CN114262381B - Recombinant baculovirus with surface displaying African swine fever virus antigen P30 protein, preparation method and application thereof - Google Patents

Abstract

The invention discloses a recombinant baculovirus with surface displaying African swine fever virus antigen P30 protein, a preparation method and application thereof. The invention inserts the gene sequence of P30 between the N-end signal peptide SP sequence of baculovirus envelope glycoprotein gp64 and fusion structural domain TMD to construct fusion gene gp64-Fp30, then connects with recombinant vector pFPG to construct recombinant expression vector pFPG-gp64-Fp30, then converts pFPG-gp64-Fp30 into DH10Bac cell to obtain recombinant Bacmid plasmid, finally transfects Sf9 cell with the recombinant Bacmid plasmid to obtain recombinant baculovirus with surface displaying African swine fever virus antigen P30 protein. The recombinant P30 protein produced by utilizing the recombinant baculovirus has a conformation similar to that of a natural protein, can induce and generate serum antibodies with excellent sensitivity and specificity, can realize efficient and rapid expression of the P30 antigen, and has wide application prospect in preparing ASFV diagnostic reagents or ASF vaccines.

Description

Recombinant baculovirus with surface displaying African swine fever virus antigen P30 protein, preparation method and application thereof

Technical Field

The invention relates to the field of recombinant protein diagnostic reagents and vaccines, in particular to a recombinant baculovirus with surface displaying African swine fever virus antigen P30 protein, a preparation method and application thereof.

Background

African swine fever (African swine fever, ASF) is a highly infectious, highly lethal acute febrile infectious disease caused by infection of domestic pigs and wild pigs with African swine fever virus (African swine fever virus, ASFV). In 1921, ASF was first reported in kenya, africa, and has been spreading to many countries in the world over the last century due to the difficulty in controlling the disease, which the world animal health Organization (OIE) lists as a legal report of animal epidemic disease.

The CP204L gene of ASFV encodes an inner envelope protein P30 with a protein size of 23.6kD, which is expressed continuously in early infection and participates in invasion of the host cell by the virus. The P30 protein has been shown to be extremely specific, sensitive and highly immunogenic and to be detectable 4h after ASFV infection, with great potential for development of early diagnostic reagents for ASFV infection or ASF vaccines ([ 1] Luan Yuxuan, zhang Hao, wang Chengbao. Progress in serodiagnosis and vaccine research of african swine fever [ J ], progress in animal medicine, 2021,42 (03): 78-82.). The OIE-recommended enzyme-linked immunosorbent assay (ELISA) detects ASFV, but this method is limited by serum sensitivity. The virus titer is low in the early stage of ASFV infection, and the general serological detection method cannot achieve the ideal diagnosis effect. In addition, ASFV has a large genome, rapid mutation, and an immune escape mechanism is not clear, so that it is necessary to develop a detection reagent having high detection specificity and high sensitivity to cope with the transmission of ASFV.

Of the various baculoviruses, the nuclear polyhedrosis virus (Autographa californica multicapsid nucleopolyhedrovirus, acMNPV) is the most widely studied, with the genes ie1, gp64 expressed in both early and late stages of AcMNPV replication and the polyhedrin gene polh expressed in late and very late stages of infection. Studies have shown that promoters Pie1 and P in AcMNPV _PH Recombinant foreign proteins displayed on the surface of baculovirus in combination not only express high levels but also induce high titer specific antibody production ([ 2)]Hu ZP,Yin J,Zhang YY,et al.Characterization of the immune responses elicited by baculovirus-based vector vaccines against influenza virus hemagglutinin[J]Acta Pharmacol sin.2012,33 (6): 783-90.) with the ability to express multiple exogenous genes allows AcMNPV applications to produce complex proteins in vitro or in vivo in eukaryotes, and also in vaccine development in conjunction with surface display technology.

