccta cgtgcccaag 11100

cagaacctgt tcagagacag gagcggcgag gagcccgagg agatgcgcgc ctcccgcttc 11160

cacgcggggc gggagctgcg gcgcggcctg gaccgaaagc gggtgctgag ggacgaggat 11220

ttcgaggcgg acgagctgac ggggatcagc cccgcgcgcg cgcacgtggc cgcggccaac 11280

ctggtcacgg cgtacgagca gaccgtgaag gaggagagca acttccaaaa atccttcaac 11340

aaccacgtgc gcacgctgat cgcgcgcgag gaggtgaccc tgggcctgat gcacctgtgg 11400

gacctgctgg aggccatcgt gcagaacccc acgagcaagc cgctgacggc gcagctgttt 11460

ctggtggtgc agcacagtcg ggacaacgag acgttcaggg aggcgctgct gaatatcacc 11520

gagcccgagg gccgctggct cctggacctg gtgaacattt tgcagagcat cgtggtgcag 11580

gagcgcgggc tgccgctgtc cgagaagctg gcggccatca acttctcggt gctgagtctg 11640

ggcaagtact acgctaggaa gatctacaag accccgtacg tgcccataga caaggaggtg 11700

aagatcgacg ggttttacat gcgcatgacc ctgaaagtgc tgaccctgag cgacgatctg 11760

ggggtgtacc gcaacgacag gatgcaccgc gcggtgagcg ccagccgccg gcgcgagctg 11820

agcgaccagg agctgatgca cagcctgcag cgggccctga ccggggccgg gaccgagggg 11880

gagagctact ttgacatggg cgcggacctg cgctggcagc ccagccgccg ggccttggaa 11940

gctgccggcg gttcccccta cgtggaggag gtggacgatg aggaggagga gggcgagtac 12000

ctggaagact gatggcgcga ccgtattttt gctagatgca gcaacagcca ccgccgccgc 12060

ctcctgatcc cgcgatgcgg gcggcgctgc agagccagcc gtccggcatt aactcctcgg 12120

acgattggac ccaggccatg caacgcatca tggcgctgac gacccgcaat cccgaagcct 12180

ttagacagca gcctcaggcc aaccggctct cggccatcct ggaggccgtg gtgccctcgc 12240

gctcgaaccc cacgcacgag aaggtgctgg ccatcgtgaa cgcgctggtg gagaacaagg 12300

ccatccgcgg tgacgaggcc gggctggtgt acaacgcgct gctggagcgc gtggcccgct 12360

acaacagcac caacgtgcag acgaacctgg accgcatggt gaccgacgtg cgcgaggcgg 12420

tgtcgcagcg cgagcggttc caccgcgagt cgaacctggg ctccatggtg gcgctgaacg 12480

ccttcctgag cacgcagccc gccaacgtgc cccggggcca ggaggactac accaacttca 12540

tcagcgcgct gcggctgatg gtggccgagg tgccccagag cgaggtgtac cagtcggggc 12600

cggactactt cttccagacc agtcgccagg gcttgcagac cgtgaacctg agccaggctt 12660

tcaagaactt gcagggactg tggggcgtgc aggccccggt cggggaccgc gcgacggtgt 12720

cgagcctgct gacgccgaac tcgcgcctgc tgctgctgct ggtggcgccc ttcacggaca 12780

gcggcagcgt gagccgcgac tcgtacctgg gctacctgct taacctgtac cgcgaggcca 12840

tcggacaggc gcacgtggac gagcagacct accaggagat cacccacgtg agccgcgcgc 12900

tgggccagga ggacccgggc aacctggagg ccaccctgaa cttcctgctg accaaccggt 12960

cgcagaagat cccgccccag tacgcgctga gcaccgagga ggagcgcatc ctgcgctacg 13020

tgcagcagag cgtggggctg ttcctgatgc aggagggggc cacgcccagc gcggcgctcg 13080

acatgaccgc gcgcaacatg gagcccagca tgtacgcccg caaccgcccg ttcatcaata 13140

agctgatgga ctacttgcat cgggcggccg ccatgaactc ggactacttt accaacgcca 13200

tcttgaaccc gcactggctc ccgccgcccg ggttctacac gggcgagtac gacatgcccg 13260

accccaacga cgggttcctg tgggacgacg tggacagcag cgtgttctcg ccgcgtccag 13320

gaaccaatgc cgtgtggaag aaagagggcg gggaccggcg gccgtcctcg gcgctgtccg 13380

gtcgcgcggg tgctgccgcg gcggtgcccg aggccgccag ccccttcccg agcctgccct 13440

tttcgctgaa cagcgtgcgc agcagcgagc tgggtcggct gacgcgaccg cgcctgctgg 13500

gcgaggagga gtacctgaac gactccttgt tgaggcccga gcgcgagaag aacttcccca 13560

ataacgggat agagagcctg gtggacaaga tgagccgctg gaagacgtac gcgcacgagc 13620

acagggacga gccccgagct agcagcgcag gcacccgtag acgccagcgg cacgacaggc 13680

agcggggact ggtgtgggac gatgaggatt ccgccgacga cagcagcgtg ttggacttgg 13740

gtgggagtgg tggtaacccg ttcgctcacc tgcgcccccg tatcgggcgc ctgatgtaag 13800

aatctgaaaa aataaaagac ggtactcacc aaggccatgg cgaccagcgt gcgttcttct 13860

ctgttgtttg tagtagtatg atgaggcgcg tgtacccgga gggtcctcct ccctcgtacg 13920

agagcgtgat gcagcaggcg gtggcggcgg cgatgcagcc cccgctggag gcgccttacg 13980

tgcccccgcg gtacctggcg cctacggagg ggcggaacag cattcgttac tcggagctgg 14040

cacccttgta cgataccacc cggttgtacc tggtggacaa caagtcggca gacatcgcct 14100

cgctgaacta ccagaacgac cacagcaact tcctgaccac cgtggtgcag aacaacgatt 14160

tcacccccac ggaggccagc acccagacca tcaactttga cgagcgctcg cggtggggcg 14220

gccagctgaa aaccatcatg cacaccaaca tgcccaacgt gaacgagttc atgtacagca 14280

acaagttcaa ggcgcgggtg atggtctcgc gcaagacccc caacggggtg gatgatgatt 14340

atgatggtag tcaggacgag ctgacctacg agtgggtgga gtttgagctg cccgagggca 14400

acttctcggt gaccatgacc atcgatctga tgaacaacgc catcatcgac aactacttgg 14460

cggtggggcg gcagaacggg gtgctggaga gcgacatcgg cgtgaagttc gacacgcgca 14520

acttccggct gggctgggac cccgtgaccg agctggtgat gccgggcgtg tacaccaacg 14580

aggccttcca ccccgacatc gtcctgctgc ccggctgcgg cgtggacttc accgagagcc 14640

gcctcagcaa cctgctgggc atccgcaagc ggcagccctt ccaggagggc ttccagatcc 14700

tgtacgagga cctggagggg ggcaacatcc ccgcgctctt ggatgtcgaa gcctacgaga 14760

aaagcaagga ggatagcacc gccgcggcga ccgcagccgt ggccaccgcc tctaccgagg 14820

tgcggggcga taattttgct agcgctgcgg cagcggccga ggcggctgaa accgaaagta 14880

agatagtcat ccagccggtg gagaaggaca gcaaggacag gagctacaac gtgctcgcgg 14940

acaagaaaaa caccgcctac cgcagctggt acctggccta caactacggc gaccccgaga 15000

agggcgtgcg ctcctggacg ctgctcacca cctcggacgt cacctgcggc gtggagcaag 15060

tctactggtc gctgcccgac atgatgcaag acccggtcac cttccgctcc acgcgtcaag 15120

ttagcaacta cccggtggtg ggcgccgagc tcctgcccgt ctactccaag agcttcttca 15180

acgagcaggc cgtctactcg cagcagctgc gcgccttcac ctcgctcacg cacgtcttca 15240

accgcttccc cgagaaccag atcctcgtcc gcccgcccgc gcccaccatt accaccgtca 15300

gtgaaaacgt tcctgctctc acagatcacg ggaccctgcc gctgcgcagc agtatccggg 15360

gagtccagcg cgtgaccgtc actgacgcca gacgccgcac ctgcccctac gtctacaagg 15420

ccctgggcgt agtcgcgccg cgcgtcctct cgagccgcac cttctaaaaa atgtccattc 15480

tcatctcgcc cagtaataac accggttggg gcctgcgcgc gcccagcaag atgtacggag 15540

gcgctcgcca acgctccacg caacaccccg tgcgcgtgcg cgggcacttc cgcgctccct 15600

