Vector system for packaging recombinant poxvirus and application thereof

Abstract

The invention discloses a vector system for packaging a recombinant poxvirus, the vector system comprises a first plasmid, a second plasmid, a third plasmid, a fourth plasmid and a fifth plasmid which respectively carry five sections which are sequentially segmented on the basis of a full-length genome of the poxvirus, the fifth plasmid carries a reverse terminal repeat (RITR) fragment and an LITR fragment of the genome of the poxvirus, and the RITR fragment and the LITR fragment are sequentially segmented on the basis of the full-length genome of the poxvirus. The two ends of each plasmid are provided with overlapping fragments which are subjected to 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 invention also discloses a method for packaging a recombinant poxvirus by using the recombinant poxvirus vector system and application of the recombinant poxvirus vector system in preparation of a poxvirus vector vaccine, and the recombinant poxvirus has the effects of large loading capacity and strong immune excitation effect.

Description

Technical Field

[0001] The present invention relates to a vector system for packaging recombinant poxvirus, in particular to a viral vector in which exogenous antigens are inserted into each backbone plasmid V1, V2, V3, and V4 and its application, which can be used in related fields such as human poxvirus vaccine research, poxvirus epidemiology, and poxvirus vector vaccine research. Background Art

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

[0003] In 1982, vaccinia virus was first used to express foreign genes. 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 fowlpox virus. Modified Vaccinia virus Ankara (MVA, ATCC deposit number VR-1508) is an attenuated variant of the replication-competent vaccinia virus Ankara produced after more than 570 passages in chicken embryo fibroblasts. During the continuous passage, about 12% of the genome is lost, so it cannot replicate in most mammalian cells. Today, MVA has been developed into a third-generation smallpox vaccine, and like canarypox virus, it is often used as a recombinant poxvirus vaccine vector. 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 the expression products can induce the body to produce a long-lasting humoral and cellular immune response. In 2020, Janssen Pharmaceuticals' Ebola vaccine Ad26.ZEBOV and MVA-BN-Filo two-dose immunization regimen were approved for marketing by the European Commission. In 2022, the MVA-BN-RSV vaccine containing five different respiratory syncytial virus antigens developed based on the MVA-BN platform was granted breakthrough drug status by the U.S. FDA for active immunization of people over 60 years old. The vaccine was approved for Phase III clinical IND application in China in November 2022.

[0004] Poxvirus vectors have a large genome sequence and can accommodate up to 25 kb of foreign gene insertion. Most genetic engineering strategies for poxviruses are to transfect homologous fragments of DNA at both ends of the insertion site into poxvirus-infected cells for homologous recombination, which is a natural process during poxvirus replication, but homologous recombination requires a complex screening process. Therefore, a fast poxvirus packaging system is needed that can facilitate the insertion of foreign gene fragments and quickly obtain recombinant poxviruses. The purpose of the present invention is to provide a plasmid of 5 MVA poxvirus genome fragments assembled based on fully chemically synthesized poxvirus DNA fragments based on synthetic biology research methods. The plasmid can be used to transfect cells to quickly obtain recombinant poxviruses, and can be used for rapid insertion of antigens and efficient gene editing of recombinant poxvirus or related vector vaccines. Summary of the invention

[0005] Based on the above purpose, the present invention first provides a recombinant MVA poxvirus vector five-plasmid system (MVA-Fast), the recombinant poxvirus vector system includes 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 fragment and LITR fragment of the poxvirus genome, and each plasmid has overlapping fragments 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. In the present invention, the first plasmid, the second plasmid, the third plasmid, the fourth plasmid, and the fifth plasmid are named V1, V2, V3, V4, and V5, respectively.

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

[0007] In a more preferred embodiment, the lengths of the four segments carried by the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid, which are sequentially divided based on the full-length genome of the poxvirus (Genbank: U94848.1), are between 30 kb and 64 kb.

[0008] Particularly preferably, the lengths of the four segments carried by the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid, which are sequentially divided based on the full-length genome of the poxvirus, are respectively about 47 kb, 64 kb, 43 kb, and 34 kb, which are respectively located between 9730 bp and 56760 bp, 52893 bp and 106124 bp, 102100 bp and 144689 bp, and 134789 bp and 168360 bp of the poxvirus genome, the LITR fragment and the RITR fragment are about 9.8 kb, LITR is located between 1 bp and 9794 bp, RITR is located between 168295 bp and the end, and is reverse complementary to LITR, V1 and V2 share a homology region of about 3.8 kb (52893-56760 bp), and V2 and V3 share a homology region of 4 kb (102100-106124 bp), V3 and V4 share a 9.9 kb homology region (134789-144689 bp), and V5 shares 65 bp (9730-9794 bp) and 66 bp (168295-168360 bp) homology regions with V1 and V4, respectively.

