Baculovirus expression vector for efficiently expressing swine foot and mouth disease virus-like particles as well as construction method and application of baculovirus expression vector

Abstract

The invention discloses a baculovirus expression vector capable of efficiently expressing swine foot-and-mouth disease virus-like particles as well as a construction method and application of the baculovirus expression vector. The baculovirus expression vector contains a segment of skeleton sequence and a segment of gene synthesis sequence of a pFastbacic-dual vector. The gene synthesis sequence comprises a first terminator, a first cloning site, an internal ribosome entry site, a transcriptional activation factor, a first promoter, an enhancer, a second promoter, a second cloning site and a second terminator which are connected in sequence, the first cloning site and the second cloning site are introduced into a gene sequence of a pig foot-and-mouth disease 3C protein and a gene sequence of a pig foot-and-mouth disease P12A protein respectively. After the baculovirus prepared by the baculovirus expression vector provided by the invention infects insect cells, the expression quantity of 3C protein can be reduced, the expression quantity of P12A protein can be increased, the expressed 3C protein can cut the P12A protein to further form swine foot-and-mouth disease virus-like particles, and the expression quantity of the foot-and-mouth disease virus-like particles can be remarkably increased.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a baculovirus expression vector for highly expressing porcine foot-and-mouth disease virus-like particles, a construction method thereof, and an application thereof. Background Art

[0002] As a powerful functional vector, the baculovirus expression vector system (BEVS) plays a significant role in the fields of biopharmaceutics, gene therapy, etc. Baculoviruses have powerful promoters, such as the polyhedrin promoter PH, P10, GP67 promoter, etc., which can drive the high expression of foreign genes in insect cells. This system can accommodate multiple foreign genes at the same time and can achieve the co-expression of multiple genes. For the expression of VLPs, the VLPs of many viruses are composed of multiple structural proteins. The baculovirus expression vector system can simultaneously introduce the genes of these structural proteins, enabling them to be expressed simultaneously in insect cells and correctly assembled into VLPs. As a eukaryotic expression system, insect cells can perform various post-translational modifications on the expressed proteins, such as glycosylation, phosphorylation, acylation, etc. These modifications are crucial for maintaining the structural stability, immunogenicity, and biological activity of VLPs. Insect cells have a perfect protein folding and assembly mechanism, which can help the expressed viral structural proteins correctly fold and assemble into VLPs.

[0003] Porcine foot-and-mouth disease virus (FMDV) is highly harmful, causing serious economic losses to the pig breeding industry and posing certain public health risks. Foot-and-mouth disease virus (FMDV) is the pathogen that causes foot-and-mouth disease in cloven-hoofed animals. Its genome is single-stranded positive-strand RNA, with a length of about 8.5 kb. The P1 region encodes the structural proteins of FMDV, including VP1, VP2, VP3, and VP4. These structural proteins are the main components of the virus capsid and are crucial for the formation of VLPs. They can self-assemble into a structure similar to the natural virus capsid.

[0004] The 3C protein is a non-structural protein of FMDV and has protease activity, playing an important role in the viral life cycle, including participating in processes such as the processing of viral polyproteins. During the formation of VLPs, the 3C protein may indirectly affect the assembly of the P1 protein by processing and cleaving the P1 protein or other related proteins, thereby influencing the formation of VLPs. The 2A protein can cleave between itself and adjacent proteins, splitting the polyprotein into different functional proteins. By affecting the cellular localization of structural proteins such as P1, it enables them to be accurately transported to the appropriate positions for assembly. At the same time, it can also affect the interactions between proteins, promoting the correct assembly of VLPs. However, in practical applications, overexpression of the 3C protein of porcine foot-and-mouth disease virus is usually toxic to cells. When the 3C protein is overexpressed, it will cleave cytoskeletal proteins, resulting in the destruction of the cytoskeletal structure. The 3C protein will cleave or interfere with some key metabolic enzymes or signal pathways in the cell, leading to apoptosis.

