Novel recombinant vaccinia virus shuttle vector and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a novel recombinant vaccinia virus shuttle vector and application thereof.The shuttle vector comprises homologous recombination arms composed of an upstream gene sequence and a downstream gene sequence of vaccinia virus thymidine kinase TK, and the homologous recombination arms are VACV-093 and VACV-095 respectively; the kit further comprises three exogenous gene expression cassettes, and the three exogenous gene expression cassettes respectively contain a pTK11 heterozygous promoter, a pSE / L promoter and a p7.5 K promoter. The invention innovatively provides a new strategy for completely removing a TK open reading frame, retaining a pTK promoter and constructing a hybrid strong promoter, and solves the problem of promoter limitation during multi-gene expression and the problem of low screening efficiency of recombinant vaccinia virus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a novel recombinant vaccinia virus shuttle vector and its application. Background Art

[0002] Oncolytic viruses (OVs) are a class of viruses that can selectively replicate in tumor cells and lyse tumor cells. Oncolytic viruses can be divided into two major categories: natural viruses and genetically engineered viruses, and most of them are genetically engineered viruses. Compared with wild viruses, genetically engineered viruses can carry therapeutic elements such as toxic proteins and immunomodulatory factors, and show significant advantages in enhancing the oncolytic ability of viruses and activating the anti-tumor immunity of the host. At present, oncolytic viruses play an important role in tumor immunotherapy and have become one of the most promising tumor immunotherapies.

[0003] Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. According to characteristics such as host range and pathogenicity, vaccinia virus can be divided into strains such as WR (Western reserve) strain, Wyeth strain, Copenhagen strain, Lister strain, and TianTan strain. VACV is a double-stranded DNA virus, and the size of its genome is about 190 kb (WR strain) and encodes more than 200 proteins. Since the entire replication cycle of vaccinia virus occurs in the cytoplasm and does not integrate into the host genome, it has high safety. Historically, wild vaccinia virus strains have been used as vaccines to eradicate smallpox. In recent years, genetically engineered recombinant vaccinia viruses have also been widely used as gene expression vectors in different fields such as vaccine development and oncolytic viruses.

[0004] Homologous recombination is a genetic information exchange process that occurs between the genetic materials of organisms. This recombination depends on the same or highly similar sequences between DNA molecules, and through intermolecular pairing and exchange, new DNA combinations are formed. Since the vaccinia virus genome is large, exogenous genes cannot be introduced into the vaccinia virus genome by direct gene cleavage and ligation methods. Therefore, using a shuttle vector containing a DNA molecule with a homologous sequence to a specific gene region of vaccinia virus for homologous recombination with vaccinia virus is the main method for constructing recombinant oncolytic vaccinia viruses.

[0005] Thymidine kinase (TK) is one of the key enzymes in the pyrimidine metabolic cycle. It catalyzes the conversion of thymidine (dTdR) to deoxythymidine monophosphate (dTMP) and plays an important role in DNA synthesis. In normal cells, the expression of TK is low, but in tumor cells, the expression of TK is significantly increased. The TK of vaccinia virus is an early expression product during the virus replication process, providing a high-level nucleotide pool for its DNA replication. Therefore, removing the TK gene of oncolytic vaccinia virus can make it more inclined to selectively replicate in tumor cells and kill tumor cells. At the same time, the TK region can also be used as a site for inserting foreign genes.

[0006] Currently, when constructing recombinant oncolytic vaccinia virus, the common strategy is to use the TK gene as a homologous recombination arm and insert the foreign expression cassette into the TK gene region, but usually the open reading frame (ORF) of the TK gene is not completely removed. Under this strategy, the TK gene promoter (pTK) of the generated recombinant vaccinia virus will still express a non-functional truncated TK protein, and the expression of foreign genes depends on an additional introduced promoter (such as pSE / L or p7.5K). However, when it is necessary to construct a recombinant vaccinia virus with co-expression of multiple genes, the available strong promoter resources are limited, which becomes a technical bottleneck restricting the construction of recombinant vaccinia virus. The present invention innovatively proposes a new strategy of completely removing the TK open reading frame while retaining the pTK promoter and constructing a hybrid strong promoter, which solves the problems of promoter limitation during multi-gene expression and low screening efficiency of recombinant vaccinia virus. Summary of the Invention

[0007] The object of the present invention is to provide a novel recombinant vaccinia virus shuttle vector and its application in view of the above existing problems.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A recombinant oncolytic vaccinia virus shuttle vector, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0010] The shuttle vector includes homologous recombination arms VACV-093 and VACV-095 composed of the gene sequences upstream and downstream of the vaccinia virus thymidine kinase (TK) gene; it also includes three foreign gene expression cassettes, and the three foreign gene expression cassettes contain pTK11 hybrid promoter, pSE / L promoter and p7.5K promoter respectively.