ggggcgccct caagggccgc gtgcgctcgc gcaccaccgt cgacgacgtg atcgaccagg 15660

tggtggccga cgcgcgcaac tacacgcccg ccgccgcgcc cgtctccacc gtggacgccg 15720

tcatcgacag cgtggtggcc gacgcgcgcc ggtacgcccg caccaagagc cggcggcggc 15780

gcatcgcccg gcggcaccgg agcacccccg ccatgcgcgc ggcgcgagcc ttgctgcgca 15840

gggccaggcg cacgggacgc agggccatgc tcagggcggc cagacgcgcg gcctccggca 15900

gcagcagcgc cggcaggacc cgcagacgcg cggccacggc ggcggcggcg gccatcgcca 15960

gcatgtcccg cccgcggcgc ggcaacgtgt actgggtgcg cgacgccgcc accggtgtgc 16020

gcgtgcccgt gcgcacccgc ccccctcgca cttgaagatg ctgacttcgc gatgttgatg 16080

tgtcccagcg gcgaggagga tgtccaagcg caaatacaag gaagagatgc tccaggtcat 16140

cgcgcctgag atctacggcc ccgcggcggc ggtgaaggag gaaagaaagc cccgcaaact 16200

gaagcgggtc aaaaaggaca aaaaggagga ggaagatgac ggactggtgg agtttgtgcg 16260

cgagttcgcc ccccggcggc gcgtgcagtg gcgcgggcgg aaagtgaaac cggtgctgcg 16320

gcccggcacc acggtggtct tcacgcccgg cgagcgttcc ggctccgcct ccaagcgctc 16380

ctacgacgag gtgtacgggg acgaggacat cctcgagcag gcggtcgagc gtctgggcga 16440

gtttgcgtac ggcaagcgca gccgccccgc gcccttgaaa gaggaggcgg tgtccatccc 16500

gctggaccac ggcaacccca cgccgagcct gaagccggtg accctgcagc aggtgctacc 16560

gagcgcggcg ccgcgccggg gcttcaagcg cgagggcggc gaggatctgt acccgaccat 16620

gcagctgatg gtgcccaagc gccagaagct ggaggacgtg ctggagcaca tgaaggtgga 16680

ccccgaggtg cagcccgagg tcaaggtgcg gcccatcaag caggtggccc cgggcctggg 16740

cgtgcagacc gtggacatca agatccccac ggagcccatg gaaacgcaga ccgagcccgt 16800

gaagcccagc accagcacca tggaggtgca gacggatccc tggatgccag caccagcttc 16860

caccagcact cgccgaagac gcaagtacgg cgcggccagc ctgctgatgc ccaactacgc 16920

gctgcatcct tccatcatcc ccacgccggg ctaccgcggc acgcgcttct accgcggcta 16980

caccagcagc cgccgccgca agaccaccac ccgccgccgt cgtcgcagcc gccgcagcag 17040

caccgcgact tccgccttgg tgcggagagt gtatcgcagc gggcgcgagc ctctgaccct 17100

gccgcgcgcg cgctaccacc cgagcatcgc catttaacta ccgcctccta cttgcagata 17160

tggccctcac atgccgcctc cgcgtcccca ttacgggcta ccgaggaaga aagccgcgcc 17220

gtagaaggct gacggggaac gggctgcgtc gccatcacca ccggcggcgg cgcgccatca 17280

gcaagcggtt ggggggaggc ttcctgcccg cgctgatccc catcatcgcc gcggcgatcg 17340

gggcgatccc cggcatagct tccgtggcgg tgcaggcctc tcagcgccac tgagacacaa 17400

aaaagcatgg atttgtaata aaaaaaaaaa tggactgacg ctcctggtcc tgtgatgtgt 17460

gtttttagat ggaagacatc aatttttcgt ccctggcacc gcgacacggc acgcggccgt 17520

ttatgggcac ctggagcgac atcggcaaca gccaactgaa cgggggcgcc ttcaattgga 17580

gcagtctctg gagcgggctt aagaatttcg ggtccacgct caaaacctat ggcaacaagg 17640

cgtggaacag cagcacaggg caggcgctga gggaaaagct gaaagaacag aacttccagc 17700

agaaggtggt tgatggcctg gcctcaggca tcaacggggt ggttgacctg gccaaccagg 17760

ccgtgcagaa acagatcaac agccgcctgg acgcggtccc gcccgcgggg tccgtggaga 17820

tgccccaggt ggaggaggag ctgcctcccc tggacaagcg cggcgacaag cgaccgcgtc 17880

ccgacgcgga ggagacgctg ctgacgcaca cggacgagcc gcccccgtac gaggaggcgg 17940

tgaaactggg cctgcccacc acgcggcccg tggcgcctct ggccaccgga gtgctgaaac 18000

ccagcagcag ccagcccgcg accctggact tgcctccgcc tcgcccctcc acagtggcta 18060

agcccctgcc gccggtggcc gtcgcgtcgc gcgccccccg aggccgcccc caggcgaact 18120

ggcagagcac tctgaacagc atcgtgggtc tgggagtgca gagtgtgaag cgccgccgct 18180

gctattaaaa gacactgtag cgcttaactt gcttgtctgt gtgtatatgt atgtccgccg 18240

accagaagga ggagtgtgaa gaggcgcgtc gccgagttgc aagatggcca ccccatcgat 18300

gctgccccag tgggcgtaca tgcacatcgc cggacaggac gcttcggagt acctgagtcc 18360

gggtctggtg cagttcgccc gcgccacaga cacctacttc agtctgggga acaagtttag 18420

gaaccccacg gtggcgccca cgcacgatgt gaccaccgac cgcagccagc ggctgacgct 18480

gcgcttcgtg cccgtggacc gcgaggacaa cacctactcg tacaaagtgc gctacacgct 18540

ggccgtgggc gacaaccgcg tgctggacat ggccagcacc tactttgaca tccgcggcgt 18600

gctggaccgg ggccctagct tcaaacccta ctctggcacc gcctacaaca gcctagctcc 18660

caagggagct cccaattcca gccagtggga gcaagcaaaa acaggcaatg ggggaactat 18720

ggaaacacac acatatggtg tggccccaat gggcggagag aatattacaa aagatggtct 18780

tcaaattgga actgacgtta cagcgaatca gaataaacca atttatgccg acaaaacatt 18840

tcaaccagaa ccgcaagtag gagaagaaaa ttggcaagaa actgaaaact tttatggcgg 18900

tagagctctt aaaaaagaca caaacatgaa accttgctat ggctcctatg ctagacccac 18960

caatgaaaaa ggaggtcaag ctaaacttaa agttggagat gatggagttc caaccaaaga 19020

attcgacata gacctggctt tctttgatac tcccggtggc accgtgaacg gtcaagacga 19080

gtataaagca gacattgtca tgtataccga aaacacgtat ttggaaactc cagacacgca 19140

tgtggtatac aaaccaggca aggatgatgc aagttctgaa attaacctgg ttcagcagtc 19200

tatgcccaac agacccaact acattgggtt cagggacaac tttatcggtc ttatgtacta 19260

caacagcact ggcaatatgg gtgtgcttgc tggtcaggcc tcccagctga atgctgtggt 19320

tgatttgcaa gacagaaaca ccgagctgtc ctaccagctc ttgcttgact ctttgggtga 19380

cagaacccgg tatttcagta tgtggaacca ggcggtggac agttatgacc ccgatgtgcg 19440

catcatcgaa aaccatggtg tggaggatga attgccaaac tattgcttcc ccttggacgg 19500

ctctggcact aacgccgcat accaaggtgt gaaagtaaaa gatggtcaag atggtgatgt 19560

tgagagtgaa tgggaaaatg acgatactgt tgcagctcga aatcaattat gtaaaggtaa 19620

cattttcgcc atggagatta atctccaggc taacctgtgg agaagtttcc tctactcgaa 19680

cgtggccctg tacctgcccg actcctacaa gtacacgccg accaacgtca cgctgccgac 19740

caacaccaac acctacgatt acatgaatgg cagagtgaca cctccctcgc tggtagacgc 19800

ctacctcaac atcggggcgc gctggtcgct ggaccccatg gacaacgtca accccttcaa 19860

ccaccaccgc aacgcgggcc tgcgctaccg ctccatgctc ctgggcaacg ggcgctacgt 19920

gcccttccac atccaggtgc cccaaaagtt tttcgccatc aagagcctcc tgctcctgcc 19980

cgggtcctac acctacgagt ggaacttccg caaggacgtc aacatgatcc tgcagagctc 20040

cctaggcaac gacctgcgca cggacggggc ctccatcgcc ttcaccagca tcaacctcta 20100