In recent years, the P30 recombinant vector constructed by the traditional pFastBac1 vector is used for producing a P30 protein antigen, the specificity of which is stronger, but the sensitivity is insufficient, and the expression quantity of the exogenous protein is limited by the action of a non-combined promoter (3 Xu Lihui, bay super, wang Yujia, etc.. The eukaryotic expression of the P30 protein of the African swine fever virus and the establishment of an indirect ELISA antibody detection method [ J ]. Chinese Protect veterinarian school report, 2021,43 (04): 382-387.). Yu Haoyang, but the screening preparation and purification steps are complicated and the period is long, thus increasing the cost of detection reagent or vaccine production ([ 4] Yu Haoyang, wang Caixia, wu Shaojiang, etc. ] the preparation of African swine fever virus p30 protein monoclonal antibody and the establishment of blocking ELISA detection method [ J/OL ]. Chinese veterinary science: 1-10[2021-12-02]. Https:// doi. Org/10.16656/J. Issn.1673-4696.2022.0001 ]. Therefore, it is necessary to construct a unique recombinant antigen vector so that the expressed ASFVP30 protein antigen has excellent immunogenicity, and the induced serum sensitivity can be comparable to that of a monoclonal antibody while having stronger specificity.

Disclosure of Invention

The invention aims at providing a recombinant protein gp64-Fp30 containing an African swine fever virus antigen P30 protein.

The recombinant protein gp64-Fp30 is composed of an N-terminal signal peptide SP of an African swine fever virus antigen P30 protein, an N-terminal signal peptide SP of a baculovirus envelope glycoprotein gp64 and a fusion domain TMD of the baculovirus envelope glycoprotein gp 64; the amino acid sequence of the African swine fever virus antigen P30 protein is shown as SEQ ID No.2, the amino acid sequence of an N-terminal signal peptide SP of baculovirus envelope glycoprotein gp64 is shown as SEQ ID No.4, and the amino acid sequence of a fusion domain TMD of baculovirus envelope glycoprotein gp64 is shown as SEQ ID No. 6.

The second object of the present invention is to provide the coding gene of the recombinant protein gp64-Fp30 containing the African swine fever virus antigen P30 protein, which is formed by sequentially connecting the coding gene of the N-terminal signal peptide SP of the baculovirus envelope glycoprotein gp64 shown in SEQ ID No.3, the coding gene of the African swine fever virus antigen P30 protein shown in SEQ ID No.1 and the coding gene of the fusion domain TMD of the baculovirus envelope glycoprotein gp64 shown in SEQ ID No. 5.

The third object of the invention is to provide a recombinant baculovirus with the surface displaying African swine fever virus antigen P30 protein, wherein the encoding gene of the recombinant protein gp64-Fp30 is inserted into a recombinant vector pFPG to construct a recombinant expression vector pFPG-gp64-Fp30, the recombinant expression vector pFPG-gp64-Fp30 is transformed into DH10Bac to obtain a recombinant Bacmid plasmid, then the recombinant Bacmid plasmid is transfected into Sf9 insect cells, and the recombinant baculovirus with the surface displaying African swine fever virus antigen P30 protein is obtained by packaging in the Sf9 insect cells.

In the invention, the insertion site of the coding gene of the recombinant protein gp64-Fp30 in the recombinant vector pFPG is after a baculovirus polyhedrin promoter Pph.

The fourth object of the present invention is to provide a method for preparing the recombinant baculovirus with the surface displaying the african swine fever virus antigen P30 protein, comprising the following steps:

(1) The coding gene of N-terminal signal peptide SP of baculovirus envelope glycoprotein gp64, the coding gene of African swine fever virus antigen P30 protein and the coding gene of fusion domain TMD of baculovirus envelope glycoprotein gp64 are respectively amplified by PCR, cloned into a pMD19-T vector to construct recombinant plasmids T-PSP, T-Fp30 and T-TMD, and then respectively subjected to double digestion by EcoRI/SaL I, saL I/XbaI and XbaI/PstI, and cloned into the pMD19-T vector to construct recombinant plasmid T-gp64-Fp30 by using T4 DNA ligase;