[0009] More preferably, the homologous region fragment adjacent to the first plasmid and the fifth plasmid is 65 bp in length (sequence shown in SEQ ID NO.1), and the homologous region fragment adjacent to the fourth plasmid and the fifth plasmid is 66 bp in length (sequence shown in SEQ ID NO.2).

[0010] In another preferred embodiment, the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid each carry or introduce restriction enzyme sites for inserting foreign genes.

[0011] In a more preferred embodiment, the restriction enzyme sites for inserting foreign genes carried or introduced on the first plasmid, the second plasmid, the third plasmid and the fourth plasmid are respectively: 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 all constructed based on the BACYAC plasmid, and the fifth plasmid is constructed based on the PET plasmid. In the present invention, 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 invention, each poxvirus genome segment is linked to the BACYAC backbone plasmid by Asis I and Asc I single restriction site connected, can be linearized by restriction digestion, each adjacent linearized fragment shares a homologous region, when the fragments are transfected into cells infected with helper poxvirus, the fragments can be homologously recombined in sequence in the host cell to splice into the complete genome sequence of MVA poxvirus. In certain embodiments, the helper poxvirus can be fowlpox virus, cowpox virus, rabbit fibroma virus.

[0014] Secondly, the present invention provides a method for constructing the above-mentioned recombinant poxvirus vector system, the method comprising the following steps: (1) The genomic segments covered by the V1-V4 plasmids were divided into 7-12 5 kb sub-segments, and the genomic segment covered by the V5 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 (BACYAC plasmid or PET plasmid), and a restriction site for linearization was introduced ( Asc I and Asis I) Using the homologous recombination method, the V1~V4 plasmids and the BACYAC plasmid are co-transfected into yeast competent cells, and a circular closed plasmid is obtained by homologous recombination in the host cells. The V5 plasmid is transformed into Escherichia coli by Gibson in vitro ligation of the two fragments contained therein and the PET plasmid; (2) Linearized plasmids V1~V5; (3) The linearized plasmids V1 to V5 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 subculture until the cells continue to become disease-prone. This will allow the recombinant MVA poxvirus to be stably subcultured.

[0015] Third, the present invention provides the use of the above-mentioned recombinant poxvirus vector system in the preparation of poxvirus vaccines. In the above-mentioned application, the antigen gene expression cassette is inserted into the restriction enzyme cutting site of the above-mentioned foreign gene as a foreign gene, for example, Eag I. Second plasmid Asis I. Plasmid 3 Pac I, or the fourth plasmid Eag I. A recombinant poxvirus carrying an antigen gene can be packaged in a host cell with the help of a helper virus, and the poxvirus can be prepared as a vaccine to achieve the purpose of inducing the body to produce a specific immune response to the antigen.

[0016] In a specific embodiment of the present invention, the restriction enzyme cleavage site of the third plasmid is PacThe expression frame 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.

[0017] 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: (1) Inserting foreign genes into the non-essential region of viral replication in the recombinant poxvirus vector system plasmids V1 to V4; (2) transfecting the recombinant poxvirus vector system obtained in step (1) into host cells, and simultaneously infecting the host cells with a helper virus; (3) culturing the host cells obtained in step (2); (4) Identify and obtain recombinant poxviruses that have been packaged with foreign genes.

[0018] 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, 37266 bp), thymidine kinase TK Genome region (75560 bp~76093 bp in Genbank: U94848.1), MVA gene A26L and A27L Between (Genbank: U94848.1, 130950 bp), hemagglutinin HA Gene region (149416 bp~150363 bp in Genbank: U94848.1).

[0019] In a specific embodiment of the present invention, in F15 TK The gene region is inserted with the "Loxp-p7.5-eGFP-Loxp" gene fragment (SEQ ID NO: 3), which can be used to express eGFP.

[0020] In a more preferred embodiment, the helper virus in step (2) is fowlpox virus (FPV for short). The present invention provides five plasmids composed of five fully artificial 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. 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 3 days after transfection. The progeny virus can be obtained by continuous passage on BHK-21 cells. The recombinant MVA poxvirus obtained by this method has consistent characteristics with the wild-type virus, including peak titer, growth curve, host range and plaque size.

