A vector system for recombination vaccinia virus packaging and use thereof

Abstract

The application discloses a carrier system for packaging recombinant poxvirus, which comprises a first plasmid, a second plasmid, a third plasmid, a fourth plasmid and a fifth plasmid respectively carrying five segments sequentially cut based on a full-length poxvirus genome, wherein the fifth plasmid carries a reverse terminal repeat sequence RITR fragment and a LITR fragment of the poxvirus genome, each plasmid has overlapping fragments at both ends for homologous recombination with adjacent plasmids, the 5' end of the first plasmid is adjacent to the 3' end of the fifth plasmid, and the 3' end of the fourth plasmid is adjacent to the 5' end of the fifth plasmid. The application further discloses a method for packaging recombinant poxvirus by using the recombinant poxvirus carrier system, and application of the recombinant poxvirus carrier system in preparation of a poxvirus carrier vaccine, and the recombinant poxvirus has the effects of large loading capacity and strong immune effect.

Description

TECHNICAL FIELD

[0001] The present application relates to a vector system for packaging recombinant poxvirus, in particular a virus vector with foreign antigens inserted in each backbone plasmid V1, V2, V3, V4 and its application, which can be used in the fields related to human poxvirus vaccine research, poxvirus epidemiology and poxvirus vector vaccine research, etc. BACKGROUND

[0002] The poxvirus genome is 130-375 kb in length, and has 10 kb long inverted terminal repeats (ITR) at both ends of the genome. The genome is huge and complex in structure, and encodes more than 200 proteins.

[0003] In 1982, vaccinia virus was first used to express foreign genes, and since then it has become a popular expression system and has gradually expanded to attenuated variants of vaccinia virus and other poxvirus strains such as avipox virus. Modified Vaccinia virus Ankara (MVA, ATCC accession number VR-1508) is a attenuated variant of the replication-competent vaccinia virus Ankara produced by more than 570 passages in chicken embryo fibroblasts. During continuous passage, about 12% of the genome was lost, so it cannot replicate in most mammalian cells. Today, MVA has been developed as a third-generation smallpox vaccine, and like canarypox virus, it is often used as a recombinant poxvirus vaccine vector research. Compared with other viral vector systems, MVA poxvirus vectors have the advantages of large capacity for inserting foreign genes, no safety risks in cytoplasmic replication, high expression level of foreign genes, and expression products that can induce the body to produce long-lasting humoral and cellular immune responses. In 2020, the Ebola vaccine Ad26.ZEBOV and MVA-BN-Filo two-dose immunization regimen developed by the Yangsen Pharmaceutical Company was approved for marketing by the European Commission. In 2022, the MVA-BN-RSV vaccine developed based on the MVA-BN platform containing 5 different respiratory syncytial virus antigens was granted Breakthrough Therapy Designation by the US FDA for active immunization of people over 60 years old. The vaccine obtained the approval of the IND application for phase III clinical trials in China in November 2022.

[0004] The poxvirus vector has a large genome sequence, which can accommodate up to 25 kb of foreign gene insertion, and most of the genetic engineering strategies for poxvirus are to transfect DNA with homologous fragments at both ends of the insertion site into poxvirus-infected cells to undergo homologous recombination, which is a natural process during poxvirus replication, but homologous recombination needs to go through a complex screening process, therefore, a fast poxvirus packaging system is needed, which can easily insert foreign gene fragments and quickly obtain recombinant poxvirus. The purpose of the present application is to provide five MVA poxvirus genome fragment plasmids assembled based on synthetic biology research methods, which are based on full-chemical artificial synthesis of poxvirus DNA fragments, and the recombinant poxvirus can be quickly obtained by transfecting cells using the plasmid, and can be used for rapid insertion of antigens and efficient editing of genes of recombinant poxvirus or related vector vaccines. SUMMARY

[0005] Based on the above purpose, the present application first provides a recombinant MVA poxvirus vector five-plasmid system (MVA-Fast), the recombinant poxvirus vector system comprises a first plasmid, a second plasmid, a third plasmid, a fourth plasmid and a fifth plasmid which respectively carry five segments sequentially divided based on the full-length genome of poxvirus, wherein the fifth plasmid carries the reverse terminal repeat sequence RITR fragment and the LITR fragment of the poxvirus genome, each plasmid has overlapping fragments at both ends for homologous recombination with the adjacent plasmid, wherein the 5' end of the first plasmid is adjacent to the 3' end of the fifth plasmid, and the 3' end of the fourth plasmid is adjacent to the 5' end of the fifth plasmid. In the present application, the first plasmid, the second plasmid, the third plasmid, the fourth plasmid and the fifth plasmid are respectively named as V1, V2, V3, V4 and V5.

[0006] In a preferred embodiment, the poxvirus is a Modified Vaccinia virus Ankara (MVA), and the ATCC accession number of the poxvirus is VR-1508.

[0007] In a more preferred embodiment, the first plasmid, the second plasmid, the third plasmid and the fourth plasmid respectively carry four segments sequentially divided based on the full-length genome of poxvirus (Genbank: U94848.1) with a length of 30 kb to 64 kb.