[0005] When using the baculovirus expression vector system to express porcine foot-and-mouth disease virus VLPs (virus-like particles), there is currently the drawback of low expression levels. The possible reason is that when expressing porcine foot-and-mouth disease virus VLPs, virus-like particles are formed only when several proteins interact and assemble correctly simultaneously. Therefore, the present invention focuses on solving the above problem by inserting different DNA elements into the baculovirus expression components. Summary of the Invention

[0006] Aiming at the problem of low expression level of porcine FMDV VLPs in the existing baculovirus expression system, the present invention provides a baculovirus expression vector for highly expressing porcine foot-and-mouth disease virus-like particles, its construction method and application, aiming to transform the baculovirus expression vector, construct a baculovirus expression vector containing the FMDV VLP antigen sequence, and further improve the expression level of the FMDV VLP foreign protein.

[0007] To achieve the above object, the technical solution of the present invention is as follows: The present invention provides a baculovirus expression vector, which includes a backbone sequence of the pFastbac-dual vector and a gene synthesis sequence. The gene synthesis sequence includes a first terminator, a first cloning site, an internal ribosome entry site, a transcriptional regulatory element, a first promoter, an enhancer, a second promoter, a second cloning site, a second terminator sequence, and a promoter sequence connected in sequence. The first cloning site and the second cloning site can respectively introduce the gene sequences of the target proteins.

[0008] Furthermore, the first terminator and the second terminator are respectively the SV40 terminator or the HSVTK terminator, the first promoter is respectively one of the GP67 promoter, the CMV promoter, the PH promoter or the P10 promoter, and the second promoter is one of the PH promoter or the P10 promoter; the transcriptional regulatory element is the transcriptional activator IE1, the enhancer is the hr1 enhancer, and the gene sequence of the internal ribosome entry site is as shown in SEQ ID NO.2.

[0009] On this basis, the present invention provides a baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles, which is characterized in that it contains the baculovirus expression vector as described in claim 2, and the gene sequences of the porcine foot-and-mouth disease 3C protein and the porcine foot-and-mouth disease P12A protein are respectively introduced into the first cloning site and the second cloning site. The gene sequence of the porcine foot-and-mouth disease virus P12A protein is as shown in SEQ ID NO:11, and the gene sequence of the porcine foot-and-mouth disease 3C protein is as shown in SEQ ID NO:10.

[0010] Furthermore, the gene sequence of the porcine foot-and-mouth disease 3C protein is tandemly expressed with the coding transcriptional regulatory element sequence IE1 through the internal ribosome entry sequence IRES and is located at the downstream gene position.

[0011] Furthermore, the first terminator is the HSVTK terminator, the first promoter is the PH promoter, the second promoter is the P10 promoter, and the second terminator is the SV40 terminator.

[0012] The construction method of the baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles provided by the present invention is as follows: Design primers to use pFastbac-dual as a template, and amplify a backbone sequence of the pFastbac-dual vector by PCR, as shown in SEQ ID NO.12. At the same time, design primers to artificially synthesize the FMDV VLP gene sequence using HSVTK-3C-IRES-IE1-PH-hr1-P10-FMDV P12A-SV40 as a template, where the HSVTK sequence is as shown in SEQ ID NO.9, the 3C sequence is as shown in SEQ ID NO.10, the IRES sequence is as shown in SEQ ID NO.2, the IE1 sequence is as shown in SEQ ID NO.3, the PH sequence is as shown in SEQ ID NO.7, the FMDV P12A sequence is as shown in SEQ ID NO.11, and the SV40 sequence is as shown in SEQ ID NO.1. Connect the amplified backbone sequence of the pFastbac-dual vector and the artificially synthesized FMDV VLP gene sequence by fusion PCR, transform, and perform clone screening to construct a baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles.

[0013] The present invention also provides a method for constructing a recombinant baculovirus, specifically: Transform the competent cell DH10Bac with the baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles as described above. After culturing at 37°C, use the blue-white screening method and perform PCR identification, shake the bacteria, extract the bacmid to obtain the recombinant bacmid; Use the transfection reagent ExpiFectamine SF to transfect the Sf9 cells in the logarithmic growth phase. After static adherent culture at 27°C in a 6-well plate for 72 h, harvest the recombinant baculovirus of the P0 generation.