[0021] The MVA-Fast poxvirus vector system constructed by the present invention comprises 5 backbone plasmids, and the poxvirus replication non-essential region of the V1-V4 plasmids comprises a single restriction site for inserting exogenous genes, and the exogenous genes are directly embedded in the restriction site of the backbone plasmid through in vitro one-step recombination to obtain a full-length recombinant plasmid for packaging. The system greatly exerts the advantage of the large capacity of the MVA poxvirus vector, provides a vector tool for rapid, efficient, multi-site gene editing and multi-region genome transformation of the viral genome, shortens the preparation cycle of the recombinant poxvirus, and uses a relatively safe and distantly related fowlpox virus as an auxiliary virus in the process of packaging the virus, thereby avoiding the occurrence of non-specific recombination, and has fast speed, high efficiency, low cost, and can be prepared in batches, has significant application advantages, and is an ideal vector system for preparing recombinant poxviruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a schematic diagram of the recombinant plasmid of the MVA-Fast system. The wild-type MVA genome (U94848.1) was divided into 37 segments for DNA synthesis, which were recombined and spliced ​​into BACYAC-F01-F10 (V1), BACYAC-F10-F21 (V2), BACYAC-F21-F30 (V3), and BACYAC-F29-F35 (V4) using TAR, and PET-LITR-RITR (V5) was spliced ​​using Gibson. The vector region and the fragments were connected by AsisI and AscI restriction sites, and the spliced ​​fragments of V1~V5 shared 65bp, 3.8kb, 4.0kb, 9.9kb, and 66bp homology regions, respectively.

[0023] Figure 2Schematic diagram of BACYAC-F01-F10 (V1) plasmid. V1 is a hybrid of chemically synthesized F01~F10 and BACYAC plasmids. Asc I and Asis I connection, F06 Eag I single restriction site is located in the MVA gene F14L and F15L (37266 bp), which can be used for the insertion of foreign genes.

[0024] Figure 3 Figure 1 is a schematic diagram of BACYAC-F10-F21 (V2) plasmid splicing. V2 was obtained by splicing chemically synthesized F10~F21 with BACYAC-Loxp-p7.5-eGFP-Loxp plasmid, where BACYAC and Loxp-p7.5-eGFP-Loxp were added by homologous primers. Asis I connected, can be used for the insertion of foreign genes, F10 and F21 through Asc I was connected to linearize the plasmid, and the "Loxp-p7.5-eGFP-Loxp" gene fragment can be used to express eGFP.

[0025] Figure 4 Schematic diagram of BACYAC-F21-F30 (V3) plasmid splicing. V3 is composed of chemically synthesized F21~F30 and BACYAC plasmids. Asc I and Asis I connection, F28 Pac I single restriction site is located in the MVA gene A26L and A27L (130950bp), which can be used for the insertion of exogenous genes.

[0026] Figure 5 Schematic diagram of BACYAC-F29-F35 (V4) plasmid splicing. V4 is composed of chemically synthesized F29~F35 and BACYAC plasmids. Asc I and Asis I connection, F32 Eag I single restriction site is located in hemagglutinin HA The gene region (149416 bp~150363bp) can be used for the insertion of exogenous genes.

[0027] Figure 6 Schematic diagram of PET-LITR-RITR (V5) plasmid splicing. V5 is composed of chemically synthesized LITR and RITR and PET vector. Asis I and Asc IConnect.

[0028] Figure 7The figure shows the agarose gel electrophoresis of five plasmids. BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), and PET-LITR-RITR (V5) were linearized using AsciI and AscI, and BACYAC-F10-F21 (V2) was linearized using AscI, and the enzyme digestion results were detected by agarose gel electrophoresis.

[0029] Figure 8 Diagram of the process of packaging into MVA poxvirus for the MVA-Fast system. Asis I and Asc The linearized plasmids V1~V5 were transfected into BHK-21 cells infected with fowlpox virus FPV, and the recombinant MVA poxvirus was packaged through homologous recombination of DNA fragments containing homologous regions.

[0030] Fig. 9 Microscope photographs showing the expression of eGFP fluorescent protein observed during continuous passage of the recombinant MVA poxvirus packaged by the MVA-Fast system on BHK-21 cells, and electron microscopy results of the separation and purification of MVA-syn in BHK-21 cells and sucrose.

[0031] Fig.10 This is the result of PCR identification of recombinant MVA poxvirus.

[0032] Fig.11 The figures are the growth curves of MVA-WT and three MVA virus plaque screening strains MVA-syn1, MVA-syn4 and MVA-syn-5 obtained after packaging by the method of the present invention on BHK-21 and DF-1 cells. MVA-syn and MVA-WT show similar growth performance in BHK-21 and DF-1 cells.