[0011] The nucleotide sequence of the homologous recombination arm VACV-093 is shown in SEQ ID NO.2; the nucleotide sequence of the homologous recombination arm VACV-095 is shown in SEQ ID NO.3.

[0012] Among them, the homologous recombination arms VACV-093 and VACV-095 can undergo homologous recombination with wild vaccinia virus, and the open reading frame sequence of the TK gene in the obtained recombinant oncolytic vaccinia virus genome is completely deleted, but the promoter pTK of the TK gene is retained.

[0013] The pTK11 hybrid promoter is formed by connecting the above-retained promoter pTK of the TK gene in series with the promoter p11.

[0014] The nucleotide sequence of the pTK11 hybrid promoter is as shown in SEQ ID NO.4.

[0015] The preparation method of the shuttle vector includes the steps:

[0016] (1) Design and synthesize the gene fragment 093-pTK-pSE / L-p7.5K-095: The gene fragment 093-pTK-pSE / L-p7.5K-095 contains the homologous recombination arm VACV-093, the pTK expression cassette, the pSE / L expression cassette, the p7.5K expression cassette and the homologous recombination arm VACV-095; the nucleotide sequence of the gene fragment 093-pTK-pSE / L-p7.5K-095 is as shown in SEQ ID NO.5;

[0017] (2) Design and synthesize the gene fragment pUCori-AmpR: The gene fragment pUCori-AmpR contains the pUCori replicon and the ampicillin resistance gene; the nucleotide sequence of the gene fragment pUCori-AmpR is as shown in SEQ ID NO.6;

[0018] (3) Use the CloneEZ "seamless" cloning technology to connect the gene fragment 093-pTK-pSE / L-p7.5K-095 and the gene fragment pUCori-AmpR to obtain a vector, and name this vector pTKmCH;

[0019] (4) Design and synthesize the p11 promoter gene fragment;

[0020] (5) Use the CloneEZ "seamless" cloning technology to connect the p11 promoter gene fragment into the pTKmCH vector to obtain the shuttle vector.

[0021] The present invention also provides the application of the shuttle vector in the preparation of recombinant oncolytic vaccinia virus.

[0022] In summary, due to the adoption of the above technical solutions, the present invention has the following beneficial effects:

[0023] 1. The recombinant oncolytic vaccinia virus shuttle vector provided by the present invention uses the upstream and downstream gene sequences VACV-093 and VACV-095 of the vaccinia virus TK gene as homologous recombination arms. After the homologous recombination of the homologous recombination arms VACV-093 and VACV-095 with the vaccinia virus, the open reading frame sequence of the TK gene in the obtained recombinant oncolytic vaccinia virus genome is completely deleted. When the open reading frame sequence of the TK gene is completely deleted, foreign genes are inserted to avoid further increase of the vaccinia virus genome as much as possible. The present invention innovatively proposes a new strategy of completely removing the TK open reading frame while retaining the pTK promoter and constructing a hybrid strong promoter, which solves the problems of promoter limitation in multi-gene expression and low screening efficiency of recombinant vaccinia virus.

[0024] 2. The vaccinia virus shuttle vector provided by the present invention contains three foreign gene expression cassettes. One of the expression cassettes contains the pTK11 hybrid promoter. After the homologous recombination of the homologous recombination arms VACV-093 and VACV-095 with the vaccinia virus, the open reading frame sequence of the TK gene in the obtained recombinant oncolytic vaccinia virus genome is completely deleted, but the promoter pTK of the TK gene is retained. The pTK11 hybrid promoter is formed by the tandem connection of the promoter pTK of the TK gene and the promoter p11. The pTK11 hybrid promoter can efficiently express the reporter gene red fluorescent protein mCherry and the screening gene hypoxanthine-guanine phosphoribosyltransferase (GPT) connected in series with the 2A peptide in the early and late stages of virus replication, which is beneficial to the screening of monoclonal recombinant oncolytic vaccinia virus. The other two expression cassettes contain the pSE / L promoter and the p7.5K promoter respectively. The pSE / L promoter and the p7.5K promoter contained in the vector can efficiently express at least two different target genes.