cgccaccttc ttccccatgg cgcacaacac cgcctccacg ctcgaggcca tgctgcgcaa 20160

cgacaccaac gaccagtcct tcaacgacta cctctcggcg gccaacatgc tctaccccat 20220

cccggccaac gccaccaacg tgcccatctc catcccctcg cgcaactggg ccgccttccg 20280

cggatggtcc ttcacgcgcc tgaagacccg cgagacgccc tcgctcggct ccgggttcga 20340

cccctacttc gtctactcgg gctccatccc ctacctagac ggcaccttct acctcaacca 20400

caccttcaag aaggtctcca tcaccttcga ctcctccgtc agctggcccg gcaacgaccg 20460

cctcctgacg cccaacgagt tcgaaatcaa gcgcaccgtc gacggagagg gatacaacgt 20520

ggcccagtgc aacatgacca aggactggtt cctggtccag atgctggccc actacaacat 20580

cggctaccag ggcttctacg tgcccgaggg ctacaaggac cgcatgtact ccttcttccg 20640

caacttccag cccatgagcc gccaggtcgt ggacgaggtc aactacaagg actaccaggc 20700

cgtcaccctg gcctaccagc acaacaactc gggcttcgtc ggctacctcg cgcccaccat 20760

gcgccagggc cagccctacc ccgccaacta cccctacccg ctcatcggca agagcgccgt 20820

cgccagcgtc acccagaaaa agttcctctg cgaccgggtc atgtggcgca tccccttctc 20880

cagcaacttc atgtccatgg gcgcgctcac cgacctcggc cagaacatgc tctacgccaa 20940

ctccgcccac gcgctagaca tgaatttcga agtcgacccc atggatgagt ccacccttct 21000

ctatgttgtc ttcgaagtct tcgacgtcgt ccgagtgcac cagccccacc gcggcgtcat 21060

cgaagccgtc tacctgcgca cgcccttctc ggccggcaac gccaccacct aagccgctct 21120

tgcttcttgc aagatgacgg cgggctccgg cgagcaggag ctcagggcca tcctccgcga 21180

cctgggctgc gggccctgct tcctgggcac cttcgacaag cgcttccctg gattcatggc 21240

cccgcacaag ctggcctgcg ccatcgtgaa cacggccggc cgcgagaccg ggggcgagca 21300

ctggctggcc ttcgcctgga acccgcgctc ccacacatgc tacctcttcg accccttcgg 21360

gttctcggac gagcgcctca agcagatcta ccagttcgag tacgagggcc tgctgcgtcg 21420

cagcgccctg gccaccgagg accgctgcgt caccctggaa aagtccaccc agaccgtgca 21480

gggtccgcgc tcggccgcct gcgggctctt ctgctgcatg ttcctgcacg ccttcgtgca 21540

ctggcccgac cgccccatgg acaagaaccc caccatgaac ttactgacgg gggtgcccaa 21600

cggcatgctc cagtcgcccc aggtggaacc caccctgcgc cgcaaccagg aagcgctcta 21660

ccgcttcctc aatgcccact ccgcctactt tcgctcccac cgcgcgcgca tcgagaaggc 21720

caccgccttc gaccgcatga atcaagacat gtaaaaaacc ggtgtgtgta tgtgaatgct 21780

ttattcataa taaacagcac atgtttatgc caccttctct gaggctctga ctttatttag 21840

aaatcgaagg ggttctgccg gctctcggca tggcccgcgg gcagggatac gttgcggaac 21900

tggtacttgg gcagccactt gaactcgggg atcagcagct tgggcacggg gaggtcgggg 21960

aacgagtcgc tccacagctt gcgcgtgagt tgcagggcgc ccagcaggtc gggcgcggag 22020

atcttgaaat cgcagttggg acccgcgttc tgcgcgcgag agttgcggta cacggggttg 22080

cagcactgga acaccatcag ggccgggtgc ttcacgcttg ccagcaccgt cgcgtcggtg 22140

atgccctcca cgtccagatc ctcggcgttg gccatcccga agggggtcat cttgcaggtc 22200

tgccgcccca tgctgggcac gcagccgggc ttgtggttgc aatcgcagtg cagggggatc 22260

agcatcatct gggcctgctc ggagctcatg cccgggtaca tggccttcat gaaagcctcc 22320

agctggcgga aggcctgctg cgccttgccg ccctcggtga agaagacccc gcaggacttg 22380

ctagagaact ggttggtggc gcagccggcg tcgtgcacgc agcagcgcgc gtcgttgttg 22440

gccagctgca ccacgctgcg cccccagcgg ttctgggtga tcttggcccg gttggggttc 22500

tccttcagcg cgcgctgccc gttctcgctc gccacatcca tctcgatagt gtgctccttc 22560

tggatcatca cggtcccgtg caggcaccgc agcttgccct cggcttcggt gcagccgtgc 22620

agccacagcg cgcagccggt gcactcccag ttcttgtggg cgatctggga gtgcgagtgc 22680

acgaagccct gcaggaagcg gcccatcatc gcggtcaggg tcttgttgct ggtgaaggtc 22740

agcgggatgc cgcggtgctc ctcgttcaca tacaggtggc agatgcggcg gtacacctcg 22800

ccctgctcgg gcatcagctg gaaggcggac ttcaggtcgc tctccacgcg gtaccggtcc 22860

atcagcagcg tcatcacttc catgcccttc tcccaggccg aaacgatcgg caggctcagg 22920

gggttcttca ccgccattgt catcttagtc gccgccgccg aggtcagggg gtcgttctcg 22980

tccagggtct caaacactcg cttgccgtcc ttctcgatga tgcgcacggg gggaaagctg 23040

aagcccacgg ccgccagctc ctcctcggcc tgcctttcgt cctcgctgtc ctggctgatg 23100

tcttgcaaag gcacatgctt ggtcttgcgg ggtttctttt tgggcggcag aggcggcggc 23160

gatgtgctgg gagagcgcga gttctcgttc accacgacta tttcttcttc ttggccgtcg 23220

tccgagacca cgcggcggta ggcatgcctc ttctggggca gaggcggagg cgacgggctc 23280

tcgcggttcg gcgggcggct ggcagagccc cttccgcgtt cgggggtgcg ctcctggcgg 23340

cgctgctctg actgacttcc tccgcggccg gccattgtgt tctcctaggg agcaacaaca 23400

agcatggaga ctcagccatc gtcgccaaca tcgccatctg cccccgccgc caccgccgac 23460

gagaaccagc agcagaatga aagcttaacc gccccgccgc ccagccccac ctccgacgcc 23520

gcggccccag acatgcaaga gatggaggaa tccatcgaga ttgacctggg ctacgtgacg 23580

cccgcggagc acgaggagga gctggcagcg cgcttttcag ccccggaaga gaaccaccaa 23640

gagcagccag agcaggaagc agagaacgag cagaaccagg ctgggcacga gcatggcgac 23700

tacctgagcg gggcagagga cgtgctcatc aagcatctgg cccgccaatg catcatcgtc 23760

aaggacgcgc tgctcgaccg cgccgaggtg cccctcagcg tggcggagct cagccgcgcc 23820

tacgagcgca acctcttctc gccgcgcgtg ccccccaagc gccagcccaa cggcacctgt 23880

gagcccaacc cgcgcctcaa cttctacccg gtcttcgcgg tgcccgaggc cctggccacc 23940

taccacctct ttttcaagaa ccaaaggatc cccgtctcct gccgcgccaa ccgcacccgc 24000

gccgacgccc tgctcaacct gggccccggc gcccgcctac ctgatatcac ctccttggaa 24060

gaggttccca agatcttcga gggtctgggc agcgacgaga ctcgggccgc gaacgctctg 24120

caaggaagcg gagaggagca tgagcaccac agcgccctgg tggagttgga aggcgacaac 24180

gcgcgcctgg cggtcctcaa gcgcacggtc gagctgaccc acttcgccta cccggcgctc 24240

aacctgcccc ccaaggtcat gagcgccgtc atggaccagg tgctcatcaa gcgcgcctcg 24300

cccctctcgg aggaggagat gcaggacccc gagagttcgg acgagggcaa gcccgtggtc 24360

agcgacgagc agctggcgcg ctggctggga gcgagtagca ccccccagag cctggaagag 24420

cggcgcaagc tcatgatggc cgtggtcctg gtgaccgtgg agctggagtg tctgcgccgc 24480

ttctttgccg acgcggagac cctgcgcaag gtcgaggaga acctgcacta cctcttcagg 24540

cacgggttcg tgcgccaggc ctgcaagatc tccaacgtgg agctgaccaa cctggtctcc 24600