[0033] Fig.12 Analysis of plaque characteristics of MVA-WT and MVA-syn 1, MVA-syn 4, and MVA-syn 5. The plaque sizes produced by MVA-syn and MVA-WT after infection of BHK-21 cells were similar, indicating that MVA-syn and MVA-WT have similar infection characteristics in the same permissive cells.

[0034] Fig.13 This is a schematic diagram of the MVA-HA recombinant vaccine packaged using the MVA-Fast system. Using poxvirus mH5 as the promoter and influenza virus H1N1 HA protein as the antigen, the entire protein expression cassette was inserted into the BACYAC-F21-F30 (V3) plasmid. Pac I site, and obtain the recombinant V3-HA plasmid by Asis I and AscI linearized plasmids were co-transfected with the other four linearized plasmids into FPV-transfected fowlpox virus (FPV)-infected BHK-21 cells, and the recombinant MVA-HA vaccine was packaged through homologous recombination of DNA fragments containing homologous regions.

[0035] Fig.14 The results of PCR identification of the MVA-HA genome and protein expression identification are shown in Figure 2. The target protein gene sequence was successfully inserted into the MVA poxvirus genome, and the expression of HA protein was detected at 12 h and 24 h after MVA-HA infected BHK-21 cells at an MOI of 1.

[0036] Fig.15 The results of antibody detection after immunization of BALB / c mice with recombinant MVA-HA candidate vaccine. MVA-HA was immunized twice on day 0 and day 21, with intramuscular injection and subcutaneous injection on the back of the neck, 100 μL each time. The high-dose group was set at 5×10 7 IFU and low dose group 5×10 6 The levels of serum-specific antibodies elicited by the candidate vaccine in mice were detected by ELISA 21 days (before the second dose) and 28 days (7 days after the second dose) after immunization. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0038] Unless otherwise specified, the starting plasmids, enzymes, and related reagents used in the following examples can all be purchased from commercial companies, and the primers used are synthesized by a synthesis company.

[0039] Example 1. Construction of the recombinant MVA poxvirus vector five-plasmid system (MVA-Fast) The poxvirus vector five-plasmid system (MVA-Fast) contains the entire MVA genome. The MVA genome U94848.1 is split into 37 chemically synthesized DNA fragments. The positions, lengths and shared homologous regions of each DNA fragment are shown in Table 1. The V1~V4 plasmids are connected to multiple adjacent fragments with the BACYAC plasmid (purchased from Invitrogen) through yeast transformation-associated recombination (TAR recombination). TAR recombination is the most efficient large-fragment DNA assembly method reported so far, and it has great advantages in synthesizing very large genomes. By designing genomic DNA with homology arms to form a circle with yeast artificial chromosome plasmids, the recombinant plasmid can replicate autonomously in yeast and Escherichia coli. It does not need to be integrated into the yeast chromosome during recombination, and can be separated from the yeast chromosome and transfected into Escherichia coli for replication. The Escherichia coli plasmid is extracted to obtain a large number of recombinant plasmids. LITR and RITR were spliced ​​by Gibson method (the kit was purchased from NEB). The kit contains T5 exonuclease, DNA polymerase and DNA ligase. T5 exonuclease digests the synthesized DNA fragments or fragments cut from the plasmid to produce 3'-single-stranded protruding DNA. The exposed homologous sequences are annealed specifically, and the exonuclease is gradually heat-inactivated. After that, DNA polymerase fills the gap and cuts off the single-stranded protruding ends, and finally DNA ligase is used to connect the growing DNA molecules (see the construction diagram). Figure 1 ). This method is convenient, fast, efficient, and can achieve seamless splicing.

[0040] Table 1. The fragment positions, lengths and shared homology region positions of the MVA genome fragments .

[0041] 1. Construction of BACYAC-F01-F10 (V1) BACYAC-F01-F10 (V1) is 56.393 kb in length and contains the genomic fragments of MVA genome (U94848.1) from 9730 bp to 56760 bp. It is composed of 10 chemically synthesized (F01~F10) MVA genomic fragments and BACYAC plasmids co-transfected into VL6-48N yeast competent cells (purchased from Shanghai Zeye Biotechnology Co., Ltd.) and homologous recombination. Asis I restriction site is provided by the primer pair of PYE-Link-F01, namely PYE-Link-F01-F (SEQ ID NO.4) and PYE-Link-F01-R (SEQ ID NO.5); F10 is homologous to BACYAC and AscI is provided by the primer pair of F10-Link-PYE, namely F10-Link-PYE-F (SEQ ID NO. 6) and F10-Link-PYE-R (SEQ ID NO. 7).