[0025] 3. The present invention solves the problems of low efficiency of previous recombinant oncolytic vaccinia virus and few inserted foreign gene fragments, laying a solid foundation for the construction of novel genetically engineered oncolytic vaccinia virus. Brief Description of the Drawings

[0026] Figure 1 It is the restriction enzyme digestion identification result and schematic diagram of the recombinant oncolytic vaccinia virus shuttle vector pTKmCH.

[0027] Figure 2 It is the restriction enzyme digestion identification result and schematic diagram of the recombinant oncolytic vaccinia virus shuttle vector pTK11mCH.

[0028] Figure 3 It is the restriction enzyme digestion identification result and schematic diagram of the recombinant oncolytic vaccinia virus shuttle vector p11mCH.

[0029] Figure 4Comparison chart of the expression efficiency of mCherry (2A peptide tandem GPT) by pTK promoter, p11 promoter and pTK11 hybrid promoter.

[0030] Figure 5 Result chart of the expression of more than 3 genes by pTK11mCH vector.

[0031] Figure 6 Screening and enrichment chart of recombinant oncolytic vaccinia viruses VACV-TK11mCH, VACV-TKmCH and VACV-11mCH.

[0032] Figure 7 Selection, identification and pattern chart of monoclonal virus of recombinant oncolytic vaccinia virus VACV-TK11mCH.

[0033] Figure 8 Sequencing chart of the pTK11 promoter region of recombinant oncolytic vaccinia virus VACV-TK11mCH. Detailed implementation manners

[0034] In order to more clearly illustrate the present invention, the following further illustrates the present invention through specific embodiments.

[0035] I. Preparation examples

[0036] Example 1

[0037] This example prepares a recombinant vaccinia virus shuttle vector pTK11mCH, including the steps of:

[0038] (1) Design and synthesize the gene fragment 093-pTK-pSE / L-p7.5K-095: The gene fragment 093-pTK-pSE / L-p7.5K-095 contains homologous recombination arms VACV-093, pTK expression cassette, pSE / L expression cassette, p7.5K expression cassette and homologous recombination arm VACV-095;

[0039] Specifically: According to the complete gene sequence of the vaccinia virus WR strain published in GenBank, partial gene sequences of the upstream gene sequence VACV-093 and the downstream gene sequence VACV-095 of the TK gene were selected as homologous recombination arms. The nucleotide sequences of the homologous recombination arms VACV-093 and VACV-095 are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively. A gene fragment 093-pTK-pSE / L-p7.5K-095 containing the homologous recombination arm VACV-093, pTK expression cassette, pSE / L expression cassette, p7.5K expression cassette and homologous recombination arm VACV-095 was designed, and its nucleotide sequence is shown in SEQ ID NO.5; then this gene fragment was synthesized artificially, and in this example, it was sent to Nanjing Genscript Biotech Co., Ltd. for synthesis;

[0040] (2) Design and synthesize the gene fragment pUCori-AmpR: The gene fragment pUCori-AmpR contains the pUCori replicon and the ampicillin resistance gene;

[0041] Specifically: A gene fragment pUCori-AmpR containing the pUCori replicon and the ampicillin resistance gene (AmpR) was designed, and its nucleotide sequence is shown in SEQ ID NO.6; then this gene fragment was synthesized artificially, and in this example, it was sent to Nanjing Genscript Biotech Co., Ltd. for synthesis; there is a 15bp overlapping sequence at the 5' and 3' ends of pUCori-AmpR and the 3' and 5' ends of the 093-pTK-pSE / L-p7.5K-095 gene fragment;

[0042] (3) Use the CloneEZ "seamless" cloning technology to ligate the gene fragment 093-pTK-pSE / L-p7.5K-095 and the gene fragment pUCori-AmpR to obtain the vector pTKmCH;

[0043] Specifically:

[0044] ① Design and synthesize primers

[0045] Primers 093-F2 for amplifying 093-pTK-pSE / L-p7.5K-095 were designed: 5'-ATGGATCACAACCAGTATCT-3' and 095-R2: 5'-CGAGTCAGTCTCATGTTCT-3';