[0008] More preferably, the lengths of the four segments of the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid, respectively, are about 47 kb, 64 kb, 43 kb, and 34 kb, which are located at 9730 bp to 56760 bp, 52893 bp to 106124 bp, 102100 bp to 144689 bp, and 134789 bp to 168360 bp of the poxvirus genome, respectively, the LITR fragment and the RITR fragment are about 9.8 kb, the LITR is located at 1 bp to 9794 bp, and the RITR is located at 168295 bp to the end and is complementary to the LITR, V1 and V2 share a homologous region of about 3.8 kb (52893-56760 bp), V2 and V3 share a homologous region of 4 kb (102100-106124 bp), V3 and V4 share a homologous region of 9.9 kb (134789-144689 bp), and V5 and V1 and V4 share homologous regions of 65 bp (9730-9794 bp) and 66 bp (168295-168360 bp), respectively.

[0009] More preferably, the lengths of the four segments of the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid, respectively, are about 47 kb, 64 kb, 43 kb, and 34 kb, which are located at 9730 bp to 56760 bp, 52893 bp to 106124 bp, 102100 bp to 144689 bp, and 134789 bp to 168360 bp of the poxvirus genome, respectively, the LITR fragment and the RITR fragment are about 9.8 kb, the LITR is located at 1 bp to 9794 bp, and the RITR is located at 168295 bp to the end and is complementary to the LITR, V1 and V2 share a homologous region of about 3.8 kb (52893-56760 bp), V2 and V3 share a homologous region of 4 kb (102100-106124 bp), V3 and V4 share a homologous region of 9.9 kb (134789-144689 bp), and V5 and V1 and V4 share homologous regions of 65 bp (9730-9794 bp) and 66 bp (168295-168360 bp), respectively.

[0010] In another preferred embodiment, the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid each have a restriction enzyme cutting site for inserting an exogenous gene.

[0011] In a more preferred embodiment, the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid each have a restriction enzyme cutting site for inserting an exogenous gene. Eag I, Asis I, Pac I and Eag I.

[0012] In a preferred embodiment, the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid are each constructed based on the BACYAC plasmid, and the fifth plasmid is constructed based on the PET plasmid. In the present application, the first plasmid, the second plasmid, the third plasmid, the fourth plasmid, and the fifth plasmid are also named BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5), respectively.

[0013] In one embodiment of the present application, each of the fragments of the poxvirus genome is connected to the BACYAC backbone plasmid through Asis I and Asc I single enzyme cutting sites, and can be linearized by enzyme cutting. Each adjacent linearized fragment shares a homologous region, and when the fragments are transfected into cells infected with a helper poxvirus, the fragments can homologously recombine in the host cells in order, splicing into the complete genomic sequence of the MVA poxvirus. In certain embodiments, the helper poxvirus can be a fowlpox virus, a vaccinia virus, a Shope fibroma virus.

[0014] Secondly, the present application provides a method for constructing the recombinant poxvirus vector system described above, the method comprising the following steps:

[0015] (1) dividing the genomic segments covered by V1~V4 plasmids into 7~12 5kb subsegments, and dividing the genomic segment covered by V5 into two subsegments of LITR and RITR, and synthesizing, respectively, introducing a homologous recombination region at the subsegment adjacent to the plasmid (BACYAC plasmid or PET plasmid), and introducing an enzyme cutting site for linearization (BamHI, XhoI, NotI, XbaI, NdeI, EcoRI, HindIII, Kpnl, Nhel, Ncol, and Xmal) Asc I and Asis I) using a homologous recombination method, co-transfecting the V1~V4 plasmids and the BACYAC plasmid into yeast competent cells, obtaining a circular closed plasmid by homologous recombination in the host cells, and obtaining the V5 plasmid by Gibson in vitro ligation of the two fragments contained in the PET plasmid and transforming E. coli;

[0016] (2) linearizing the plasmids V1~V5;

[0017] (3) transfecting the linearized plasmids V1~V5 into BHK-21 cells infected with fowlpox virus FPV;

[0018] (4) maintaining culture for 3~5 days, freeze-thawing the cell culture solution, and continuously passing the cells to maintain the cytopathic effect, so as to obtain the stably passed recombinant MVA poxvirus.

[0019] Thirdly, the present application provides the use of the recombinant poxvirus vector system described above in the preparation of a poxvirus vaccine. In the use, an antigen gene expression frame is inserted into the restriction enzyme cutting site of the exogenous gene described above, for example, the Eag I of the first plasmid, the Asis I of the second plasmid, the Pac I of the third plasmid, or the Eag I of the fourth plasmid, so as to package the recombinant poxvirus carrying the antigen gene in the host cells with the help of the helper virus, and the poxvirus can be prepared into a vaccine to induce the body to produce a specific immune response against the antigen.

[0020] In a specific embodiment of the present invention, the restriction enzyme cutting site of the third plasmid is Pac The expression cassette of the HA protein of influenza virus H1N1 was inserted into I, and the MVA-HA recombinant vaccine was prepared by packaging the recombinant poxvirus using a recombinant poxvirus vector system.