(2) The recombinant plasmid T-gp64-Fp30 and the recombinant vector pFPG are subjected to double enzyme digestion by EcoRI/PstI, and then the DH10Bac competent strain is subjected to enzyme ligation and transformation, and the successfully constructed recombinant expression vector pFPG-gp64-Fp30 is obtained through blue-white spot screening and PCR verification;

(3) Transforming a recombinant expression vector pFPG-gp64-Fp30 into DH10Bac to obtain a recombinant Bacmid plasmid;

(4) And (3) transfecting the Sf9 insect cells with the recombinant Bacmid plasmid, culturing, and collecting the supernatant to obtain the recombinant baculovirus with the surface displaying the African swine fever virus antigen P30 protein.

The fourth object of the present invention is to provide the application of the recombinant baculovirus with the surface displaying the African swine fever virus antigen P30 protein in preparing ASFV diagnostic reagent or ASF vaccine.

Compared with the prior art, the invention has the following advantages:

(1) The gene sequence of the African swine fever conserved structural protein P30 is inserted between the N-terminal signal peptide SP sequence of the baculovirus envelope glycoprotein gp64 and the fusion structural domain TMD to construct a fusion gene gp64-Fp30, which is beneficial to the successful transfer of the Fp30 gene into Sf9 cells and can realize the mass expression of Fp30;

(2) After the fusion gene gp64-Fp30 is constructed on the baculovirus polyhedrin promoter Pph, the promoter Pph has strong effect in Sf9 cells, so that a large amount of expression of the recombinant protein gp64-Fp30 is realized;

(3) The Sf9 cell surface display of the P30 protein can be realized by transfecting and infecting Sf9 cells after DH10Bac is transformed by the constructed specific expression vector pFPG-gp64-Fp30, the serum titer of a P30 protein antigen immunized mouse prepared by surface display can reach 1:12800, and the Westernblot and IFA test of serum show that the serum antibody produced by a P30 protein antigen stimulus test animal prepared by surface display has excellent specificity for the P30 protein; meanwhile, indirect ELISA proves that the serum sensitivity of the antigen is superior to that of the protein antigen prepared by the traditional method, and the antigen is suitable for preparing ASFV diagnostic reagents or ASF vaccines;

(4) The method is simple and quick, and is suitable for large-scale industrial production.

Drawings

FIG. 1 is a schematic diagram of the construction of recombinant expression vector pFPG-gp64-Fp30. A: physical mass spectrogram of fusion gene gp64-Fp30, the front and the back of the fusion gene are respectively provided with EcoRI and PstI restriction sites; b, empty expression vector pFPG physical mass spectrogram, ecoRI and PstI are respectively arranged at the front end and the rear end of MCS; c: and constructing a physical mass spectrum of the successfully constructed recombinant expression vector PFPG-gp64-Fp30.

FIG. 2 is a diagram showing the double-restriction identification electrophoresis after each target fragment was ligated into the T vector. Wherein M: DNA Marker (DL-5000); 1-4: ecoRI/SaL I double-digested T-PSP;5-8: saL I/XbaI double-enzyme cutting T-TMD;9-11: xbaI/PstI double cleavage T-Fp30.

FIG. 3 shows PCR identification of bacterial liquid with fusion genes after gene fragments PSP, fp30 and TMD are connected into a T vector after the action of T4 DNA ligase. Wherein M: DNA Marker (DL-5000); 1-4: and (3) PCR electrophoresis results with PSPF/TMDR as a primer and bacterial liquid as a template.

FIG. 4 shows the identification of the recombinant expression vector pFPG-gp64-Fp30 by EcoRI/PstI double cleavage. Wherein M: DNA Marker (DL-10000); 1-2: ecoRI/PstI double cuts pFPG-gp64-Fp30.

FIG. 5 is a microscopic view of Sf9 cells transfected with recombinant virus REAcMNPV-gp64-Fp 30. Wherein A: healthy Sf9 cell growth status; b: the volume of the Sf9 cells transfected by the recombinant viruses is enlarged, black particles are observed in the cells, and the arrows show; c: expression of a large amount of green fluorescent protein was observed under an inverted fluorescent microscope.