[0046] Primers 095-F for amplifying pUCori-AmpR were designed: 5'-CATGAGACTGACTCGGGC-3' and 093-R: 5'-CTGGTTGTGATCCATTTATTGAT-3';

[0047] Then, the above primers were synthesized artificially. In this example, they were sent to Nanjing GenScript Biotech Corporation for synthesis;

[0048] ② Amplify and purify gene fragments

[0049] Using the gene fragment 093 - pTK - pSE / L - p7.5K - 095 synthesized in step (1) and the gene fragment pUCori - AmpR synthesized in step (2) as templates, the above primers were used to perform PCR amplification on them respectively; the PCR reaction conditions were: pre - denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 68°C for 15 s / kb, for 30 cycles; extension at 68°C for 5 min. Then, a PCR product purification kit (purchased from CW Biotech Co., Ltd.) was used to purify the amplified products to obtain the purified gene fragment 093 - pTK - pSE / L - p7.5K - 095 and the purified gene fragment pUCori - AmpR;

[0050] ③ Obtain a recombinant vector mixture using CloneEZ "seamless" cloning technology

[0051] Mix 0.1 pmol of the purified gene fragment 093 - pTK - pSE / L - p7.5K - 095, 0.1 pmol of the purified gene fragment pUCori - AmpR, and GenBuilder 2x Master Mix (purchased from Nanjing GenScript Biotech Corporation), add deionized water to make up to 20 μl, mix gently, and then incubate the reaction solution at 50°C for 15 minutes in a thermal cycler to obtain a recombinant vector mixture;

[0052] ④ Transform the recombinant vector mixture into Escherichia coli and identify the vector

[0053] Transform 2 μl of the recombinant vector mixture into competent Escherichia coli TOP10 cells by the conventional method, then spread them on an LB agar plate containing 100 mg / ml ampicillin and culture overnight at 37°C; pick positive clones, shake the bacteria and extract plasmids, and then perform enzyme digestion identification using NcoI and HindIII. The enzyme digestion results are as Figure 1 shown in A; the vector that was correctly identified by sequencing was named pTKmCH, and its map is as Figure 1 shown in B.

[0054] (4) Design and synthesize the p11 promoter gene fragment;

[0055] Specifically: According to the complete gene sequence of the vaccinia virus WR strain published in GenBank, the p11 promoter sequence: 5’-ATTTAGAATATATGTATGTAAAAATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAA-3’ was selected, and the p11 promoter gene fragment was artificially synthesized. In this example, it was sent to Nanjing Genscript Biotech Co., Ltd. for synthesis.

[0056] (5) The p11 promoter gene fragment was ligated into the pTKmCH vector by using CloneEZ "seamless" cloning technology, and the shuttle vector pTK11mCH was obtained.

[0057] Specifically:

[0058] ① Design and synthesize primers

[0059] Primers p11-F1: 5’-TTCTTTATTGTCATCATTTAGAATATATGTATGTAAAAAT-3’ and p11-R1: 5’-GCCCTTGCTCACCATTTATAGCATAGAAAAAAACAAAAT-3’ for amplifying the p11 promoter gene fragment were designed;

[0060] Primers mCH-F1: 5’-ATGGTGAGCAAGGGCGAG-3’ and pTK-R1: 5’-GATGACAATAAAGAATTAATTATTGT-3’ for amplifying the pTKmCH gene fragment were designed;

[0061] Then the above primers were artificially synthesized. In this example, they were sent to Nanjing Genscript Biotech Co., Ltd. for synthesis.

[0062] ② Amplify and purify gene fragments

[0063] Using the p11 promoter gene fragment synthesized in step (4) and the pTKmCH vector constructed in step (3) as templates, PCR amplification was performed on them respectively with the above primers; The PCR reaction conditions were: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 15 s, annealing at 55℃ for 15 s, extension at 68℃ for 15 s / kb, 30 cycles; extension at 68℃ for 5 min; Then a PCR product purification kit (purchased from CW Biotech Co., Ltd.) was used to purify the amplification products to obtain the purified p11 promoter gene fragment and the purified pTK11mCH gene fragment.