[0021] Finally, the present invention provides a method for packaging a recombinant poxvirus using the above-mentioned recombinant poxvirus vector system, the method comprising the following steps:

[0022] (1) Inserting the exogenous gene into the non-essential region of viral replication in the recombinant poxvirus vector system plasmids V1 to V4;

[0023] (2) transfecting the recombinant poxvirus vector system obtained in step (1) into host cells, and simultaneously infecting the host cells with a helper virus;

[0024] (3) culturing the host cells obtained in step (2);

[0025] (4) Identify and obtain recombinant poxviruses packaged with exogenous genes.

[0026] In a preferred embodiment, the non-essential region for viral replication in step (1) is located in the MVA gene F14L and F15L between (Genbank: U94848.1, bp 37266), thymidine kinase TK Genome region (75560 bp~76093 bp in Genbank: U94848.1), MVA gene A26L and A27L Between (Genbank: U94848.1 in the 130950 bp), hemagglutinin HA Gene region (149416 bp~150363 bp in Genbank: U94848.1).

[0027] In a specific embodiment of the present invention, in F15 TK The "Loxp-p7.5-eGFP-Loxp" gene fragment (SEQ ID NO: 3) was inserted into the gene region and can be used to express eGFP.

[0028] In a more preferred embodiment, the helper virus in step (2) is Fowlpox virus (FPV). The present application provides five plasmids based on the full artificial chemical synthesis of MVA genome fragments, including BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5), which contain the entire genome of MVA. When packaging the recombinant MVA virus, the linearized five fragments are transfected into BHK-21 cells infected with fowlpox virus, and the virus is collected after 3 days of transfection. The recombinant MVA virus obtained using this method has consistent characteristics with the wild-type virus, including peak titer, growth curve, host range, and plaque size.

[0029] The MVA-Fast poxvirus vector system constructed in the present application contains five backbone plasmids, and the poxvirus replication non-essential region of V1-V4 plasmids contains a single enzyme digestion site for insertion of exogenous genes. The exogenous genes are directly inserted into the enzyme digestion site of the backbone plasmid through in vitro one-step recombination, and the full-length recombinant plasmid for packaging is obtained. This system greatly takes advantage of the large capacity of MVA poxvirus vector, provides a vector tool for rapid, efficient, multi-site gene editing, and multi-region genome modification of the viral genome, shortens the preparation period of recombinant poxvirus, and uses a safer and more distant relative fowlpox virus as a helper virus during virus packaging, avoiding the occurrence of non-specific recombination. It is fast, efficient, low-cost, and can be mass-produced, has significant application advantages, and is an ideal vector system for preparing recombinant poxvirus. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The schematic diagram of MVA-Fast system recombinant plasmid. The wild-type MVA genome (U94848.1) is divided into 37 segments of DNA synthesis, and is spliced into BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4) using TAR recombination, and PET-LITR-RITR (V5) using Gibson splicing. The vector region and the fragments are connected by AsisI and AscI enzyme digestion sites, and V1-V5 each splicing fragment shares 65bp, 3.8kb, 4.0kb, 9.9kb, and 66bp homologous regions, respectively.

[0031] Figure 2Figure 1 is a schematic diagram of the BACYAC-F01-F10 (V1) plasmid. V1 is obtained by ligating F01~F10 chemically synthesized and BACYAC plasmid through Asc I and Asis I, the Eag I single enzyme digestion site of F06 is located between MVA gene F14L and F15L (37266 bp), which can be used for the insertion of exogenous genes.

[0032] Figure 3 Figure 2 is a schematic diagram of the BACYAC-F10-F21 (V2) plasmid splicing. V2 is obtained by ligating F10~F21 chemically synthesized and BACYAC-Loxp-p7.5-eGFP-Loxp plasmid, wherein BACYAC and Loxp-p7.5-eGFP-Loxp are connected through Asis I, which can be used for the insertion of exogenous genes, F10 and F21 are connected through Asc I, which is used for the linearization of the plasmid, and the "Loxp-p7.5-eGFP-Loxp" gene fragment, which can be used for the expression of eGFP.

[0033] Figure 4 Figure 3 is a schematic diagram of the BACYAC-F21-F30 (V3) plasmid splicing. V3 is obtained by ligating F21~F30 chemically synthesized and BACYAC plasmid through Asc I and Asis I, the Pac I single enzyme digestion site of F28 is located between MVA gene A26L and A27L (130950 bp), which can be used for the insertion of exogenous genes.

[0034] Figure 5 Figure 4 is a schematic diagram of the BACYAC-F29-F35 (V4) plasmid splicing. V4 is obtained by ligating F29~F35 chemically synthesized and BACYAC plasmid through Asc I and Asis I, the Eag I single enzyme digestion site of F32 is located in the hemagglutinin HA gene region (149416 bp~150363bp), which can be used for the insertion of exogenous genes.

[0035] Figure 6 Figure 5 is a schematic diagram of the PET-LITR-RITR (V5) plasmid splicing. V5 is obtained by ligating LITR and RITR chemically synthesized and PET vector through Asis I and Asc I.

[0036] Figure 7Figure 1 is a photograph of an agarose gel electrophoresis of five plasmids. The BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), PET-LITR-RITR (V5) were linearized with AsiSI and AscI, and BACYAC-F10-F21 (V2) was linearized with AscI. The results of the enzyme digestion were detected by agarose gel electrophoresis.