[0014] The present invention also provides a method for preparing porcine foot-and-mouth disease virus-like particles, specifically: Infect H5 cells with the recombinant baculovirus obtained by the method for constructing a recombinant baculovirus as described above according to an inoculation amount of 0.1 MOI. The cell density is 2.5×10 6 cells / ml, the culture temperature is 27°C, the rotation speed is 120 r / min, culture for 6 d, the cells are naturally lysed, centrifuged, and the supernatant is the protein solution; For the harvested protein supernatant, perform sucrose gradient centrifugation. Add 5 ml of the obtained protein solution to an ultracentrifuge tube, and then sequentially add 3.5 ml of 15% and 3.5 ml of 45% sucrose to the centrifuge tube. When adding, use a long needle to add from the bottom upwards. Centrifuge for 4 h, collect the protein band into the centrifuge tube with a long needle, appropriately dilute the purified virus with PBS buffer, then centrifuge for 3 h, and suspend the precipitate with 500 μl of PBS buffer to finally obtain the purified porcine foot-and-mouth disease virus-like particles.

[0015] Compared with the prior art, the progress of a baculovirus expression vector for highly expressing porcine foot-and-mouth disease virus-like particles, its construction method and application disclosed by the present invention lies in the following aspects:

[0016] 1. The hr1 enhancer and the transcriptional activator IE1 are introduced into the expression vector. The hr1 enhancer is a homologous region rich in AT repeats or palindromic structures, and EcoRI restriction enzyme sites often appear in this region. Adding an enhancer to the baculovirus promoter can greatly increase the protein yield. IE1, with a size of 67 kD, can bind to DNA and stimulate transcription. IE1 is a transcriptional activator with broad-spectrum and non-specificity. After IE1 binds to hr1 in the form of a dimer, it will enhance DNA transcription and increase the protein expression level.

[0017] 2. Two multiple cloning sites are designed in the expression vector, which can carry the gene sequences of two functional proteins simultaneously, and are particularly suitable for increasing the expression that requires the cooperation of multiple protein functions.

[0018] 3. The internal ribosome entry sequence IRES is introduced into the expression vector. The internal ribosome entry site can recruit ribosomes, enabling the ribosomes to directly bind to specific positions inside the mRNA, thereby initiating the translation of downstream genes. The gene sequence of the porcine foot-and-mouth disease 3C protein in the expression vector is tandemly expressed with the sequence IE1 encoding the transcriptional regulatory element through the internal ribosome entry sequence IRES and is located at the downstream gene position. Due to the action of the internal ribosome entry sequence IRES, ribosomes preferentially bind to the 5' end of the mRNA to initiate the translation of the upstream gene, while the efficiency of binding ribosomes through IRES to initiate the translation of the downstream gene is relatively low, resulting in an obvious difference in the expression levels of the upstream and downstream genes, and the expression level of the downstream gene is lower than that of the upstream gene. Therefore, when the expression vector expresses foot-and-mouth disease virus-like particles (FMDV VLP) in cells, the expression level of the 3C protein is reduced, and the expression level of the P1 protein is increased, avoiding the damage to cells caused by the overexpression of the 3C protein, while ensuring the appropriate processing and cleavage of the P1 protein by the 3C protein and promoting the assembly of the P1 protein. Description of the Drawings

[0019] Figure 1 Schematic diagram of a synthetic sequence provided by the present invention. It includes a transcriptional regulatory region, an enhancer region, an IRES region, a promoter region, a terminator region, a P12A region, and a 3C region.

[0020] Figure 2 Schematic diagram of the construction process of a baculovirus expression vector for highly expressing porcine foot-and-mouth disease virus-like particles VLP.

[0021] Figure 3Schematic diagram of the baculovirus expression vector pHPP10SP12A3C for highly efficient expression of foot-and-mouth disease virus-like particles VLP in pigs.