FIG. 6 is a Western blot of cell lysates and cell membrane protein extracts of recombinant virus-infected Sf9 cells. Wherein M: protein Marker; s: a supernatant; E1-E4: cell membrane extracts; L1-L4: cell lysates. The Flag mouse monoclonal antibody is used as a primary antibody, and the AP marked goat anti-mouse IgG is used as a secondary antibody.

FIG. 7 is confocal cellular localization of antigen P30 protein. Wherein A: when the recombinant virus infects the Sf9 cells for 96 hours, observing by an inverted fluorescence microscope; b: detecting Cy3 red light of the confocal microscope; c: confocal microscopy DAPI blue light detection; d: confocal microscopy red and blue light co-detection.

FIG. 8 shows the Western-blot detection results of immune serum with murine serum as primary antibody and AP-labeled goat anti-mouse IgG as secondary antibody.

FIG. 9 shows the results of indirect ELISA for determining immune serum antibody titers. At 5min after addition of stop solution, the ratio of OD values (P/N) of positive serum-coated wells to negative control serum-coated wells at 450 nm: p <0.05.

FIG. 10 is an indirect Immunofluorescence (IFA) analysis (100X) of immune serum. Healthy Sf9 cells, observed with light microscopy; b, the obvious pathological change of the cells occurs after the recombinant baculovirus particles infect the cells for 4 d; the result of IFA when the mouse negative control serum antibody of the cell disruption liquid of the Sf9 cells infected with the wild virus is treated by sedimentation to be primary antibody and the Cy3 (red) marked goat anti-mouse antibody is secondary antibody; d, performing inverted fluorescence microscopy on the cell infected by the recombinant virion with gfp gene for 4D; E. f, IFA results when the serum antibody settled by the immunized mice is used as a primary antibody and the Cy3 (red) -marked goat anti-mouse antibody is used as a secondary antibody.

Detailed Description

The invention is further described below with reference to the detailed description and the accompanying drawings. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1 expression and analysis of antigen P30 protein

1. Construction of recombinant expression vector pFPG-gp64-Fp30

According to NCBI, query and obtain the gene CP204L sequence 561bp (AF 462271.1) of ASFV p30 and gp64 gene sequence 1539bp (KF 022001.1) in the whole genome sequence of AcMNPV, artificial synthesis analysis is carried out on p30 gene fragments, 3 groups of specific primers are designed by using Oligo7 software, 50 mu L of PCR reaction system is adopted by adopting a PCR method, pre-deformation is carried out for 8min at 94 ℃, denaturation is carried out for 40s at 94 ℃, annealing is carried out for 40s at 56 ℃, extension is carried out for 1min at 72 ℃, amplification is carried out for 30 cycles, target fragments are amplified at 72 ℃, PSP with gp64 with a promoter sequence and TMD (fusion domain TM containing gp64 gene and cytoplasmic domain CTD) are amplified from AcMNPV DNA, the target fragments are cloned into pMD19-T vectors respectively, recombinant plasmids T-PSP, T-Fp30 and T-TMD are constructed, after sequencing verification, the plasmids are extracted respectively through EcoRI/SaL I, xbaI/PsaI and PsaI/T are connected to the plasmids through double-restriction enzyme digestion (FIG. 4) and PCR is carried out for the specific cloning of the plasmids such as that the plasmids are cut into the plasmid PmP 1-T19 (FIG. 4, and the PCR is verified). The commercialized pFastBac1 vector is taken as a framework, P is taken as a reaction product _PH The homologous recombination of the specific sequence before the gene sequence is the tandem sequence of Pie1 and EGFP, specifically, the homologous recombination of the tandem sequence of Pie1 and EGFP and a segment between restriction sites BamHI and HindIII of a commercial pFastBac1 vector is carried out to construct a recombinant vector pFPG (shown in figure 1B), and the nucleotide sequence is shown in SEQ ID No. 7. And (3) carrying out double enzyme digestion on the T-gp64-Fp30 and the recombinant vector pFPG by EcoRI/PstI, then carrying out enzyme ligation to transform the DH10Bac competent strain, and carrying out blue-white spot screening and PCR verification to obtain a successful recombinant expression vector pFPG-gp64-Fp30 (shown in figure 1C).