[0037] Figure 8 Figure 2 is a flow chart of the process of packaging MVA-Fast system into MVA vaccinia virus. The linearized plasmids V1-V5 were transfected into BHK-21 cells infected with fowlpox virus FPV, and the recombinant MVA vaccinia virus was packaged by homologous recombination of the DNA fragments containing homologous regions. Asis I and Asc Figure 3 is a photograph of the microscope observation of the expression of eGFP fluorescent protein of the recombinant MVA vaccinia virus packaged by MVA-Fast system in BHK-21 cells, and the results of electron microscope detection of MVA-syn in BHK-21 cells and sucrose separation and purification.

[0038] Figure 9 Figure 4 is the results of PCR identification of the recombinant MVA vaccinia virus.

[0039] Figure 10 Figure 5 is the growth curves of MVA-WT and three MVA virus plaque screening strains MVA-syn1, MVA-syn4, MVA-syn-5 obtained by the method of the present application on BHK-21 and DF-1 cells. MVA-syn and MVA-WT showed similar growth performance on BHK-21 and DF-1 cells.

[0040] Figure 11 Figure 6 is the plaque property analysis of MVA-WT and MVA-syn1, MVA-syn4, MVA-syn5. The plaque sizes produced by MVA-syn and MVA-WT after infecting BHK-21 cells were similar, indicating that MVA-syn and MVA-WT had similar properties in the same permissive cells.

[0041] Figure 12 Figure 7 is a schematic diagram of the MVA-HA recombinant vaccine packaged by MVA-Fast system. The entire protein expression frame of the influenza virus H1N1 HA protein was inserted into the

[0042] Figure 13 Figure 8 is a schematic diagram of the MVA-HA recombinant vaccine packaged by MVA-Fast system. The entire protein expression frame of the influenza virus H1N1 HA protein was inserted into the Pac I site of the BACYAC-F21-F30 (V3) plasmid, and the recombinant V3-HA plasmid was obtained by Asis I and AscI linearization and other linearization of four plasmid co-transfection of FPV transfection of fowlpox virus FPV infected BHK-21 cells, by containing the homologous region of DNA fragments homologous recombination package out recombinant MVA-HA vaccine.

[0043] Figure 14 PCR identification of MVA-HA genome and protein expression identification results. The target protein gene sequence was successfully inserted into the MVA poxvirus genome, and HA protein expression was detected at 12 h and 24 h after MVA-HA infection of BHK-21 cells at MOI = 1.

[0044] Figure 15 Results of detection of binding antibodies after immunization of BALB / c mice with recombinant MVA-HA candidate vaccine. MVA-HA was immunized twice at 0 days and 21 days, with a muscle injection and a subcutaneous injection on the neck and back, 100 μL each time, and the high-dose group was set to 5 x 10 7 IFU and the low-dose group was set to 5 x 10 6 IFU. At 21 days after immunization (before the second immunization) and 28 days after immunization (7 days after the second immunization), the serum specific antibody level induced in the mice by the candidate vaccine was detected by ELISA. DETAILED DESCRIPTION

[0045] The advantages and features of the present application will become more apparent with the description of the specific embodiments. However, these embodiments are only exemplary and do not constitute any limitation on the protection scope defined by the claims of the present application.

[0046] Unless otherwise specified, the starting plasmids, enzymes, and related reagents used in the following examples can be purchased from commercially available companies, and the primers are synthesized by a synthesis company.

[0047] Example 1. Construction of recombinant MVA poxvirus vector five-plasmid system (MVA-Fast)

[0048] The poxvirus vector five-plasmid system (MVA-Fast) comprises the entire MVA genome, the MVA genome U94848.1 is split into 37 chemically synthesized DNA fragments, the position, length and shared homologous region of each DNA fragment are shown in Table 1, V1~V4 plasmids are connected to the BACYAC plasmid (purchased from Invitrogen Company) by yeast transformation-associated recombination (TAR recombination), TAR recombination is the most efficient large fragment DNA assembly method reported at present, which has great advantages in synthesizing super large genomes, by designing genome DNA with homologous arms and yeast artificial chromosome plasmid into a ring, the recombination plasmid can be self-replicated in yeast and E. coli, and can be separated from the yeast chromosome and then transfected into E. coli for replication, and the E. coli plasmid is extracted to obtain a large amount of recombination plasmid. LITR and RITR are spliced by Gibson method (kit purchased from NEB Company), the kit contains T5 exonuclease, DNA polymerase and DNA ligase, T5 exonuclease digests the synthesized DNA fragments or the fragments cut from the plasmid to produce 3'-single-stranded overhanging DNA, the exposed homologous sequence specifically anneals, and the exonuclease is gradually heat inactivated. Then, the DNA polymerase fills the gap and cuts off the single-stranded overhanging end, and finally the DNA ligase connects the long DNA molecule (construction schematic diagram is shown in Figure 1 ). This method is convenient, fast and efficient, and can realize scarless splicing.

[0049] Table 1. Position, length and shared homologous region information table of MVA genome split fragments

[0050] .