[0022] Figure 4 WB detection diagram of foot-and-mouth disease virus-like particles VLP. The figure shows the WB detection results of combination 2 and combinations 13, 14.

[0023] Figure 5 Schematic diagram of sucrose gradient centrifugation purification of foot-and-mouth disease virus-like particles VLP. The arrow indicates the foot-and-mouth disease virus-like particle protein.

[0024] Figure 6 Electron microscopy image of foot-and-mouth disease virus-like particles VLP in pigs.

[0025] Figure 7 OD280 measurement diagram of protein concentration. After VLP production using different baculovirus expression vectors for expressing foot-and-mouth disease virus-like particles VLP in pigs, the OD280 measurement diagram of protein concentration.

[0026] Figure 8 Immune response diagram of foot-and-mouth disease virus-like particles VLP in pigs. After VLP production using different baculovirus expression vectors for expressing foot-and-mouth disease virus-like particles VLP in pigs, the diagram after immune response detection of foot-and-mouth disease virus-like particles VLP. Detailed implementation methods

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and do not limit the present invention.

[0028] The reagents used in the embodiments of the present invention are all commercially available products.

[0029] The list of sources of reagents, strains and plasmids of the present invention is as follows:

[0030] The pFastbac-dual baculovirus expression vector used in the present invention was purchased from Invitrogen;

[0031] The insect cells SF9 and H5 were purchased from Invitrogen;

[0032] The insect cell culture medium IB905 was all purchased from Yishengke (Shenzhen) Co., Ltd.;

[0033] The DH10Bac competent cells were purchased from Bomed Biotechnology Co., Ltd.;

[0034] The restriction enzymes were purchased from NEB;

[0035] PrimeSTAR HS DNA Polymerase was purchased from Takara Biotechnology (Beijing) Co., Ltd.

[0036] The In-Fusion HD Cloning Kits were purchased from Baoruiyi Biotechnology (Beijing) Co., Ltd.

[0037] Sequence optimization and sequence synthesis were completed by Genewiz (Suzhou) Inc.

[0038] The O-type foot-and-mouth disease antibody liquid-phase blocking ELISA detection kit was purchased from Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0039] Example 1

[0040] Sequence synthesis and vector construction

[0041] 1. Gene synthesis: Synthesize a gene sequence, which is different combinations (see Table 2) of different elements (see Table 1). The schematic diagrams of different combinations are shown in Figure 1 , and these sequence combinations are ligated to the pUC57 vector.

[0042] Table 1 Information of different elements

[0043] Serial number Component name Base sequence 1 SV40 polyA SEQ ID NO.1 2 IRES SEQ ID NO.2 3 IE1 SEQ ID NO.3 4 hr1 SEQ ID NO.4 5 GP67 promoter SEQ ID NO.5 6 CMV promoter SEQ ID NO.6 7 PH promoter SEQ ID NO.7 8 P10 promoter SEQ ID NO.8 9 HSV TK polyA SEQ ID NO.9 10 FMDV 3C SEQ ID NO.10 11 FMDV P12A SEQ ID NO.11 12 pFastdual backbone sequence SEQ ID NO.12 13 pHPP10SP12A3C SEQ ID NO.13