[0064] ③ Obtain a recombinant vector mixture by CloneEZ "seamless" cloning technology

[0065] Mix 0.1 pmol of the purified p11 promoter gene fragment, 0.1 pmol of the purified pTK11mCH gene fragment, and GenBuilder 2x Master Mix (purchased from Nanjing Genscript Biotech Co., Ltd.), supplement deionized water to 20 μl, mix gently, and incubate the reaction solution in a thermal cycler at 50 °C for 15 minutes to tandemly link the pTK promoter and p11 promoter sequences to obtain a recombinant vector mixture;

[0066] ④ Transformation of the recombinant vector mixture into Escherichia coli and vector identification

[0067] Transform 2 μl of the recombinant vector mixture into competent Escherichia coli TOP10 cells by a conventional method, then spread it on an LB agar plate containing 100 mg / ml ampicillin and culture overnight at 37 °C. Pick positive clones, shake the bacteria and extract plasmids, and then perform restriction enzyme digestion identification using NcoI and HindIII. The restriction enzyme digestion results are as Figure 2 shown in A. The vector correctly identified by sequencing was named pTK11mCH, and its map is as Figure 2 shown in B.

[0068] Comparative Example 1

[0069] This comparative example prepared the recombinant vaccinia virus shuttle vector p11mCH. The specific steps were as follows:

[0070] ① Design and synthesis of primers

[0071] Design the upstream primer p11-F2 for amplifying the p11 promoter gene fragment: 5’-GTGAACAATAATTAAGAATTCATTTAGAATATATGTATGT-3’;

[0072] Design the downstream primer pTK-R2 for amplifying the pTKmCH vector gene fragment: 5’-TTAATTATTGTTCACTTTATTCGACT-3’;

[0073] Then synthesize the above primers artificially. In this comparative example, they were sent to Nanjing Genscript Biotech Co., Ltd. for synthesis.

[0074] ② Amplification and purification of gene fragments

[0075] Using the p11 promoter gene fragment synthesized in step (4) of Example 1 as a template, the p11 promoter gene fragment was amplified using the synthesized primer p11-F2 and the primer p11-R1 synthesized in step (5) of Example 1; using the pTKmCH vector constructed in step (3) of Example 1 as a template, the pTKmCH vector gene fragment was amplified using the primer mCH-F1 synthesized in step (5) of Example 1 and the synthesized primer pTK-R2; the PCR reaction conditions were: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 68°C for 15 s / kb, 30 cycles; extension at 68°C for 5 min.

[0076] ③ Construction of vector p11mCH using CloneEZ "seamless" cloning technology

[0077] The p11mCH was constructed using a CloneEZ "seamless" cloning technology strategy similar to that for constructing pTK11mCH;

[0078] ④ Identification of p11mCH vector

[0079] The p11mCH vector was digested and identified using XbaI and HindIII, and the digestion results are as Figure 3 shown in A. The vector with correct sequencing was named p11mCH, and its map is as Figure 3 shown in B. In this vector, the p11 promoter was used to replace the partial sequence TTCTTTATTGTCATC of the pTK promoter, and an EcoRI restriction site (GAATTC) was added in front of the p11 promoter sequence.

[0080] II. Performance testing

[0081] 1. Comparison of the efficiencies of expressing mCherry-GPT by pTK promoter, p11 promoter and pTK11 hybrid promoter

[0082] HEK-293A cells were seeded in a 6-well plate and cultured routinely in an incubator at 37°C and 5% CO2. When the cell confluence was greater than 90%, wild vaccinia virus (WR strain, purchased from ATCC, accession number: VR-1354) was added to the cell culture medium at an MOI of 0.1. After 2 hours of infection, (Purchased from Polyplus-transfection) pTKmCH, p11mCH, and pTK11mCH plasmids were transfected into HEK-293A cells respectively. At 24, 48, and 72 hours of culture, the expression of red fluorescent protein mCherry was observed under a fluorescence microscope. Since the selection gene hypoxanthine-guanine phosphoribosyltransferase (GPT) is tandemly linked with mCherry by a 2A peptide and their expression levels are consistent, the expression level of mCherry can directly reflect the expression level of GPT. As Figure 4 shown, the pTK promoter expressed weak red fluorescence at 24, 48, and 72 hours. The p11 promoter and the pTK11 hybrid promoter showed stronger red fluorescence than the pTK promoter at 24, 48, and 72 hours of culture, but the pTK11 hybrid promoter showed higher red fluorescence intensity. The above results indicate that the pTK11mCH vector containing the pTK11 hybrid promoter can efficiently express the reporter gene red fluorescent protein mCherry and the selection gene hypoxanthine-guanine phosphoribosyltransferase (GPT) in the early and late stages of virus replication, which is more conducive to the screening of recombinant oncolytic vaccinia virus.