tacatgggca tcctgcacga gaaccgcctg gggcaaaacg tgctgcacac caccctgcgc 24660

ggggaggccc gccgcgacta catccgcgac tgcgtctacc tgtacctctg ccacacctgg 24720

cagacgggca tgggcgtgtg gcagcagtgc ctggaggagc agaacctgaa agagctctgc 24780

aagctcctgc agaagaacct caaggccctg tggaccgggt tcgacgagcg taccaccgcc 24840

tcggacctgg ccgacctcat cttccccgag cgcctgcggc tgacgctgcg caacgggctg 24900

cccgacttta tgagccaaag catgttgcaa aactttcgct ctttcatcct cgaacgctcc 24960

gggatcctgc ccgccacctg ctccgcgctg ccctcggact tcgtgccgct gaccttccgc 25020

gagtgccccc cgccgctctg gagccactgc tacttgctgc gcctggccaa ctacctggcc 25080

taccactcgg acgtgatcga ggacgtcagc ggcgagggtc tgctggagtg ccactgccgc 25140

tgcaacctct gcacgccgca ccgctccctg gcctgcaacc cccagctgct gagcgagacc 25200

cagatcatcg gcaccttcga gttgcaaggc cccggcgacg gcgagggcaa ggggggtctg 25260

aaactcaccc cggggctgtg gacctcggcc tacttgcgca agttcgtgcc cgaggactac 25320

catcccttcg agatcaggtt ctacgaggac caatcccagc cgcccaaggc cgagctgtcg 25380

gcctgcgtca tcacccaggg ggccatcctg gcccaattgc aagccatcca gaaatcccgc 25440

caagaatttc tgctgaaaaa gggccacggg gtctacttgg acccccagac cggagaggag 25500

ctcaacccca gcttccccca ggatgccccg aggaagcagc aagaagctga aagtggagct 25560

gccgccgccg gaggatttgg aggaagactg ggagagcagt caggcagagg aggaggagat 25620

ggaagactgg gacagcactc aggcagagga ggacagcctg caagacagtc tggaggagga 25680

agacgaggtg gaggaggcag aggaagaagc agccgccgcc agaccgtcgt cctcggcgga 25740

gaaagcaagc agcacggata ccatctccgc tccgggtcgg ggtcgcggcg gccgggccca 25800

cagtaggtgg gacgagaccg ggcgcttccc gaaccccacc acccagaccg gtaagaagga 25860

gcggcaggga tacaagtcct ggcgggggca caaaaacgcc atcgtctcct gcttgcaagc 25920

ctgcgggggc aacatctcct tcacccggcg ctacctgctc ttccaccgcg gggtgaactt 25980

cccccgcaac atcttgcatt actaccgtca cctccacagc ccctactact gtttccaaga 26040

agaggcagaa acccagcagc agcagaaaac cagcggcagc agcagctaga aaatccacag 26100

cggcggcagg tggactgagg atcgcggcga acgagccggc gcagacccgg gagctgagga 26160

accggatctt tcccaccctc tatgccatct tccagcagag tcgggggcag gagcaggaac 26220

tgaaagtcaa gaaccgttct ctgcgctcgc tcacccgcag ttgtctgtat cacaagagcg 26280

aagaccaact tcagcgcact ctcgaggacg ccgaggctct cttcaacaag tactgcgcgc 26340

tcactcttaa agagtagccc gcgcccgccc acacacggaa aaaggcggga attacgtcac 26400

cacctgcgcc cttcgcccga ccatcatgag caaagagatt cccacgcctt acatgtggag 26460

ctaccagccc cagatgggcc tggccgccgg cgccgcccag gactactcca cccgcatgaa 26520

ctggctcagt gccgggcccg cgatgatctc acgggtgaat gacatccgcg cccaccgaaa 26580

ccagatactc ctagaacagt cagcgatcac cgccacgccc cgccatcacc ttaatccgcg 26640

taattggccc gccgccctgg tgtaccagga aattccccag cccacgaccg tactacttcc 26700

gcgagacgcc caggccgaag tccagctgac taactcaggt gtccagctgg ccggcggcgc 26760

cgccctgtgt cgtcaccgcc ccgctcaggg tataaagcgg ctggtgatcc gaggcagagg 26820

cacacagctc aacgacgagg tggtgagctc ttcgctgggt ctgcgacctg acggagtctt 26880

ccaactcgcc ggatcgggga gatcttcctt cacgcctcgt caggccgtcc tgactttgga 26940

gagttcgtcc tcgcagcccc gctcgggcgg catcggcact ctccagttcg tggaggagtt 27000

cactccctcg gtctacttca accccttctc cggctccccc ggccactacc cggacgagtt 27060

catcccgaac ttcgacgcca tcagcgagtc ggtggacggc tacgattgaa tgtcccatgg 27120

tggcgcagct gacctagctc ggcttcgaca cctggaccac tgccgccgct tccgctgctt 27180

cgctcgggat ctcgccgagt ttgcctactt tgagctgccc gaggagcacc ctcagggccc 27240

agcccacgga gtgcggatca tcgtcgaagg gggcctcgac tcccacctgc ttcggatctt 27300

cagccagcga ccgatcctgg tcgagcgcga acaaggacag acccttctta ctttgtactg 27360

catctgcaac caccccggcc tgcatgaaag tctttgttgt ctgctgtgta ctgagtataa 27420

taaaagctga gatcagcgac tactccggac tcgattgtgg tgttcctgct atcaaccggt 27480

ccctgttctt caccgggaac gagaccgagc tccagctcca gtgtaagccc cacaagaagt 27540

acctcacctg gctgttccag ggctccccga tcgccgttgt caaccactgc gacaacgacg 27600

gagtcctgct gagcggccct gccaacctta ctttttccac ccgcagaagc aagctccagc 27660

tcttccaacc cttcctcccc gggacctatc agtgcgtctc aggaccctgc catcacacct 27720

tccacctgat cccgaatacc acagcgccgc tccccgctac taacaaccaa actacccacc 27780

aacgccaccg tcgcgacctt tcctctgaat ctaataccac taccggaggt gagctccgag 27840

gtcgaccaac ctctgggatt tactacggcc cctgggaggt ggtggggtta atagcgctag 27900

gcctagttgc gggtgggctt ttggttctct gctacctata cctcccttgc tgttcgtact 27960

tagtggtgct gtgttgctgg tttaagaaat ggggaagatc accctagtga gctgcggtgc 28020

gctggtggcg gtgttgcttt cgattgtggg actgggcggc gcggctgtag tgaaggagaa 28080

ggccgatccc tgcttgcatt tcaatcccaa caaatgccag ctgagttttc agcccgatgg 28140

caatcggtgc gcggtactga tcaagtgcgg atgggaatgc gagaacgtga gaatcgagta 28200

caataacaag actcggaaca atactctcgc gtccgtgtgg cagcccgggg accccgagtg 28260

gtacaccgtc tctgtccccg gtgctgacgg ctccccgcgc accgtgaata atactttcat 28320

ttttgcgcac atgtgcaaca cggtcatgtg gatgagcaag cagtacgata tgtggccccc 28380

cacgaaggag aacatcgtgg tcttctccat cgcttacagc ctgtgcacgg cgctaatcac 28440

cgctatcgtg tgcctgagca ttcacatgct catcgctatt cgccccagaa ataatgccga 28500

gaaagagaaa cagccataac acgttttttc acacaccttg tttttacaga caatgcgtct 28560

gttaaatttt ttaaacattg tgctcagtat tgcttatgcc tctggttatg caaacataca 28620

gaaaaccctt tatgtaggat ctgatggtac actagagggt acccaatcac aagccaaggt 28680

tgcatggtat ttttatagaa ccaacactga tccagttaaa ctttgtaagg gtgaattgcc 28740

gcgtacacat aaaactccac ttacatttag ttgcagcaat aataatctta cacttttttc 28800

aattacaaaa caatatactg gtacttatta cagtacaaac tttcatacag gacaagataa 28860

atattatact gttaaggtag aaaatcctac cactcctaga actaccacca ccaccactac 28920

tgcaaagccc actgtgaaaa ctacaactag gaccaccaca actacagaaa ccaccaccag 28980

cacaacactt gctgcaacta cacacacaca cactaagcta accttacaga ccactaatga 29040

tttgatcgcc ctgctgcaaa agggggataa cagcaccact tccaatgagg agatacccaa 29100