[0044] Table 2 Different combinations of different elements

[0045] Combination 1 HGP10SP12A3C - pUC57 HSV TK - 3C - IRES - IE1 - GP67 - hr1 - P10 - FMDV P12A - SV40 Combination 2 HPP10SP12A3C - pUC57 HSV TK - 3C - IRES - IE1 - PH - hr1 - P10 - FMDV P12A - SV40 Combination 3 HCP10SP12A3C - pUC57 HSV TK - 3C - IRES - IE1 - CMV - hr1 - P10 - FMDV P12A - SV40 Combination 4 SGP10HP12A3C - pUC57 SV40 - 3C - IRES - IE1 - GP67 - hr1 - P10 - FMDV P12A - HSV TK Combination 5 SPP10HP12A3C - pUC57 SV40 - 3C - IRES - IE1 - PH - hr1 - P10 - FMDV P12A - HSV TK Combination 6 SCP10HP12A3C - pUC57 SV40 - 3C - IRES - IE1 - CMV - hr1 - P10 - FMDV P12A - HSV TK Combination 7 HGPSP12A3C - pUC57 HSV TK - 3C - IRES - IE1 - GP67 - hr1 - PH - FMDV P12A - SV40 Combination 8 HPPSP12A3C - pUC57 HSV TK - 3C - IRES - IE1 - PH - hr1 - PH - FMDV P12A - SV40 Combination 9 HCPSP12A3C - pUC57 HSV TK - 3C - IRES - IE1 - CMV - hr1 - PH - FMDV P12A - SV40 Combination 10 SGPHP12A3C - pUC57 SV40 - 3C - IRES - IE1 - GP67 - hr1 - PH - FMDV P12A - HSV TK Combination 11 SPPHP12A3C - pUC57 SV40 - 3C - IRES - IE1 - PH - hr1 - PH - FMDV P12A - HSV TK Combination 12 SCPHP12A3C - pUC57 SV40-3C-IRES-IE1-CMV-hr1-PH-FMDVP12A-HSVTK Combination 13 SPP10HP12A3C△IE1△hr1-pUC57 SV40-3C-PH-P10-FMDVP12A-HSVTK Combination 14 HPP10SP12A3C△IE1△hr1-pUC57 HSVTK-3C-PH-P10-FMDVP12A-SV40

[0046] 2. Amplification of vector backbone sequence and gene sequence: Using pFastbac dual as the template, amplify a backbone sequence on pFastbac dual with primers Bone-TN7L-FP and Bone-RP; using the vectors of combinations 1, 2, 3, 7, 8, 9, 14 in Table 2 as templates, amplify the FMDV VLP sequences of different combined elements with primers VLP-SV40-FP and VLP-TSK-RP, and using the vectors of combinations 4, 5, 6, 10, 11, 12, 13 in Table 2 as templates, amplify the FMDV VLP sequences of different combined elements with primers VLP-HSV-FP and VLP-SV40-RP. Use Prime STAR HS DNA Polymerase to amplify the gene sequence, and purify the amplification products with a PCR product purification kit. The primers and primer sequences are shown in Table 3, and the amplification system and reaction conditions are shown in 4.

[0047] Table 3 Fusion PCR primers

[0048]

[0049] Table 4 Amplification system and reaction conditions

[0050]

[0051]

[0052] 3. Construction of expression vector by fusion PCR: Since the amplified vector backbone sequence and the gene sequence containing FMDV VLP have about 15 bp homologous arms at both ends, the amplified backbone sequence was ligated to the FMDV VLP sequences of different combined elements respectively using the operation method of In-Fusion HD Cloning Kits. The reaction system and reaction conditions are shown in Table 5.

[0053] Table 5 Fusion PCR system and reaction conditions

[0054] Component Volume Purified backbone vector PCR fragment 10 - 50 ng PCR fragment containing the gene sequence of FMDV VLPs 50 - 200 ng 5×In-Fusion HD Enzyme Premix 2 μl Sterilized water Up to 10 μl Reaction conditions 50 °C, 8 min.

[0055] The above reaction products were transfected into competent peptide DH5a, cultured on LB medium containing ampicillin resistance, single colonies were picked, plasmids were extracted, and molecular cloning methods such as sequencing were used to obtain different baculovirus expression vectors (see Table 6). Restriction enzyme sites were added at the connection positions of different elements, and sequence replacement could be carried out. The plasmid map of the baculovirus expression vector constructed to express FMDV VLP of porcine foot-and-mouth disease with combination 2 as an example is shown in Figure 3 , and its sequence is SEQ ID NO.13. This figure shows all restriction enzyme sites. If sequence replacement of VLP or element replacement is required, it can be carried out using restriction enzyme sites.