[0042] The specific method of TAR recombination is as follows: 100 ng of purified linearized BACYAC vector, 200 ng of 10 PCR-amplified purified DNA fragments (F01-F10), 500 ng of two pairs of primers, and 20 μL of Yeastmaker Carrier DNA were added to 200 μL of VL6-48N yeast competent cells (purchased from Shanghai Zeye Biotechnology Co., Ltd.), gently mixed, 800 μL of PEG / Li Ac was added, gently mixed, incubated at 30°C for 45 min, gently shaken and mixed every 15 min, 160 μL of fresh DMSO was added, incubated at 42°C water bath for 20 min, gently shaken and mixed every 10 min, centrifuged at 700 g at room temperature for 5 min, resuspended the cells with 3 mL of YPD Plus medium (purchased from Beijing Coolbo Technology Co., Ltd.), incubated at 30°C shaking for 90 min, centrifuged at 700 g at room temperature for 5 min, discarded the supernatant, resuspended with 500 μL of 0.9% (w / v) NaCl solution, and 100 μL of DMSO was added. µL was coated on SD-trp agar plates (purchased from Takara) and incubated at 30°C for 3–5 days. The yeast DNA mixture identified as positive by PCR was extracted from yeast colonies and transformed into DH10B competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electric stimulation. A large amount of recombinant plasmids were obtained after plasmid extraction. The intersections between homologous arms were introduced into Asis I and Asc I restriction site, a single restriction site connected to both ends of the genome by the BACYAC plasmid when packaging the virus Asis I and Asc I linearization (see Figure 2 ).

[0043] 2. Construction of BACYAC-F10-F21 (V2) BACYAC-F10-F21 (V2) is 63.724 kb in length and contains the MVA genome (U94848.1) fragments from 52893 bp to 106124 bp. It is produced by co-transfection of 12 chemically synthesized (F10-F21) MVA genome fragments with BACYAC plasmids into VL6-48N yeast competent cells for homologous recombination. AscThe I restriction site is provided by the primer pair of F21-LINK-F10, namely F21-LINK-F10-F (SEQ ID NO.8) and F21-LINK-F10-R (SEQ ID NO.9).

[0044] The homology regions of F14 and BACYAC were provided by the primer pair of F14-LINK-PYE, namely F14-LINK-PYE-F (SEQ ID NO. 10) and F14-LINK-PYE-R (SEQ ID NO. 11).

[0045] The homology region between F15 and BACYAC was provided by the primer pair F15-LINK-PYE, F15-LINK-PYE-F (SEQ ID NO.12), F15-LINK-PYE-R (SEQ ID NO.13), wherein F15 contains TK The "Loxp-p7.5-eGFP-Loxp" sequence was inserted at 275 bp in the gene region. The sequence is shown in SEQ ID NO: 3. The early and late promoter P7.5 of the poxvirus was added in front of eGFP. The two loxP sites were located at both ends of the eGFP expression frame and in the same direction. The sequence between the LoxP sites could be knocked out by Cre recombinase. One end of BACYAC was connected to F14, and the other end was connected to a single restriction site. Asis I is linked to the "Loxp-p7.5-eGFP-Loxp" sequence, with a single restriction site introduced by the primer pair providing the homology region Asis I can be used to insert the foreign gene expression frame, F10 and F21 are connected head to tail, and the virus is packaged through Asc I cuts the junction between F10 and F21, linearizes the plasmid and exposes the homologous fragment (see Figure 3 ).

[0046] 3. Construction of BACYAC-F21-F30 (V3) BACYAC-F21-F30 (V3) is 51.952 kb in length and contains the MVA genome (U94848.1) genomic fragments from 102100 bp to 144689 bp. It is composed of 10 chemically synthesized (F21~F30) MVA genome fragments and BACYAC plasmid through TAR recombination splicing.

[0047] The homology regions of F21 and BACYAC were provided by the primer pair of F21-Link-PYE, namely F21-Link-PYE-F (SEQ ID NO. 14) and F21-Link-PYE-R (SEQ ID NO. 15).

[0048] The homology region between F30 and BACYAC was provided by the primer pair F30-Link-PYE, which are F30-Link-PYE-F (SEQ ID NO.16) and F30-Link-PYE-R (SEQ ID NO.17). Asis I is connected to F21, and the other end is connected to Asc I is connected to F30, and when packaging the virus, the single restriction site is connected to both ends of the genome through the BACYAC plasmid Asis I and Asc I linearization (see Figure 4 ).