[0051] 1. Construction of BACYAC-F01-F10 (V1)

[0052] BACYAC-F01-F10 (V1) has a length of 56.393 kb, which comprises the genome fragment from 9730 bp to 56760 bp of MVA genome (U94848.1), and is obtained by co-transfecting 10 chemically synthesized (F01~F10) MVA genome fragments and BACYAC plasmid into VL6-48N yeast competent cells (purchased from Shanghai Ze Ye Biotechnology Co., Ltd.) for homologous recombination, the homologous region of F01 and BACYAC and AsisI The enzyme cutting site is provided by the primer pair of PYE-Link-F01, PYE-Link-F01-F (SEQ ID NO. 4) and PYE-Link-F01-R (SEQ ID NO. 5); F10 and the homologous region of BACYAC, and Asc I The enzyme cutting site is provided by the primer pair of F10-Link-PYE, F10-Link-PYE-F (SEQ ID NO. 6) and F10-Link-PYE-R (SEQ ID NO. 7).

[0053] The method of TAR recombination is as follows: 100 ng of purified linearized BACYAC vector, 200 ng of 10 PCR amplified and purified (F01~F10) DNA fragments, 500 ng of 2 pairs of primers, and 20 μL of Yeastmaker Carrier DNA are mixed with 200 μL of VL6-48N yeast competent cells (purchased from Shanghai Zeyebio Technology Co., Ltd.), mixed gently, 800 μL of PEG / Li Ac is added, mixed gently, incubated at 30°C for 45 min, mixed gently every 15 min, 160 μL of fresh DMSO is added, 20 min of 42°C water bath, mixed gently every 10 min, centrifuged at room temperature for 5 min at 700 g, resuspend the cells with 3 mL of YPD Plus medium (purchased from Beijing Coolabio Technology Co., Ltd.), incubate at 30°C for 90 min, centrifuge at room temperature for 5 min at 700 g, discard the supernatant, resuspend with 500 μL of 0.9% (w / v) NaCl solution, take 100 μL and spread on a SD-trp agar plate (purchased from Takara), incubate at 30°C for 3-5 days, extract the yeast colony PCR positive yeast DNA mixture, and transform DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electroporation. A large amount of recombinant plasmid is obtained after large-scale plasmid extraction, and the intersection position between the homologous arms will be introduced into Asis I and Asc I enzyme cutting site, and the single enzyme cutting site connecting the two ends of the genome when packaging the virus Asis I and Asc I linearization (see Figure 2 ).

[0054] 2. Construction of BACYAC-F10-F21 (V2)

[0055] BACYAC-F10-F21 (V2) is 63.724 kb in length, which contains the MVA genome (U94848.1) from 52893 bp to 106124 bp genome fragment, which is formed by 12 chemically synthesized (F10~F21) MVA genome fragments and BACYAC plasmid co-transfected VL6-48N competent cells homologous recombination, the homologous region of F21 and F10 and Asc I The restriction site is provided by the primer pair of F21-LINK-F10, F21-LINK-F10-F (SEQ ID NO. 8) and F21-LINK-F10-R (SEQ ID NO. 9).

[0056] The homologous region of F14 and BACYAC is provided by the primer pair of F14-LINK-PYE, F14-LINK-PYE-F (SEQ ID NO. 10) and F14-LINK-PYE-R (SEQ ID NO. 11).

[0057] The homologous region of F15 and BACYAC is provided by the primer pair of F15-LINK-PYE, F15-LINK-PYE-F (SEQ ID NO. 12) and F15-LINK-PYE-R (SEQ ID NO. 13), wherein F15 contains TK The 275 bp insertion of "Loxp-p7.5-eGFP-Loxp" sequence in the gene region, the sequence is SEQ ID NO: 3, the P7.5 early and late promoter of poxvirus is added in front of eGFP, and the two loxP sites are located at both ends of the eGFP expression frame and have the same direction, which can knock out the sequence between LoxP by Cre recombinase, one end of BACYAC is connected with F14, and the other end is connected with F15 through a single enzyme cutting site Asis I connected with "Loxp-p7.5-eGFP-Loxp" sequence, the single enzyme cutting site introduced by the primer pair providing the homologous region Asis I can be used for the insertion of the exogenous gene expression frame, F10 and F21 are connected head to tail, and when packaging the virus, the Asc I cuts the F10 and F21 connection site to linearize the plasmid, which exposes the homologous fragment (see Figure 3 ).

[0058] 3. Construction of BACYAC-F21-F30 (V3)

[0059] BACYAC-F21-F30 (V3) is 51.952 kb in length, which contains the MVA genome (U94848.1) from 102100 bp to 144689 bp genome fragment, which is spliced by 10 chemically synthesized (F21~F30) MVA genome fragments and BACYAC plasmid through TAR recombination.

[0060] The homologous region of F21 and BACYAC is provided by the primer pair of F21-Link-PYE, which is F21-Link-PYE-F (SEQ ID NO. 14) and F21-Link-PYE-R (SEQ ID NO. 15).

[0061] The homologous region of F30 and BACYAC is provided by the primer pair of F30-Link-PYE, which is F30-Link-PYE-F (SEQ ID NO. 16) and F30-Link-PYE-R (SEQ ID NO. 17). One end of BACYAC is connected to F21, and the other end is connected to F30. Asis Asc I and F30, and the single enzyme digestion site for connecting the two ends of the genome of the BACYAC plasmid when packaging the virus Asis I and F21, and the other end is connected to F30. Asc I linearization (see Figure 4 ).