[0083] 2. Construction and identification of the vector pTK11mCH-dLuc expressing mCherry, GPT, and dual luciferase

[0084] (1) Artificial synthesis of the dLuc gene fragment

[0085] Restriction enzyme cleavage sites PacI (TTAATTAA) and SacI (GAGCTC) were added to the 5' and 3' ends of the gene fragment dLuc containing Renilla luciferase (Lucia), pSE / L promoter, p7.5K promoter, and Firefly luciferase respectively, and its nucleotide sequence is as shown in SEQ ID NO.7; among them, the pSE / L promoter is used to drive the expression of Lucia, and the p7.5K promoter is used to drive the expression of Firefly luciferase. The above DNA fragment was sent to Nanjing Genscript Biotech Co., Ltd. for artificial synthesis.

[0086] (2) Restriction enzyme digestion and ligation of the target fragment and the vector

[0087] The synthesized dLuc gene fragment and the pTK11mCH vector constructed in step (5) of Example 1 were digested with PacI and SacI respectively. The digested products were subjected to 1% agarose gel electrophoresis, and the gel was cut under ultraviolet irradiation. The digested dLuc gene fragment and pTK11mCH vector fragment were recovered using a DNA gel recovery kit (purchased from ComWin Biotech Co., Ltd.). Then, the recovered digested products were ligated using T4 DNA ligase (purchased from Nanjing Novozymes Co., Ltd.) to obtain a ligation product.

[0088] (3) Transformation of the ligation product into Escherichia coli and vector identification

[0089] 4 μl of the ligation product was transformed into competent Escherichia coli TOP10 cells by a conventional method and then spread on an LB agar plate containing 100 mg / ml ampicillin and cultured overnight at 37°C. Positive clones were picked, cultured in liquid medium, and plasmids were extracted. The correctly identified vector by sequencing was pTK11mCH-dLuc, and its map is as Figure 5 shown in A.

[0090] (4) Transfection of HEK-293A cells with the pTK11mCH-dLuc vector and determination of mCherry expression and luciferase activity

[0091] HEK-293A cells were seeded in 6-well plates and cultured routinely in an incubator at 37°C with 5% CO2. When the cell confluence was greater than 90%, wild vaccinia virus (WR strain) was added to the cell culture medium for infection at an MOI of 0.1. After 2 hours, the medium was replaced with fresh medium, and the pTK11mCH-dLuc vector was transfected into HEK-293A cells using (purchased from Polyplus-transfection).

[0092] Forty-eight hours after transfection, the expression of the red fluorescent protein mCherry was observed using a fluorescence microscope. As Figure 5 shown in B, the pTK11mCH-dLuc vector was able to express a high level of mCherry.

[0093] After 48 hours of transfection, the culture medium supernatant was removed, 1 ml of serum-free medium was added to each well, and then the cells in the 6-well plate were repeatedly frozen and thawed 3 times to fully lyse the cells. The lysate was collected and centrifuged at 3000 g for 5 min, and the supernatant was aspirated for later use. 100 μl of the supernatant was taken, and the substrate QUANTI-LucTM of Lucia (purchased from Invivogen) and the fluorescent substrate D-Luciferin of Firefly luciferase (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) were added respectively, and the luciferase activity was immediately measured using a multifunctional microplate reader. As Figure 5 shown in C, both the pSE / L and p7.5K promoters can function and express high levels of Lucia and Firefly luciferase. In this example, the pTK11 promoter of pTK11mCH, the pSE / L promoter, and the p7.5K promoter successfully co-expressed 4 foreign proteins at the same time. Among them, the pTK11 promoter successfully expressed mCherry and GPT tandemly connected by 2A peptide, the pSE / L promoter successfully expressed Lucia, and the p7.5K promoter successfully expressed Firefly luciferase.