[0049] 4. Construction of BACYAC-F29-F35 (V4) BACYAC-F29-F35 (V4) is 42.934 kb in length and contains the MVA genome (U94848.1) genomic fragments from 102100 bp to 144689 bp. It is composed of 7 chemically synthesized (F29~F35) MVA genomic fragments and BACYAC plasmid through TAR recombination splicing. The homologous regions of F29 and BACYAC are provided by the primer pair of F29-Link-PYE, namely F29-Link-PYE-F (SEQ ID NO.18) and F29-Link-PYE-R (SEQ ID NO.19).

[0050] The homology region between F35 and BACYAC was provided by the primer pair PYE-Link-F35, F35-Link-PYE-F (SEQ ID NO.20), F35-Link-PYE-R (SEQ ID NO.21). Asis I is connected to F29, and the other end is connected to Asc I is connected to F35, and when packaging the virus, the single restriction site is connected to both ends of the genome through the BACYAC plasmid Asis I and Asc I linearization (see Figure 5 ).

[0051] 5. Construction of PET-LITR-RITR (V5) PET-LITR-RITR (V5) was assembled by Gibson ligation. V5 was composed of chemically synthesized F36 (LITR) and F37 (RITR) and PET plasmid (stored in our laboratory) by Asis I and AscI connection, the 3' end of the chemically synthesized RITR sequence added a 21bp sequence at the 5' end of the LITR sequence to provide the necessary homologous region for homologous recombination during Gibson splicing, prepare the linearized fragments of LITR and RITR, and use the reserved single restriction site at the end of the chemically synthesized LITR and RITR genes X I and Asis I linearized the fragments and amplified the PET vector fragment. The amplification primer for adding the homology arm of the 3' end of LITR to the PET vector fragment was PET-LITR-F (SEQ ID NO.22), and the amplification primer for adding the homology arm of the 5' end of RITR was PET-RITR-R (SEQ ID NO.23). The length of the amplified product was 2540 bp. After the above enzyme digestion and PCR amplification fragments were purified, they were spliced ​​using the Gibson recombination system (NEB). The specific method was 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 was controlled to be 2:1), 10 μL of Gibson Mix assembly solution, 20 μL of sterile water, incubated at 50°C for 15 min, and ice bathed for about 3 min. Take 3 μL of the recombination system to transform into 50 μL of TOP10 chemically competent cells, incubate for 40-60 min, spread on a plate, culture overnight, and extract the recombinant plasmid after the colony is identified as positive. After the splicing is completed, the intersection between the homology arms will introduce Asis I and Asc I restriction site for plasmid linearization (see Figure 6 ).

[0052] The present invention provides five plasmids based on the splicing of five MVA genome fragments synthesized by full artificial chemical synthesis, 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 Figure 2-Figure 6 ).

[0053] Figure 7 Agarose gel electrophoresis of five plasmids is shown. Asis I and Asc I linearized BACYAC-F01-F10 (V1), BACYAC-F21-F30 (V3), BACYAC-F29-F35 (V4), PET-LITR-RITR (V5), using Asc I linearized BACYAC-F10-F21 (V2), and the enzyme digestion was detected using 1% agarose. The obtained plasmid length was consistent with the expected value.

[0054] Example 2. Packaging of recombinant MVA-syn poxvirus Figure 8 A packaging schematic diagram of the present invention is given, in which linearized plasmids V1~V5 are transfected into BHK-21 cells infected with fowlpox virus FPV, and recombinant MVA poxvirus is packaged through homologous recombination of DNA fragments containing homologous regions. Among them, BHK-21 cells (stored in this laboratory) are selected as the packaging cell line, and FPV (fowlpox virus) is infected with BHK-21 cells (stored in this laboratory) at MOI=0.1~1 for 1 hour, and then the linearized fragments are transfected into BHK-21 cells using Turbofect transfection reagent, and the cells are maintained in culture for 3~5 days, and the cell culture medium is frozen and thawed. The recombinant MVA-syn virus is continuously passaged until the cells continue to be pathological, and a stably passaged recombinant MVA-syn virus can be obtained. We have completed the packaging of the recombinant MVA-syn poxvirus of the MVA-Fast vector system, and used the MVA-syn recombinant virus to infect BHK-21 cells, and found that eGFP can be sustainably expressed ( Fig. 9 A). The structure of the recombinant virus particles was detected using an electron microscope. The diameter of the virus particles was 220-450 nm, with a typical oval or brick-shaped structure, which was consistent with the expected results ( Fig. 9 B), indicating that this system can obtain recombinant MVA-syn poxvirus with normal structure and stable expression of foreign genes.