[0062] 4. Construction of BACYAC-F29-F35 (V4)

[0063] BACYAC-F29-F35 (V4) is 42.934 kb in length, which contains the MVA genome (U94848.1) from 102100 bp to 144689 bp genome fragment, which is spliced by 7 chemically synthesized (F29~F35) MVA genome fragments and BACYAC plasmid through TAR recombination, and the homologous region of F29 and BACYAC is provided by the primer pair of F29-Link-PYE, which is F29-Link-PYE-F (SEQ ID NO. 18) and F29-Link-PYE-R (SEQ ID NO. 19).

[0064] The homologous region of F35 and BACYAC is provided by the primer pair of PYE-Link-F35, which is F35-Link-PYE-F (SEQ ID NO. 20) and F35-Link-PYE-R (SEQ ID NO. 21). One end of BACYAC is connected to F29, and the other end is connected to F35. Asis Asc I and F35, and the single enzyme digestion site for connecting the two ends of the genome of the BACYAC plasmid when packaging the virus​​Asis I and Asc I linearization (see Figure 5 ).

[0065] 5. Construction of PET-LITR-RITR (V5)

[0066] PET-LITR-RITR (V5) was assembled by Gibson assembly, V5 was connected by chemically synthesized F36 (LITR) and F37 (RITR) and PET plasmid (preserved by the laboratory) through Asis I and Asc I The 3' end of the chemically synthesized RITR sequence was added with the sequence of 21 bp at the 5' end of the LITR sequence to provide the homologous region necessary for homologous recombination when Gibson assembly, linearized fragments of LITR and RITR were prepared, and the reserved single enzyme cutting site at the end of the chemically synthesized LITR and RITR genes Xma I and Asis I Linearize the fragments, amplify the PET vector fragment, the amplification primer for increasing the homologous arm at the 3' end of the LITR is PET-LITR-F (SEQ ID NO. 22), and the amplification primer for increasing the homologous arm at the 5' end of the RITR is PET-RITR-R (SEQ ID NO. 23), the length of the amplification product is 2540 bp, after the above enzyme cutting and PCR amplification, the fragments are purified, and then spliced using the Gibson recombination system (NEB company), the specific method is as follows: 50 ng of the purified vector fragment, about 100 ng of the purified multiple fragments for integration (the molar ratio with the vector is controlled to be 2:1), 10 μL of Gibson Mix assembly solution, 20 μL of sterile water, 50°C incubation for 15 min, ice bath for about 3 min. 3 μL of the recombination system is transformed into 50 μL of TOP10 chemically competent cells, incubated for 40-60 min, plated, and incubated overnight, and the positive colonies are identified to obtain the recombinant plasmid. After splicing, the intersection position between the homologous arms will introduce Asis I and Asc I enzyme cutting site for plasmid linearization (see Figure 6 ).

[0067] The present application provides five five-plasmids based on five artificially chemically synthesized MVA genome fragments, including BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5), which contain the entire genome of MVA (see Figures 2-6 ).

[0068] Figure 7 The agarose gel electrophoresis figure of five plasmids is given. The linearized BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), PET-LITR-RITR (V5) are used Asis I and Asc I linearized BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), PET-LITR-RITR (V5) are used Asc I linearized BACYAC-F10-F21 (V2) is used, and 1% agarose is used to detect the enzyme cutting condition, and the length of the obtained plasmid is consistent with the expected.

[0069] Example 2. Packaging of recombinant MVA-syn virus

[0070] Figure 8 The packaging schematic of the application is given, and the linearized plasmids V1-V5 are transfected into BHK-21 cells infected with fowlpox virus FPV, and the recombinant MVA virus is packaged by homologous recombination of the DNA fragments containing the homologous regions. Among them, BHK-21 cells (preserved by the laboratory) are selected as the packaging cell line, FPV (fowlpox virus) is used to infect BHK-21 cells (preserved by the laboratory) at MOI=0.1-1 for 1h, and then the linearized fragments are transfected into BHK-21 cells using Turbofect transfection reagent, and maintained for 3-5 days, and the freeze-thawed cell culture fluid is continuously passaged to the cells until the cytopathic effect is sustained, and the stably passaged recombinant MVA-syn virus is obtained. We completed the packaging of MVA-Fast vector system recombinant MVA-syn virus, and found that eGFP can be continuously expressed by using the MVA-syn recombinant virus to infect BHK-21 cells (Fig. 1 Figure 9 A). The structure of the recombinant virus particles is detected by electron microscope, and the virus particle size is 220-450 nm, which is a typical oval or brick structure, consistent with the expected result (Fig. 1 Figure 9 B), which shows that the system can obtain recombinant MVA-syn virus with normal structure and stable expression of exogenous genes.