atccatgatt ggcattattg ttgctgtagt ggtgtgcatg ttgatcatcg ccttgtgcat 29160

ggtgtactat gccttctgct acagaaagca cagactgaac gacaagctgg aacacttact 29220

aagtgttgaa ttttaatttt ttagaaccat gaagatccta ggccttttta gtttttctat 29280

cattacctct gctctttgtg aatcagtgga tagagatgtt actattacca ctggttctaa 29340

ttatacactg aaagggccac cctcaggtat gctttcgtgg tattgctatt ttggaactga 29400

cactgatcaa actgaattat gcaattttca aaaaggcaaa acctcaaact ctaaaatctc 29460

taattatcaa tgcaatggca ctgatctgat actactcaat gtcacgaaag catatggtgg 29520

cagttattat tgccctggac aaaacactga agaaatgatt ttttacaaag tggaagtggt 29580

tgatcccact acaccaccca ccaccacaac tattcatacc acacacacag aacaaacacc 29640

agaggcaaca gaagcagagt tggccttcca ggttcacgga gattcctttg ctgtcaatac 29700

ccctacaccc gatcagcggt gtccggggcc gctagtcagc ggcattgtcg gtgtgctttc 29760

gggattagca gtcataatca tctgcatgtt catttttgct tgctgctata gaaggcttta 29820

ccgacaaaaa tcagacccac tgctgaacct ctatgtttaa ttttttccag agccatgaag 29880

gcagttagcg ctctagtttt ttgttctttg attggcattg tttttaatag taaaattacc 29940

agagttagct ttattaaaca tgttaatgta actgaaggag ataacatcac actagcaggt 30000

gtagaaggtg ctcaaaacac cacctggaca aaataccatc taggatggag agatatttgc 30060

acctggaatg taacttatta ttgcatagga gttaatctta ccattgttaa cgctaaccaa 30120

tctcagaatg ggttaattaa aggacagagt gttagtgtga ccagtgatgg gtactatacc 30180

cagcatagtt ttaactacaa cattactgtc ataccactgc ctacgcctag cccacctagc 30240

actaccacac agacaaccac atacagtaca tcaaatcagc ctaccaccac tacagcagca 30300

gaggttgcca gctcgtctgg ggtccgagtg gcatttttga tgttggcccc atctagcagt 30360

cccactgcta gtaccaatga gcagactact gaatttttgt ccactgtcga gagccacacc 30420

acagctacct ccagtgcctt ctctagcacc gccaatctct cctcgctttc ctctacacca 30480

atcagccccg ctactactcc tagccccgct cctcttccca ctcccctgaa gcaaacagac 30540

ggcggcatgc aatggcagat caccctgctc attgtgatcg ggttggtcat cctggccgtg 30600

ttgctctact acatcttctg ccgccgcatt cccaacgcgc accgcaagcc ggcctacaag 30660

cccatcgtta tcgggcagcc ggagccgctt caggtggaag ggggtctaag gaatcttctc 30720

ttctctttta cagtatggtg attgaactat gattcctaga caattcttga tcactattct 30780

tatctgcctc ctccaagtct gtgccaccct cgctctggtg gccaacgcca gtccagactg 30840

tattgggccc ttcgcctcct acgtgctctt tgccttcgtc acctgcatct gctgctgtag 30900

catagtctgc ctgcttatca ccttcttcca gttcattgac tggatctttg tgcgcatcgc 30960

ctacctgcgc caccaccccc agtaccgcga ccagcgagtg gcgcagctgc tcaggctcct 31020

ctgataagca tgcgggctct gctacttctc gcgcttctgc tgttagtgct cccccgtccc 31080

gtcgaccccc ggtcccccac tcagtccccc gaggaggttc gcaaatgcaa attccaagaa 31140

ccctggaaat tcctcaaatg ctaccgccaa aaatcagaca tgcatcccag ctggatcatg 31200

atcattggga tcgtgaacat tctggcctgc accctcatct cctttgtgat ttacccctgc 31260

tttgactttg gttggaactc gccagaggcg ctctatctcc cgcctgaacc tgacacacca 31320

ccacagcagc aacctcaggc acacgcacta ccaccaccac agcctaggcc acaatacatg 31380

cccatattag actatgaggc cgagccacag cgacccatgc tccccgctat tagttacttc 31440

aatctaaccg gcggagatga ctgacccact ggccaataac aacgtcaacg accttctcct 31500

ggacatggac ggccgcgcct cggagcagcg actcgcccaa cttcgcattc gtcagcagca 31560

ggagagagcc gtcaaggagc tgcaggacgg catagccatc caccagtgca agagaggcat 31620

cttctgcctg gtgaaacagg ccaagatctc ctacgaggtc acccagaccg accatcgcct 31680

ctcctacgag ctcctgcagc agcgccagaa gttcacctgc ctggtcggag tcaaccccat 31740

cgtcatcacc cagcagtcgg gcgataccaa ggggtgcatc cactgctcct gcgactcccc 31800

cgactgcgtc cacactctga tcaagaccct ctgcggcctc cgcgacctcc tccccatgaa 31860

ctaatcaccc ccttatccag tgaaataaag atcatattga tgatgattta aataaaaaaa 31920

ataatcattt gatttgaaat aaagatacaa tcatattgat gatttgagtt taacaaaaat 31980

aaagaatcac ttacttgaaa tctgatacca ggtctctgtc catgttttct gccaacacca 32040

cctcactccc ctcttcccag ctctggtact gcaggccccg gcgggctgca aacttcctcc 32100

acacgctgaa ggggatgtca aattcctcct gtccctcaat cttcatttta tcttctatca 32160

gatgtccaaa aagcgcgtcc gggtggatga tgacttcgac cccgtctacc cctacgatgc 32220

agacaacgca ccgaccgtgc ccttcatcaa cccccccttc gtctcttcag atggattcca 32280

agagaagccc ctgggggtgt tgtccctgcg actggctgac cccgtcacca ccaagaacgg 32340

ggaaatcacc ctcaagctgg gagagggggt ggacctcgac tcgtcgggaa aactcatctc 32400

caacacggcc accaaggccg ccgcccctct cagtatttca aacaacacca tttcccttaa 32460

aactgctgcc cctttctaca acaacaatgg aactttaagc ctcaatgtct ccacaccatt 32520

agcagtattt cccacattta acactttagg cataagtctt ggaaacggtc ttcagacttc 32580

aaataagttg ttgactgtac aactaactca tcctcttaca ttcagctcaa atagcatcac 32640

agtaaaaaca gacaaagggc tatatattaa ctccagtgga aacagaggac ttgaggctaa 32700

tataagccta aaaagaggac tagtttttga cggtaatgct attgcaacat atattggaaa 32760

tggcttagac tatggatctt atgatagtga tggaaaaaca agacccgtaa ttaccaaaat 32820

tggagcagga ttaaattttg atgctaacaa agcaatagct gtcaaactag gcacaggttt 32880

aagttttgac tccgctggtg ccttgacagc tggaaacaaa caggatgaca agctaacact 32940

ttggactacc cctgacccaa gccctaattg tcaattactt tcagacagag atgccaaatt 33000

tactctctgt cttacaaaat gcggtagtca aatactaggc actgtggcag tggcggctgt 33060

tactgtagga tcagcactaa atccaattaa tgacacagtc aaaagcgcca tagttttcct 33120

tagatttgat tccgatggtg tactcatgtc aaactcatca atggtaggtg attactggaa 33180

ctttagggag ggacagacca ctcaaagtgt agcctataca aatgctgtgg gattcatgcc 33240

aaatataggt gcatatccaa aaacccaaag taaaacacct aaaaatagca tagtcagtca 33300

ggtatattta actggagaaa ctactatgcc aatgacacta accataactt tcaatggcac 33360

tgatgaaaaa gacacaaccc cagttagcac ctactctatg acttttacat ggcagtggac 33420

tggagactat aaggacaaaa atattacctt tgctaccaac tcattctctt tttcctacat 33480

cgcccaggaa taatcccacc cagcaagcca accccttttc ccaccacctt tgtctatatg 33540

gaaactctga aacagaaaaa taaagttcaa gtgttttatt gaatcaacag ttttacagga 33600