[0055] Example 3. PCR identification results of recombinant MVA poxvirus In order to identify the recombinant MVA-syn poxvirus genome, five recombinant MVA-syn poxvirus monoclones (MVA-syn1~5) were isolated by plaque purification. The viral genomic DNA / RNA rapid extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the MVA-syn1~5, MVA-WT and FPV fowlpoxvirus genomes. The fragment-specific amplification primers of BACYAC, F01, F06, F16, F26 and F35 and FPV were designed, respectively. The primers for detecting BACYAC plasmid (SEQ ID NO.24-25), primers for detecting F01 (SEQ ID NO.26-27), primers for detecting F16 (SEQID NO.28-29), primers for detecting F26 (SEQ ID NO.30-31), primers for detecting F35 (SEQ ID NO.32-33), and primers for detecting MVA ITR (SEQ ID NO.34-35) were designed. The PCR products were analyzed by agarose gel electrophoresis. Fig.10) revealed that the recombinant MVA-syn poxvirus contained the BACYAC plasmid and a PCR fragment with the same length as MVA-WT, but did not contain the PCR fragment of FPV fowlpox virus, indicating that the five plasmid fragments of the MVA-Fast system had completed the recombination, and the recombinant MVA-syn virus could be separated from the FPV helper virus by passaging or plaque purification.

[0056] Example 4. Growth curve of recombinant MVA poxvirus MVA poxvirus cannot replicate on most mammalian cells, but can replicate on BHK-21 and DF-1 cells. Three PCR-positive recombinant MVA-syn poxviruses, MVA-syn1, MVA-syn4 and MVA-syn5, were randomly selected for comparison with MVA-WT. BHK-21 and DF-1 cells (purchased from Beina Biotechnology Co., Ltd.) were infected with MOI=0.01, respectively. Virus growth curves were drawn at 0, 12, 24 and 72 h to compare the growth characteristics of MVA-syn and MVA-WT. The growth curve results showed that MVA-syn and MVA-WT exhibited similar growth kinetics on BHK-21 and DF-1 cells (see Fig.11 ).

[0057] Example 5. Analysis of plaque characteristics of recombinant MVA poxvirus MVA-WT and MVA-syn were used to infect BHK-21 cells in a 6-well plate at an MOI of 0.001. After infection, the culture medium was discarded, the cells were washed three times with PBS, covered with cold agar, and cultured at 37°C for 72 h. The culture medium was discarded, and a sufficient amount of 4% PFA tissue cell fixative (purchased from Beijing Regen Biotechnology Co., Ltd.) was slowly added along the well wall. The cells were fixed at room temperature for 12 h, stained with crystal violet, and the well plates were scanned using a Celigo full-view cell analyzer (Nexcelom). The plaque area was measured using the scanned digital images. The results showed that the plaque sizes produced by MVA-syn and MVA-WT after infection of BHK-21 cells were similar, and the two had similar abilities to spread in the same permissive cells (see Fig.12 ).

[0058] Example 6. Construction and characterization of recombinant MVA poxvirus vector MVA-HA vaccine like Fig.13 As shown, the HA protein expression cassette was inserted into the BACYAC-F21-F30 (V3) plasmid using the Gibson method. Pac In site I, the specific method is as follows: PacI digested the V3 plasmid, purified the digested product by ethanol precipitation (Takara), and PCR amplified the HA protein expression cassette (sequence shown in SEQ ID NO: 36), with primers HA-F (sequence shown in SEQ ID NO: 37) and HA-R (sequence shown in SEQ ID NO: 38). The two fragments were spliced ​​by Gibson in vitro one-step recombination method (see Example 1 for the specific method), and the V3-HA recombinant plasmid with HA protein successfully inserted was obtained, and the recombinant MVA-HA influenza vaccine was packaged by the MVA-Fast system (see Example 2 for the specific method). The viral genomes of MVA-HA, MVA-WT and MVA-syn4 were extracted (see Example 3 for the specific method), and the identification primers HA-Part-F (sequence shown in SEQ ID NO: 39) and HA-Part-R (sequence shown in SEQ ID NO: 40) of the HA protein gene were designed. The agarose gel electrophoresis results of the PCR amplification product showed that MVA-HA successfully inserted the HA protein gene sequence (see Fig.14 ). BHK-21 cells were infected with MVA-HA at an MOI of 1, and cell samples were collected after 12 h and 24 h of culture. Western blot results showed that BHK-21 cells infected with MVA-HA could normally express HA protein within 12 h and 24 h.