[0071] Example 3. PCR identification results of recombinant MVA virus

[0072] To identify the recombinant MVA-syn virus genome, five recombinant MVA-syn virus monoclonal (MVA-syn1~5) were isolated using the plaque purification method, the MVA-syn1~5, MVA-WT and FPV fowlpox virus genome were extracted using the virus genome DNA / RNA rapid extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the fragment-specific amplification primers of BACYAC, F01, F06, F16, F26 and F35 and FPV were designed, respectively, the primers for detecting the BACYAC plasmid (SEQ ID NO. 24-25), the primers for detecting F01 (SEQ ID NO. 26-27), the primers for detecting F16 (SEQ ID NO. 28-29), the primers for detecting F26 (SEQ ID NO. 30-31), the primers for detecting F35 (SEQ ID NO. 32-33), and the primers for detecting MVA ITR (SEQ ID NO. 34-35). The PCR products were analyzed by agarose gel electrophoresis, and the PCR results (see Figure 10 ) revealed that the recombinant MVA-syn virus contained the BACYAC plasmid and contained the PCR fragments consistent with the length of MVA-WT, and did not contain the PCR fragments of FPV fowlpox virus, indicating that the five-plasmid fragment of the MVA-Fast system was completed, and the recombinant MVA-syn virus could be separated from the FPV helper virus by passage or plaque purification.

[0073] Example 4. Growth curve of recombinant MVA virus

[0074] MVA virus cannot replicate on most mammalian cells, but can replicate on BHK-21 and DF-1 cells, and three recombinant MVA-syn viruses MVA-syn1, MVA-syn4 and MVA-syn5 randomly selected by PCR identification were compared with MVA-WT, and were infected with MOI = 0.01 to infect BHK-21 and DF-1 cells (purchased from Beina), and the virus growth curves at 0, 12, 24 and 72 h were drawn, and the growth characteristics of MVA-syn and MVA-WT were compared, and the growth curve results showed that MVA-syn and MVA-WT showed similar growth kinetics on BHK-21 and DF-1 cells (see Figure 11 ).

[0075] Example 5. Analysis of plaque characteristics of recombinant MVA virus

[0076] MVA-WT and MVA-syn were used to infect monolayer BHK-21 cells in 6-well plates at MOI = 0.001, the culture medium was discarded after infection, and the cells were washed with PBS for 3 times, then covered with low temperature agar, and cultured at 37°C for 72 h. The culture medium was discarded, and enough 4% PFA tissue cell fixation solution (purchased from Beijing Regen Biotechnology Co., Ltd.) was slowly added along the wall of the well, and fixed at room temperature for 12 h. Crystal violet staining was used, and the well plate was scanned using Celigo full field cell analyzer (Nexcelom). The scanned digital image was used to measure the plaque area, and the results showed that the plaque size produced by MVA-syn and MVA-WT after infecting BHK-21 cells was similar, and the propagation ability of the two in the same permissive cells was similar (see Figure 12 ).

[0077] Example 6. Construction and characterization of recombinant MVA vaccinia virus vector MVA-HA vaccine

[0078] As shown in Figure 13 , the Gibson method was used to insert the HA protein expression frame into the BACYAC-F21-F30 (V3) plasmid Pac I site, and the specific method was as follows: Pac The V3 plasmid was digested with I, and the digestion product was purified by ethanol precipitation (Takara). The HA protein expression frame (the sequence is shown in SEQ ID NO: 36) was amplified by PCR, and the primers were HA-F (the sequence is shown in SEQ ID NO: 37) and HA-R (the sequence is shown in SEQ ID NO: 38). The Gibson in vitro one-step recombination method was used to splice the two fragments (for the specific method, see Example 1), and the V3-HA recombinant plasmid successfully inserted with the HA protein was obtained. The recombinant MVA-HA influenza vaccine was packaged by the MVA-Fast system (for the specific method, see Example 2). The viral genomes of MVA-HA, MVA-WT and MVA-syn4 were extracted (for the specific method, see Example 3), and the identification primers HA-Part-F (the sequence is shown in SEQ ID NO: 39) and HA-Part-R (the sequence is shown in SEQ ID NO: 40) of the HA protein gene were designed. The agarose gel electrophoresis result of the PCR amplification product showed that MVA-HA successfully inserted the HA protein gene sequence (see Figure 14 ). MVA-HA was used to infect BHK-21 cells at MOI = 1, and cell samples were collected at 12 h and 24 h, respectively. The Western blot result showed that the BHK-21 infected with MVA-HA could normally express HA protein within 12 h and 24 h.