ctcgagcagt tatttttcct ccaccctccc aggacatgga atacaccacc ctctcccccc 33660

gcacagcctt gaacatctga atgccattgg tgatggacat gcttttggtc tccacgttcc 33720

acacagtttc agagcgagcc agtctcggat cggtcaggga gatgaaaccc tccgggcact 33780

cccgcatctg cacctcacag ctcaacagct gaggattgtc ctcggtggtc gggatcacgg 33840

ttatctggaa gaagcagaag agcggcggtg ggaatcatag tccgcgaacg ggatcggccg 33900

gtggtgtcgc atcaggcccc gcagcagtcg ctgccgccgc cgctccgtca agctgctgct 33960

cagggggttc gggtccaggg actccctcag catgatgccc acggccctca gcatcagtcg 34020

tctggtgcgg cgggcgcagc agcgcatgcg aatctcgctc aggtcactgc agtacgtgca 34080

acacaggacc accaggttgt tcaacagtcc atagttcaac acgctccagc cgaaactcat 34140

cgcgggaagg atgctaccca cgtggccgtc gtaccagatc ctcaggtaaa tcaagtggcg 34200

ctccctccag aagacgctgc ccatgtacat gatctccttg ggcatgtggc ggttcaccac 34260

ctcccggtac cacatcaccc tctggttgaa catgcagccc cggatgatcc tgcggaacca 34320

cagggccagc accgccccgc ccgccatgca gcgaagagac cccggatccc ggcaatgaca 34380

atggaggacc caccgctcgt acccgtggat catctgggag ctgaacaagt ctatgttggc 34440

acagcacagg catatgctca tgcatctctt cagcactctc agctcctcgg gggtcaaaac 34500

catatcccag ggcacgggga actcttgcag gacagcgaac cccgcagaac agggcaatcc 34560

tcgcacataa cttacattgt gcatggacag ggtatcgcaa tcaggcagca ccgggtgatc 34620

ctccaccaga gaagcgcggg tctcggtctc ctcacagcgt ggtaaggggg ccggccgata 34680

cgggtgatgg cgggacgcgg ctgatcgtgt tctcgaccgt gtcatgatgc agttgctttc 34740

ggacattttc gtacttgctg tagcagaacc tggtccgggc gctgcacacc gatcgccggc 34800

ggcggtctcg gcgcttggaa cgctcggtgt taaagttgta aaacagccac tctctcagac 34860

cgtgcagcag atctagggcc tcaggagtga tgaagatccc atcatgcctg atagctctga 34920

tcacatcgac caccgtggaa tgggccaggc ccagccagat gatgcaattt tgttgggttt 34980

cggtgacggc gggggaggga agaacaggaa gaaccatgat taacttttaa tccaaacggt 35040

ctcggagcac ttcaaaatga aggtcacgga gatggcacct ctcgcccccg ctgtgttggt 35100

ggaaaataac agccaggtca aaggtgatac ggttctcgag atgttccacg gtggcttcca 35160

gcaaagcctc cacgcgcaca tccagaaaca agacaatagc gaaagcggga gggttctcta 35220

attcctcaac catcatgtta cactcctgca ccatccccag ataattttca tttttccagc 35280

cttgaatgat tcgaactagt tcctgaggta aatccaagcc agccatgata aaaagctcgc 35340

gcagagcacc ctccaccggc attcttaagc acaccctcat aattccaaga tattctgctc 35400

ctggttcacc tgcagcagat tgacaagcgg aatatcaaaa tctctgccgc gatccctgag 35460

ctcctccctc agcaataact gtaagtactc tttcatatcg tctccgaaat ttttagccat 35520

aggaccccca ggaataagag aagggcaagc cacattacag ataaaccgaa gtccccccca 35580

gtgagcattg ccaaatgtaa gattgaaata agcatgctgg ctagacccgg tgatatcttc 35640

cagataactg gacagaaaat cgggtaagca atttttaaga aaatcaacaa aagaaaaatc 35700

ttccaggtgc acgtttaggg cctcgggaac aacgatggag taagtgcaag gggtgcgttc 35760

cagcatggtt agttagctga tctgtaaaaa aacaaaaaat aaaacattaa accatgctag 35820

cctggcgaac aggtgggtaa atcgttctct ccagcaccag gcaggccacg gggtctccgg 35880

cgcgaccctc gtaaaaattg tcgctatgat tgaaaaccat cacagagaga cgttcccggt 35940

ggccggcgtg aatgattcga gaagaagcat acacccccgg aacattggag tccgtgagtg 36000

aaaaaaagcg gccgaggaag caatgaggca ctacaacgct cactctcaag tccagcaaag 36060

cgatgccatg cggatgaagc acaaaatttt caggtgcgta aaaaatgtaa ttactcccct 36120

cctgcacagg cagcgaagct cccgatccct ccagatacac atacaaagcc tcagcgtcca 36180

tgcccgggca agcttaccga gcggcagcag cagcggcaca caacaggcgc aagagtcaga 36240

gaaaagactg agctctaacc tgtccgcccg ctctctgctc aatatatagc cccagatcta 36300

cactgacgta aaggccaaag tctaaaaata cccgccaaat aatcacacac gcccagcaca 36360

cgcccagaaa ccggtgacac actcagaaaa atacgcgcac ttcctcaaac ggccaaactg 36420

ccgtcatttc cgggttccca cgctacgtca tcaaaacacg actttcaaat tccgtcgacc 36480

gttaaaaaca tcacccgccc cgcccctaac ggtcgccgct cccgcagcca atcaccttcc 36540

tccctcccca aattcaaaca gctcatttgc atattaacgc gcaccaaaag tttgaggtat 36600

attattgatg atgccttaat taagaattca ctggccgtcg ttttacaacg tcgtgactgg 36660

gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 36720

cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 36780

gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 36840

tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 36900

ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 36960

gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 37020

gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 37080

gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 37140

tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 37200

caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 37260

ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 37320

gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 37380

aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 37440

ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 37500

atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 37560

gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 37620

gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 37680

atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 37740

aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 37800

actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 37860

aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 37920

tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 37980

ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 38040

agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 38100

tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 38160

aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 38220

gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 38280

atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 38340

gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 38400

gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 38460

tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 38520

accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 38580

ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 38640

cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 38700

agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 38760

ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 38820

tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 38880

ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 38940

cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 39000

gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 39060

tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 39120

cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 39180

cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 39240

tatgaccatg attacgccaa gcttgcatgc ctgcaggttt aaacttaatt aagg 39294

<210> 2

<211> 1440

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

tcacagaacc ctagtattca acctgccacc tccctcccaa cacacagagt acacagtcct 60

ttctccccgg ctggccttaa aaagcatcat atcatgggta acagacatat tcttaggtgt 120

tatattccac acggtttcct gtcgagccaa acgctcatca gtgatattaa taaactcccc 180

gggcagctca cttaagttca tgtcgctgtc cagctgctga gccacaggct gctgtccaac 240

ttgcggttgc ttaacgggcg gcgaaggaga agtccacgcc tacatggggg tagagtcata 300

atcgtgcatc aggatagggc ggtggtgctg cagcagcgcg cgaataaact gctgccgccg 360

ccgctccgtc ctgcaggaat acaacatggc agtggtctcc tcagcgatga ttcgcaccgc 420

ccgcagcata aggcgccttg tcctccgggc acagcagcgc accctgatct cacttaaatc 480

agcacagtaa ctgcagcaca gcaccacaat attgttcaaa atcccacagt gcaaggcgct 540

gtatccaaag ctcatggcgg ggaccacaga acccacgtgg ccatcatacc acaagcgcag 600

gtagattaag tggcgacccc tcataaacac gctggacata aacattacct cttttggcat 660

gttgtaattc accacctccc ggtaccatat aaacctctga ttaaacatgg cgccatccac 720

caccatccta aaccagctgg ccaaaacctg cccgccggct atacactgca gggaaccggg 780

actggaacaa tgacagtgga gagcccagga ctcgtaacca tggatcatca tgctcgtcat 840

gatatcaatg ttggcacaac acaggcacac gtgcatacac ttcctcagga ttacaagctc 900

ctcccgcgtt agaaccatat cccagggaac aacccattcc tgaatcagcg taaatcccac 960

actgcaggga agacctcgca cgtaactcac gttgtgcatt gtcaaagtgt tacattcggg 1020

cagcagcgga tgatcctcca gtatggtagc gcgggtttct gtctcaaaag gaggtagacg 1080

atccctactg tacggagtgc gccgagacaa ccgagatcgt gttggtcgta gtgtcatgcc 1140

aaatggaacg ccggacgtag tcatatttcc tgaagcaaaa ccaggtgcgg gcgtgacaaa 1200

cagatctgcg tctccggtct cgccgcttag atcgctctgt gtagtagttg tagtatatcc 1260

actctctcaa agcatccagg cgccccctgg cttcgggttc tatgtaaact ccttcatgcg 1320

ccgctgccct gataacatcc accaccgcag aataagccac acccagccaa cctacacatt 1380

cgttctgcga gtcacacacg ggaggagcgg gaagagctgg aagaaccatg tttttttttt 1440

<210> 3

<211> 170

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

taactataac ggtcctaagg tagcgagaga cccaagctgg ctagcgttta aacgggccct 60

ctagactcga gcggccgcca ctgtgctgga tgatccgagc tcggtaccaa gcttaagttt 120

aaaccgctga tcaatctatg tcgggtgcgg agaaagaggt aatgaaatgg 170

<210> 4

<211> 252

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

actataacgg tcctaaggta gcgaatacac tttggttaac ccagtggatt cgcgggcaca 60

gacgcccagg accgcgctcc ccacgtggcg gagggactgg ggacccgggc acccgtcctg 120

ccccttcacc ttccagctcc gcctcctccg cgcggacccc gccccgtccc gacccctccc 180

gggtccccgg cccagccccc tccgggccct cccagcccct ccccttcctt tccgcggccc 240

cgccctgtta ac 252

<210> 5

<211> 1394

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atttcgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta 60

gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc 120

tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg 180

ccaataggga ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg 240

gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa 300

tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac 360

atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg 420

cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg 480

agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca 540

ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg 600

ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag 660

acccaagctg gctagcgcca ccatgtggct gcagagcctg ctgctcttgg gcactgtggc 720

ctgcagcatc tctgcacccg cccgctcgcc cagccccagc acgcagccct gggagcatgt 780

gaatgccatc caggaggccc ggcgtctcct gaacctgagt agagacactg ctgctgagat 840

gaatgaaaca gtagaagtca tctcagaaat gtttgacctc caggagccga cctgcctaca 900

gacccgcctg gagctgtaca agcagggcct gcggggcagc ctcaccaagc tcaagggccc 960

cttgaccatg atggccagcc actacaagca gcactgccct ccaaccccgg aaacttcctg 1020

tgcaacccag attatcacct ttgaaagttt caaagagaac ctgaaggact ttctgcttgt 1080

catccccttt gactgctggg agccagtcca ggagtaatct agactcgagc ggccgccact 1140

gtgctggatg atccgagctc ggtaccaagc ttaagtttaa accgctgatc agcctcgact 1200

gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 1260

gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg 1320

agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 1380

gaagacaata gcag 1394

Claims (10)

1. A method for constructing a recombinant adenovirus vector, comprising: the method is to transform chimpanzee adenovirus AdC6 to obtain a recombinant adenovirus vector, wherein the transformation comprises the following steps: the E1 gene sequence of AdC6 is completely or partially deleted, the E3 gene sequence of AdC6 is partially deleted, and the E4 gene sequence of AdC6 is modified by the E4 gene sequence of the C subtype human adenovirus genome.