[0056] Table 6 Plasmids constructed using different combinations

[0057] Combination 1 pHGP10SP12A3C Combination 2 pHPP10SP12A3C Combination 3 pHCP10SP12A3C Combination 4 pSGP10HP12A3C Combination 5 pSPP10HP12A3C Combination 6 pSCP10HP12A3C Combination 7 pHGPSP12A3C Combination 8 pHPPSP12A3C Combination 9 pHCPSP12A3C Combination 10 pSGPHP12A3C Combination 11 pSPPHP12A3C Combination 12 pSCPHP12A3C Combination 13 pSPP10HP12A3C△IE1△hr1 Combination 14 pHPP10SP12A3C△IE1△hr1

[0058] Example 2

[0059] Baculogeneration

[0060] The baculovirus FMDV VLP expression vector constructed in Table 6 of Example 1 was transformed into competent cell DH10Bac. After culturing at 37 °C, blue-white screening method was used and PCR identification was carried out. The bacteria were shaken and baculogeneration was extracted to obtain recombinant baculogeneration.

[0061] Example 3

[0062] Baculovirus preparation

[0063] The recombinant bacmid obtained in Example 2 was transfected into Sf9 cells in the logarithmic growth phase using the transfection reagent ExpiFectamine SF. After static adherent culture at 27°C for 72 h in a 6-well plate, the recombinant baculovirus of P0 generation was harvested. The harvested P0 generation virus was inoculated with the P1 generation virus at an inoculation amount of 5%, and after suspension culture at 27°C for 72 h, the P1 generation virus was harvested. The harvested P1 generation virus was inoculated with the P2 generation virus at an inoculation amount of 5%, and after suspension culture at 27°C for 72 h, the P2 generation virus was harvested. The supernatant was the virus solution, and the virus titer of the virus solution was measured by indirect immunofluorescence.

[0064] Example 4

[0065] Protein expression

[0066] According to an inoculation amount of 0.1 MOI, the H5 cells were infected with the P2 generation recombinant baculovirus. The cell density was 2.5×10 6 cells / ml, the culture temperature was 27°C, the rotation speed was 120 r / min, and the cells were cultured for 6 d. The cells were naturally lysed, centrifuged, and the supernatant was the protein solution. At the same time, the harvested protein solution was detected by WB. The WB images of combination 2, combination 13, and combination 14 are shown in Figure 4 .

[0067] Example 5

[0068] Sucrose gradient centrifugation

[0069] For the harvested protein supernatant, sucrose gradient centrifugation was performed. 5 ml of the obtained protein solution was added to an ultracentrifugation tube, and then 3.5 ml of 15% and 3.5 ml of 45% sucrose were sequentially added to the centrifugation tube. When adding, a long needle was used to add from the bottom upwards. Centrifuge at 110,000 g for 4 h, and collect the protein band into the centrifugation tube with a long needle (see the state of the protein after centrifugation in Figure 5 ). Dilute the purified virus with an appropriate amount of PBS buffer, and then centrifuge at 110,000 g for 3 h. Suspend the precipitate with 500 μl of PBS buffer, and finally the purified virus was obtained. Store it at -20°C for later use.

[0070] Example 6

[0071] Negative staining electron microscopy observation

[0072] Take 20 μl of the FMDV VLP sample purified by sucrose gradient centrifugation for electron microscopy observation. Drop 1 drop onto the Formvar film of a 400-mesh copper grid and adsorb for about 1 - 2 min. Gently suck off the excess solution from the edge of the copper grid with a filter paper. Drop 2% phosphotungstic acid salt (pH 7.2) onto the copper grid for negative staining for about 2 min, and suck off the excess residual liquid with a filter paper. Let it dry slightly, and then electron microscopy observation (Philips CMl0 electron microscope, working voltage 40 kV) can be carried out, and take pictures.

[0073] Electron microscopy observation of the purified VLP showed that VLP virus-like particles with a diameter of 30 nm could be observed. The electron microscopy images of the VLP virus-like particles in combination 2 are shown in Figure 6 .