[0079] Example 7. Immunogenicity of MVA-HA vaccine

[0080] To determine the immunogenicity of HA antigens expressed using MVA poxvirus, we used 5 × 10 7 IFU / piece and 5×10 6 Balb / c mice were immunized with a dose of 100 μL / well of HA protein (IFU / mouse). The mice received two immunizations on days 0 and 21, respectively, by intramuscular injection and subcutaneous immunization at the back of the neck, with a volume of 100 μL / mouse. Sera were collected on days 21 and 28, and the levels of HA protein-binding antibodies were detected by ELISA. The specific method is as follows: HA protein (purchased from Sino Biological Biotechnology Co., Ltd.) was diluted to 1 μg / mL, and 100 μL / well was added to a microplate. The plate was coated overnight at 4°C. After washing the plate three times with washing solution, 100 μL / well of blocking solution was added, and the plate was blocked at 37°C for 1 hour. The liquid in the plate was discarded and washed three times with a plate washer. The serum sample was diluted at a specific initial dilution (1:100, 1.5 μL + 148.5 μL), followed by a 4-fold serial dilution. Seven dilutions were set for each sample, and 12 control wells without serum were set per plate. After serum dilution was completed, the plate was incubated at 37°C for 1 h. After washing, a 1:20,000 dilution of HRP-conjugated anti-mouse IgG secondary antibody (Santa Cruz, sc-2005) was added and incubated for another 1 h. The plate was developed using TMB one-component colorimetric solution (solarbio, PR1200) for 6 min. 50 μL of 2 M sulfuric acid was added to each well to terminate the reaction. The absorbance at 450 nm was measured on a microplate reader (MolecμLar Devices, Spectra Max). Figure 15 ), after two immunizations, both intramuscular and subcutaneous immunizations can stimulate mice to produce high-titer antibody responses, and the antibody response level has a significant dose-dependent relationship, indicating that using this vector to deliver antigens can stimulate excellent immune effects.

Claims

1. A vector system for packaging a recombinant poxvirus, characterized in that: The vector system for packaging the recombinant poxvirus comprises a first plasmid, a second plasmid, a third plasmid, a fourth plasmid, and a fifth plasmid, each carrying five segments sequentially divided based on the full-length poxvirus genome, wherein the fifth plasmid carries the inverted terminal repeat sequence RITR segment and LITR segment of the poxvirus genome, and each plasmid has homologous regions at its two ends for homologous recombination with adjacent plasmids, wherein the 5' end of the first plasmid is adjacent to the 3' end of the fifth plasmid, and the 3' end of the fourth plasmid is adjacent to the 5' end of the fifth plasmid, and the lengths of the four segments sequentially divided based on the full-length poxvirus genome carried by the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid are 47 kb, 64 kb, 43 kb, and 34 kb, respectively, and are located between 9730 bp and 56760 bp, 52893 bp and 106124 bp, respectively, of the poxvirus genome. bp, 102100 bp to 144689 bp, and 134789 bp to 168360 bp. The LITR fragment and RITR fragment are both 9.8 kb, LITR is located from 1 bp to 9794 bp, and RITR is located from 168295 bp to the end, and is reverse complementary to LITR. The first plasmid and the second plasmid share a 3.8 kb homology region, the second plasmid and the third plasmid share a 4 kb homology region, and the third plasmid and the fourth plasmid share a 9.9 kb homology region. The sequence of the homology region between the first plasmid and the fifth plasmid is shown in SEQ ID NO.1, and the sequence of the homology region fragment length between the fourth plasmid and the fifth plasmid is shown in SEQ ID NO.

2. Restriction enzyme sites for inserting exogenous genes are respectively provided on the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid, respectively: Eag I. Asis I. Pac I and Eag I. The poxvirus is the poxvirus Ankara strain with ATCC deposit number VR-1508.

2. The method for constructing a vector system for packaging a recombinant poxvirus according to claim 1, characterized in that: The method comprises the following steps: (1) The genomic segments covered by the first plasmid to the fourth plasmid were divided into 7 to 12 5 kb sub-segments, and the genomic segment covered by the fifth plasmid was divided into two sub-segments, LITR and RITR, and synthesized separately. A homologous recombination region was introduced into the sub-segment adjacent to the plasmid, and an enzyme cutting site for linearization was introduced. Using the homologous recombination method, the first plasmid to the fourth plasmid and the BACYAC plasmid were co-transfected into yeast competent cells, and a circular closed plasmid was obtained by homologous recombination in the host cells. The fifth plasmid was obtained by transforming the two fragments contained therein with the PET plasmid into Escherichia coli through Gibson in vitro ligation; (2) Linearize plasmids 1 to 5 by enzyme digestion; (3) Linearized plasmid 1-plasmid 5 were transfected into BHK-21 cells infected with fowlpox virus FPV (4) Maintain the culture for 3 to 5 days, freeze and thaw the cell culture medium, and continuously passage the cells until they become persistently diseased. This will yield a stably passaged recombinant MVA poxvirus.

3. Use of the vector system for packaging recombinant poxvirus according to claim 1 in the preparation of poxvirus vector vaccines.

4. The use according to claim 3, characterized in that The poxvirus vector vaccine is a poxvirus vector vaccine of the HA protein of influenza virus H1N1.

5. A method for packaging a recombinant poxvirus using the vector system for packaging a recombinant poxvirus according to claim 1, characterized in that: The method comprises the following steps: (1) Inserting the exogenous gene into the non-essential region of viral replication in the recombinant poxvirus vector system, wherein the non-essential region of viral replication is located in the MVA gene F14L and F15L Thymidine kinase TK Gene region, MVA gene A26L and A27L between, or hemagglutinin HA Gene coding region; (2) transfecting the recombinant poxvirus vector system obtained in step (1) into host cells, and simultaneously infecting the host cells with a helper virus, wherein the helper virus is fowlpox virus; (3) culturing the host cells obtained in step (2); (4) Identify and obtain recombinant poxviruses that have been packaged and inserted with foreign genes.

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