2. The method of constructing a recombinant adenoviral vector according to claim 1, wherein:

the E3 gene sequence of the partially deleted AdC6 is A1 or A2:

a1, reserving ORF1 sequence of AdC6E 3 gene, and partially or completely deleting other sequences of AdC6E 3 gene;

a2, reserving ORF1 sequence and ORF9 sequence of AdC6E 3 gene, and partially or totally deleting other sequences of AdC6E 3 gene;

the E4 gene sequence of AdC6 transformed by the E4 gene sequence of the C subtype human adenovirus genome is any one of B1-B3:

b1, replacing an ORF6 sequence and/or an ORF6/7 sequence of the AdC6E 4 gene by a corresponding sequence of a C subtype human adenovirus genome;

b2, replacing an ORF4 sequence of the AdC6E 4 gene by a corresponding sequence of a C subtype human adenovirus genome on the basis of the B1;

b3, deleting the ORF1 sequence and/or the ORF2 sequence of the AdC6E 4 gene on the basis of B1 or B2.

3. The method for constructing a recombinant adenoviral vector according to claim 1 or 2, wherein: the recombinant adenovirus vector is a non-replicative adenovirus vector, and the E1 gene sequence of the completely or partially deleted AdC6 is a completely deleted AdC6E1 gene sequence.

4. The method for constructing a recombinant adenoviral vector according to claim 3, wherein: the recombinant adenovirus vector is any one of the following vectors 1) to 5):

1) The recombinant adenovirus vector is named pAdC6XY2, and the nucleotide sequence of the pAdC6XY2 is a nucleotide sequence obtained by deleting 563-3413 th sites of a sequence 1 in a sequence table, deleting 27441-31864 th sites of the sequence 1 in the sequence table and replacing 33841-34746 th sites of the sequence 1 in the sequence table with 280-1164 th sites of the sequence 2 in the sequence table;

2) The recombinant adenovirus vector is named pAdC6XY3, and the nucleotide sequence of the pAdC6XY3 is a nucleotide sequence obtained by deleting 563-3413 th sites of a sequence 1 in a sequence table, deleting 27441-31864 th sites of the sequence 1 in the sequence table and replacing 33593-35026 th sites of the sequence 1 in the sequence table with 1-1440 th sites of the sequence 2 in the sequence table;

3) The recombinant adenovirus vector is named pAdC6XY4, and the nucleotide sequence of the pAdC6XY4 is obtained by deleting 563-3413 th sites of a sequence 1 in a sequence table, deleting 27441-31864 th sites of the sequence 1 in the sequence table, replacing 33593-35026 th sites of the sequence 1 in the sequence table with 1-1440 th sites of the sequence 2 in the sequence table, and deleting 35381-36190 th sites of the sequence 1 in the sequence table;

4) The recombinant adenovirus vector is named pAdC6XY5, wherein the nucleotide sequence of the pAdC6XY5 is a nucleotide sequence obtained by deleting 563-3413 th sites of a sequence 1 in a sequence table, deleting 27441-31456 th sites of the sequence 1 in the sequence table, and replacing 33593-35026 th sites of the sequence 1 in the sequence table with 1-1440 th sites of the sequence 2 in the sequence table;

5) The recombinant adenovirus vector is named pAdC6XY6, wherein the nucleotide sequence of pAdC6XY6 is obtained by deleting 563-3413 th sites of a sequence 1 in a sequence table, deleting 27441-31456 th sites of the sequence 1 in the sequence table, replacing 33593-35026 th sites of the sequence 1 in the sequence table with 1-1440 th sites of the sequence 2 in the sequence table, and deleting 35381-36190 th sites of the sequence 1 in the sequence table.

5. The method for constructing a recombinant adenoviral vector according to claim 1 or 2, wherein: the recombinant adenovirus vector is a conditionally replicating oncolytic adenovirus vector, the E1 gene sequence of the completely or partially deleted AdC6 is an E1A sequence of a reserved AdC6E1 gene, and other sequences of the AdC6E1 gene are partially or completely deleted;

and the method further comprises the step of placing a tumor specific promoter in front of the E1A gene of the AdC6 to regulate the expression of E1A.

6. The method for constructing a recombinant adenoviral vector according to claim 5, wherein: the method further comprises the step of inserting an exogenous gene that promotes oncolytic in the AdC6E1 region or E3 region or E4 or other regions.

7. The method for constructing a recombinant adenoviral vector according to claim 5 or 6, wherein: the recombinant adenovirus vector is any one of the following 6) to 7):

6) A recombinant adenovirus vector named pAdC6XYR, wherein the nucleotide sequence of pAdC6XYR is obtained by deleting 1905-3413 th sites in a sequence 1 in a sequence table, inserting the sequence 4 in the sequence table between 564 th site and 574 th site, deleting 27896-31904 th sites in the sequence table, and replacing 33841-34746 th sites in the sequence table 1 with 280-1164 th sites in the sequence table 2;

7) The recombinant adenovirus vector named AdC6XYR-CSF2 is a nucleotide sequence obtained by deleting 1905-3413 th bits from a sequence 1 in a sequence table, inserting a sequence 4 in the sequence table between 564 th bit and 574 th bit, deleting 27896-31904 th bits from the sequence 1 in the sequence table to replace the sequence 5 in the sequence table, and replacing 33841-34746 th bits from the sequence 1 in the sequence table with 280-1164 th bits from the sequence 2 in the sequence table.

8. A recombinant adenoviral vector constructed by the method of any one of claims 1-7.

9. Introducing the recombinant adenovirus vector of claim 8 into a packaging cell and packaging the resulting recombinant adenovirus.

10. Any one of the following applications:

use of P1, the method of any one of claims 1-7, or the recombinant adenoviral vector of claim 8 in the preparation of a recombinant adenovirus;

use of P2, a method according to any one of claims 3 to 4, a recombinant adenoviral vector constructed according to any one of claims 3 to 4, or a recombinant adenovirus packaged with a recombinant adenoviral vector constructed according to any one of claims 3 to 4 for the preparation of a vaccine vector;

use of P3, a method according to any one of claims 3 to 4, a recombinant adenoviral vector constructed according to any one of claims 3 to 4, or a recombinant adenovirus packaged with a recombinant adenoviral vector constructed according to any one of claims 3 to 4 for the preparation of a vaccine;

use of P4, the method of any one of claims 3 to 4, the recombinant adenoviral vector constructed according to any one of claims 3 to 4, or the recombinant adenovirus packaged with the recombinant adenoviral vector constructed according to any one of claims 3 to 4 for the manufacture of a medicament for gene therapy;

use of P5, the method of any one of claims 5 to 7, the recombinant adenoviral vector constructed according to any one of claims 5 to 7, or the recombinant adenovirus packaged with the recombinant adenoviral vector constructed according to any one of claims 5 to 7 in the preparation of a medicament for inhibiting tumor cells;

use of P6, the method of any one of claims 5 to 7, the recombinant adenoviral vector constructed according to any one of claims 5 to 7, or the recombinant adenovirus packaged with the recombinant adenoviral vector constructed according to any one of claims 5 to 7 in the manufacture of a medicament for the prevention or treatment of a tumor.

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