[0074] Example Seven

[0075] Measuring the concentration of FMDV VLP by OD280nm

[0076] After diluting the FMDV VLP protein solution prepared in Example Five by 10 times, 2 μl was taken and the protein concentration was measured using Nanodrop according to the absorbance at OD280nm.

[0077] The present invention demonstrated the influence of combinations of different elements on the expression of FMDV VLP in insect cells. Among them, the combination of HSVTK-3C-IRES-IE1-PH-hr1-P10-FMDV P12A-SV40 (HPP10SP12A3C) had a protein concentration that could reach 50 mg / ml, significantly higher than other combinations. Followed by the combination of HSVTK-3C-IRES-IE1-GP67-hr1-P10-FMDV P12A-SV40 (HGP10SP12A3C) with a protein concentration that could reach 42 mg / ml. See Table 7 and Figure 7 .

[0078] Table 7 Measurement of protein concentration expressed by baculovirus prepared with different combinations

[0079]

[0080]

[0081] Example Eight

[0082] Detecting the immunogenicity of FMDV-VLP by Elisa

[0083] Elisa detection: After diluting the protein solution prepared in Example Five by 100 times, 50 μl was taken and added to the Elisa plate coated with foot-and-mouth disease type O rabbit antibody, 50 μl / well, sealed the plate, and incubated at 37°C for 60 min. Continuously wash the plate 3 - 5 times with PBST, pat dry, add the working solution of foot-and-mouth disease type O guinea pig antibody (red) at 50 μl / well, seal the plate, and incubate at 37°C for 30 min. Continuously wash the plate 3 - 5 times with PBST, pat dry, add the working solution of rabbit anti-guinea pig IgG-HRP (blue) at 50 μl / well, seal the plate, and incubate at 37°C for 30 min. Continuously wash the plate 3 - 5 times with PBST, pat dry. Mix the TMB substrate solution A and B in a 1:1 ratio, add the mixed substrate solution at 50 μl / well, and incubate at 37°C for color development for 15 min. After the color development reaction ended, add the termination solution at 50 μl / well to terminate the reaction, and read OD450nm on the microplate reader.Figure 8 The results of the immune response showed that the combination of HSVTK-3C-IRES-IE1-PH-hr1-P10-FMDV P12A-SV40 (HPP10SP12A3C) had a significantly higher OD450nm value than other combinations. Followed by the OD450nm value of HSVTK-3C-IRES-IE1-GP67-hr1-P10-FMDV P12A-SV40 (HGP10SP12A3C). This result was consistent with the protein concentration result.

[0084] The baculovirus expression vector expressing porcine foot-and-mouth disease virus-like particle protein described above is only an embodiment of the present invention, and the scope of implementation of the present invention cannot be limited thereby. That is, all simple equivalent changes and modifications made according to the claims of the present invention and the description of the invention still fall within the scope covered by the present invention.

Claims

1. A baculovirus expression vector, characterized in that, The baculovirus expression vector includes a backbone sequence of the pFastbac-dual vector and a gene synthesis sequence. The gene synthesis sequence includes a first terminator, a first cloning site, an internal ribosome entry site, a transcriptional regulatory element, a first promoter, an enhancer, a second promoter, a second cloning site, and a second terminator that are sequentially connected. The first cloning site and the second cloning site can respectively carry the gene sequences of the target proteins.

2. The baculovirus expression vector according to claim 1, characterized in that, The first terminator and the second terminator are respectively the SV40 terminator or the HSV TK terminator. The first promoter is one of the GP67 promoter, the CMV promoter, the PH promoter, or the P10 promoter. The second promoter is one of the PH promoter or the P10 promoter. The transcriptional regulatory element is the transcriptional activator IE1. The enhancer is the hr1 enhancer. The gene sequence of the internal ribosome entry sequence IRES is as shown in SEQ ID NO.

2.