[0059] Example 7. Immunogenicity of MVA-HA vaccine To determine the immunogenicity of HA antigens expressed using MVA poxvirus, we used 5 × 10 7 IFU / piece and 5×10 6Balb / c mice were immunized with a dose of 100 μL / well of HA protein. The mice received two vaccinations 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. Serum was collected on days 21 and 28, and the level of HA protein binding antibodies was detected by ELISA. The specific method was as follows: HA protein (purchased from Sino Biological Biotechnology Co., Ltd.) was diluted to 1 μg / mL, 100 μL / well was added to the microplate, coated overnight at 4°C, the plate was washed 3 times with washing solution, and then 100 μL / well of blocking solution was added, and the plate was blocked at 37°C for 1h. The liquid in the plate was shaken off and washed 3 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 gradient dilution. Seven dilutions were set for each sample, and 12 control wells without serum were set for each plate. After the serum was diluted, it was incubated at 37°C for 1 h. After washing, a 1:20000 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 for 6 min using TMB single-component colorimetric solution (solarbio, PR1200). 50 μL of 2M sulfuric acid was added to each well to terminate the reaction. The absorbance at 450 nm was detected on a microplate reader (MolecμLar Devices, Spectra Max). From the results of the binding antibody detection (see Fig.15 ), after two immunizations, both intramuscular and subcutaneous immunizations could stimulate mice to produce high-titer antibody responses, and the antibody response levels had 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 of which carries five segments sequentially divided based on the full-length genome of the poxvirus, wherein the fifth plasmid carries the inverted terminal repeat sequence RITR fragment and LITR fragment 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.

2. The vector system for packaging recombinant poxvirus according to claim 1, characterized in that: The poxvirus is the poxvirus Ankara strain with ATCC deposit number VR-1508.

3. The vector system for packaging recombinant poxvirus according to claim 2, characterized in that: The lengths of the four segments carried by the first plasmid, the second plasmid, the third plasmid and the fourth plasmid, which are sequentially divided based on the full-length genome of the poxvirus, are between 30 kb and 64 kb.

4. The vector system for packaging recombinant poxvirus according to claim 3, characterized in that: The lengths of the four segments carried by the first plasmid, the second plasmid, the third plasmid, and the fourth plasmid, which are sequentially divided based on the full-length genome of the poxvirus, 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, 102100 bp and 144689 bp, and 134789 bp and 168360 bp of the poxvirus genome, respectively. The LITR fragment and the RITR fragment are both 9.8 kb, LITR is located between 1 bp and 9794 bp, and RITR is located from 168295 bp to the end, and is reverse complementary to LITR.

5. The vector system for packaging recombinant poxvirus according to claim 4, characterized in that: 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, 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.

6. The vector system for packaging recombinant poxvirus according to claim 2, characterized in that: The first plasmid, the second plasmid, the third plasmid and the fourth plasmid are each provided with a restriction enzyme cutting site for inserting foreign genes.

7. The vector system for packaging recombinant poxvirus according to claim 6, characterized in that: The restriction enzyme sites for inserting foreign genes are respectively provided on the first plasmid, the second plasmid, the third plasmid and the fourth plasmid: Eag I. Asis I. Pac I and Eag I.

8. The method for constructing a vector system for packaging a recombinant poxvirus according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) The genomic segments covered by the first plasmid to the fourth plasmid are divided into 7 to 12 5 kb sub-segments, and the genomic segment covered by the fifth plasmid is divided into two sub-segments, LITR and RITR, and synthesized separately. A homologous recombination region is introduced into the sub-segment adjacent to the plasmid, and a restriction site for linearization is introduced. Using the homologous recombination method, the first plasmid to the fourth plasmid and the BACYAC plasmid are co-transfected into yeast competent cells, and a circular closed plasmid is obtained by homologous recombination in the host cells. The fifth plasmid is obtained by transforming the two fragments contained therein with the PET plasmid into Escherichia coli through Gibson in vitro ligation; (2) Linearizing 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 subculture until the cells continue to become disease-prone. This will allow the recombinant MVA poxvirus to be stably subcultured.

9. Use of the vector system for packaging recombinant poxvirus according to any one of claims 1 to 7 in the preparation of poxvirus vector vaccines.

10. The use according to claim 9, characterized in that: The poxvirus vector vaccine is a poxvirus vector vaccine of the HA protein of influenza virus H1N1.

11. A method for packaging a recombinant poxvirus using the vector system for packaging a recombinant poxvirus according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: (1) Inserting foreign genes into the non-essential region of viral replication in the recombinant poxvirus vector system; (2) transfecting the recombinant poxvirus vector system obtained in step (1) into host cells, and simultaneously infecting the host cells with a helper 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.

12. The method according to claim 11, characterized in that The non-essential region for viral replication in step (1) 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.

13. The method according to claim 11, characterized in that The helper virus in step (2) is fowlpox virus.

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