[0094] 3. Preparation, screening, and enrichment of recombinant oncolytic vaccinia viruses VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH

[0095] (1) Preparation of recombinant oncolytic vaccinia viruses VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH

[0096] ① HEK-293A cells were seeded in a 6-well plate and cultured routinely in an incubator at 37 °C and 5% CO2. When the cell confluence was greater than 90%, wild vaccinia virus (WR strain, purchased from ATCC, accession number: VR-1354) was added to the cell culture medium. After 2 hours of infection, (purchased from Polyplus-transfection) was used to transfect HEK-293A cells with pTK11mCH, pTKmCH, and p11mCH vectors respectively, so that the pTK11mCH, pTKmCH, and p11mCH vectors underwent homologous recombination with wild vaccinia virus in HEK-293A cells to generate oncolytic vaccinia viruses VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH.

[0097] ② After 72 hours of transfection, remove the cell culture medium, add 500 μl of serum-free medium, and pipette to harvest HEK-293A cells. Freeze-thaw the cells 3 times to release the virus into the serum-free medium. Centrifuge at 3000 g to remove cell debris, and collect the serum-free medium containing VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH viruses.

[0098] (2) Screening and enrichment of recombinant oncolytic vaccinia viruses VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH

[0099] ① Add the above-collected serum-free medium containing VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH viruses to HELA-S3 cells that are more than 90% confluent, and perform screening and enrichment under the condition of a screening medium containing 25 μg / ml mycophenolic acid (MPA), 250 μg / ml xanthine, and 15 μg / ml hypoxanthine. After 72 h of virus infection, observe the expression of red fluorescent protein and the formation of virus plaques under a fluorescence microscope. Remove the cell culture medium, add 500 μl of serum-free medium, and pipette to harvest HEK-293A cells. Freeze-thaw the cells 3 times to release the virus into the serum-free medium. Centrifuge at 3000 g to remove cell debris, and collect the serum-free medium containing VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH viruses.

[0100] ② Repeat step ① to repeatedly screen and enrich the recombinant oncolytic vaccinia viruses VACV-TK11mCH, VACV-TKmCH, and VACV-11mCH.

[0101] As Figure 6 shown, VACV-11mCH requires the 5th round of screening and enrichment to observe virus plaques under a fluorescence microscope, and the red fluorescence intensity is relatively high; although VACV-TKmCH can observe virus plaques under a fluorescence microscope after the 3rd round of screening and enrichment, the red fluorescence intensity is relatively weak; VACV-TK11mCH can observe virus plaques under a fluorescence microscope after 2 rounds of screening and enrichment, and the red fluorescence intensity is relatively strong. The above results indicate that the efficiency of preparing recombinant vaccinia viruses by homologous recombination of the pTK11mCH vector containing the pTK11 hybrid promoter with the wild-type vaccinia virus WR strain is significantly improved.

[0102] 4. Selection and identification of monoclonal viruses of recombinant oncolytic vaccinia virus VACV-TK11mCH

[0103] Add the VACV-TK11mCH virus solution obtained by the above two rounds of screening and enrichment to the HELA-S3 cells with more than 90% confluence, and use the plaque purification method to select monoclonal viruses under a fluorescence microscope. As Figure 7 shown in A, the four monoclonal viruses Clone1, Clone2, Clone3, and Clone4 obtained can all express high levels of the red fluorescent protein mCherry in HELA-S3 cells.

[0104] Design and artificially synthesize primers TK-F: 5’-TGTGAAGACGATAAATTAATGATC-3’ and TK-R: 5’-GTTTGCCATACGCTCACAG-3’ for amplifying the wild-type vaccinia virus TK gene, and send them to Nanjing Genscript Biotech Co., Ltd. for artificial synthesis.

[0105] Then use a viral genome extraction kit (purchased from CWBIO) to extract the genome of the oncolytic vaccinia virus, and use a 2X Hot Start PCR Premix (purchased from ThermoFisher) and the above primers to amplify the wild-type vaccinia virus TK gene; the PCR reaction conditions are: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 60°C for 15 s, extension at 68°C for 15 s / kb, 30 cycles; extension at 68°C for 5 min, and the amplified product is subjected to 1% agarose gel electrophoresis. As Figure 7 shown in B, Clone1, Clone3, and Clone4 without the wild-type vaccinia virus TK gene band in the PCR amplification are the successfully screened monoclonal recombinant oncolytic vaccinia viruses VACV-TK11mCH, and their schematic diagrams are as Figure 7 shown in C.