3. A baculovirus expression vector capable of efficiently expressing porcine foot-and-mouth disease virus-like particles, characterized in that, The baculovirus expression vector as described in claim 2 is included. The first cloning site and the second cloning site respectively carry the gene sequence of the porcine foot-and-mouth disease 3C protein and the gene sequence of the porcine foot-and-mouth disease P12A protein. The gene sequence of the porcine foot-and-mouth disease virus P12A protein is as shown in SEQ ID NO:11, and the gene sequence of the porcine foot-and-mouth disease 3C protein is as shown in SEQ ID NO:

10.

4. A baculovirus expression vector capable of efficiently expressing porcine foot-and-mouth disease virus-like particles according to claim 3, characterized in that, The gene sequence of the porcine foot-and-mouth disease 3C protein is tandemly expressed with the coding transcriptional regulatory element sequence IE1 through the internal ribosome entry sequence IRES and is located at the downstream gene position.

5. The baculovirus expression vector capable of efficiently expressing porcine foot-and-mouth disease virus-like particles according to claim 4, wherein The first terminator is the HSV TK terminator, the first promoter is the PH promoter, the second promoter is the P10 promoter, and the second terminator is the SV40 terminator.

6. A method for constructing a baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles as described in claim 5, characterized in that: Primers are designed to amplify a backbone sequence of the pFastbac-dual vector, as shown in SEQ ID NO.12, by PCR using pFastbac-dual as a template. At the same time, primers are designed to artificially synthesize the FMDV VLP gene sequence using HSVTK-3C-IRES-IE1-PH-hr1-P10-FMDV P12A-SV40 as a template, where the HSVTK sequence is as shown in SEQ ID NO.9, the 3C sequence is as shown in SEQ ID NO.10, the IRES sequence is as shown in SEQ ID NO.2, the IE1 sequence is as shown in SEQ ID NO.3, the PH sequence is as shown in SEQ ID NO.7, the FMDV P12A sequence is as shown in SEQ ID NO.11, and the SV40 sequence is as shown in SEQ IDNO.

1. The amplified backbone sequence of the pFastbac-dual vector and the artificially synthesized FMDV VLP gene sequence are connected, transformed, and cloned and screened by the method of fusion PCR to construct a baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles.

7. A method for constructing a recombinant baculovirus, characterized in that, The baculovirus expression vector capable of highly expressing porcine foot-and-mouth disease virus-like particles described in claim 5 was transformed into competent cells DH10Bac. After culturing at 37 °C, blue-white screening method was used and PCR identification was carried out. The bacteria were shaken, bacmids were extracted, and recombinant bacmids were obtained; the recombinant bacmids were transfected into Sf9 cells in the logarithmic growth phase by using the transfection reagent ExpiFectamine SF. After static adherent culture at 27 °C in a 6-well plate for 72 h, the recombinant baculovirus of P0 generation was harvested.

8. A method for preparing porcine foot-and-mouth disease virus-like particles, characterized in that, Using the recombinant baculovirus obtained by the method for constructing a recombinant baculovirus according to claim 7 with an inoculum amount of 0.1 MOI to infect H5 cells, with a cell density of 2.5×10 6 cells / ml, a culture temperature of 27°C, a rotation speed of 120 r / min, cultured for 6 days, the cells were lysed naturally, centrifuged, and the supernatant protein solution was harvested; for the harvested protein solution, sucrose gradient centrifugation was performed. 5 ml of the obtained protein solution was added to an ultracentrifugation tube, and then 3.5 ml of 15% sucrose and 3.5 ml of 45% sucrose were successively added to the centrifugation tube. When adding, a long needle was used to add from the bottom upwards. Centrifuged for 4 h, the protein band was collected into the centrifugation tube with a long needle, the purified virus was diluted appropriately with PBS buffer, then centrifuged for 3 h, and the precipitate was suspended with 500 μl PBS buffer, thus finally obtaining purified foot-and-mouth disease virus-like particles of pigs.

9. Application of the method for preparing porcine foot-and-mouth disease virus-like particles as described in claim 8 in the production of porcine foot-and-mouth disease virus vaccines or diagnostic reagents.

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