[0106] 5. Sequencing of the pTK11 promoter region of the recombinant oncolytic vaccinia virus VACV-TK11mCH

[0107] Design primers pTK11-F: 5’-GACAATTGACAAAATTCACAGACT-3’ and 095-R: 5’-ATCTAACGACACAACATCCAT-3’ for amplifying the pTK11 promoter region, and send them to Nanjing Genscript Biotech Co., Ltd. for artificial synthesis.

[0108] The genome of the selected monoclonal recombinant oncolytic vaccinia virus VACV-TK11mCH was extracted using a viral genome extraction kit (purchased from CWBIO). PCR amplification was performed using a 2X Hot Start PCR Premix (purchased from ThermoFisher), primers pTK11-F and 095-R. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, extension at 68°C for 15 s / kb, for 30 cycles; and extension at 68°C for 5 min. The amplification product was subjected to DNA sequencing using primer pTK11-F. As Figure 8 shown, the DNA sequencing results showed that the TK gene expression cassette of the recombinant vaccinia virus VACV-TK11mCH was completely deleted, and the remaining pTK promoter and p11 promoter formed a pTK11 hybrid promoter to drive the expression of the red fluorescent protein mCherry and GPT.

[0109] The above description is a detailed description of the preferred and feasible embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications made under the technical spirit disclosed by the present invention shall fall within the scope of the patent covered by the present invention.

Claims

1. A recombinant oncolytic vaccinia virus shuttle vector, characterized in that, Its nucleotide sequence is as shown in SEQ ID NO.

1.

2. The shuttle vector according to claim 1, wherein It includes homologous recombination arms composed of the upstream gene sequence and the downstream gene sequence of vaccinia virus thymidine kinase TK, and the homologous recombination arms are VACV-093 and VACV-095 respectively; it also includes three foreign gene expression cassettes, and the three foreign gene expression cassettes contain the pTK11 hybrid promoter, the pSE / L promoter and the p7.5K promoter respectively.

3. The shuttle vector according to claim 2, wherein, The nucleotide sequence of the homologous recombination arm VACV-093 is as shown in SEQ ID NO.2; the nucleotide sequence of the homologous recombination arm VACV-095 is as shown in SEQ ID NO.

3.

4. The shuttle vector according to claim 2, wherein, The pTK11 hybrid promoter is formed by the tandem connection of the promoter pTK of the TK gene and the promoter p11.

5. The shuttle vector according to any one of claims 2-4, characterized in that, The nucleotide sequence of the pTK11 hybrid promoter is as shown in SEQ ID NO.

4.

6. A method for preparing the shuttle vector according to any one of claims 1-4, characterized in that, It includes the steps: (1) Design and synthesize the gene fragment 093-pTK-pSE / L-p7.5K-095: The gene fragment 093-pTK-pSE / L-p7.5K-095 contains the homologous recombination arm VACV-093, the pTK expression cassette, the pSE / L expression cassette, the p7.5K expression cassette and the homologous recombination arm VACV-095; the nucleotide sequence of the gene fragment 093-pTK-pSE / L-p7.5K-095 is as shown in SEQ ID NO.5; (2) Design and synthesize the gene fragment pUCori-AmpR: The gene fragment pUCori-AmpR contains the pUCori replicon and the ampicillin resistance gene; the nucleotide sequence of the gene fragment pUCori-AmpR is as shown in SEQ ID NO.6; (3) Use the CloneEZ "seamless" cloning technology to connect the gene fragment 093-pTK-pSE / L-p7.5K-095 and the gene fragment pUCori-AmpR to obtain a vector, and name this vector pTKmCH; (4) Design and synthesize the p11 promoter gene fragment: Select the p11 promoter sequence 5’-ATTTAGAATATATGTATGTAAAAATATAGTAGAATTTCATTTTGTTTTTTTCTATGCTATAA-3’, and artificially synthesize the p11 promoter gene fragment; (5) Use the CloneEZ "seamless" cloning technology to connect the p11 promoter gene fragment into the pTKmCH vector to obtain the shuttle vector pTK11mCH.

7. Use of the shuttle vector according to any one of claims 1-4 in the preparation of recombinant oncolytic vaccinia virus.

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