The designed primer sequence is as follows:

FP30F(SEQID No.8)：

FP30R(SEQIDNo.9)：5’-CTCTAGATTTTTTTTTTAAAAGTTTAATGACCATGAG-3’，

PSPF(SEQIDNo.10)：5’-GGAATTCGAGCGTCCGTGTTCATGATC-3’，

PSPR(SEQIDNo.11)：5’-CGTCGACCATTTGCGCGTTGCAGTG-3’，

TMDF(SEQIDNo.12)：5’-CTCTAGACTCATAACCACCATGGAG-3’，

TMDR(SEQIDNo.13)：5’-ACTGCAGTTAATATTGTCTATTACGGTTTCTAAT-3’。

the thick underline is the restriction enzyme cleavage site, the double thin underline is the Flag tagged DNA sequence; PSPF and PSPR are respectively the upstream and downstream amplification primers of PSP gene, and the introduced enzyme cutting sites are respectively EcoRI and SaL I; fp30F, fp R is an upstream and downstream amplification primer of the Fp30 gene, and the introduced enzyme digestion sites are SaL I and XbaI respectively; TMDF and TMDR are respectively upstream and downstream amplification primers of TMD genes, and the introduced enzyme digestion sites are respectively XbaI and PstI.

2. Expression and identification of antigen P30 protein

Double enzyme digestion verification is carried out on the recombinant expression vector pFPG-gp64-Fp30 by using EcoRI and PstI (as shown in figure 4), then DH10Bac competent strains are transformed by using the successfully constructed recombinant expression vector, shaking culture is carried out for 4 hours at 37 ℃, LB solid plates containing antibiotics and IPTG and X-gal are streaked and coated, white single colonies are picked up after standing for 48 hours in a constant temperature incubator at 37 ℃, PCR identification is carried out on the colonies to be correct and purified, finally purified recombinant Bacmid is obtained, amplification and shaking are further carried out, and a large amount of recombinant Bacmid plasmid DNA is extracted. The recombinant plasmid was transfected into Sf9 insect cells with DOTAP eukaryotic cell transfection reagent and the cell status was observed under an inverted fluorescent microscope (see fig. 5). After 4d of transfection and infection, the cultured cell liquid is collected, cells are lysed by using Western and IP cell lysate, the supernatant and the cell precipitate of the lysed cell liquid are respectively collected by centrifugation at 2000rpm and 5min, SDS-PAGE identification and Western-blot experiments (shown in FIG. 6) are carried out, a clear protein band of 23.6kDa is obtained, the size of the clear protein band is consistent with that of the expected P30 protein band, and the highest expression level of fusion protein is verified when the Sf9 cells are infected by recombinant viruses for 96 h.

3. Antigen P30 protein concentration assay

The protein concentration of the recombinant virus at 96h infection of Sf9 cells was determined by Bradford protein concentration assay. A stock solution of 20mg/mL Bovine Serum Albumin (BSA) was prepared as a 1mg/mL stock solution. 10mL of a 1mg/mL mother solution was prepared, and PBS diluent was added to the mother solution. The mother solution and PBS were added dropwise to a 4mL EP tube in the following volume scheme:

adding 100 mu L of each tube into 2mL G250 Bradford dyeing working solution, gently reversing and uniformly mixing, standing at room temperature for 5min, diluting 0.2mL of Fp30 protein solution to be detected to 20mL by using PBS, adding the solution into 2mL G250 Bradford dyeing working solution, measuring absorbance of A595nm by using an ultraviolet spectrophotometer, and measuring OD595 = 0.251 of fusion protein gp64-Fp30, wherein the corresponding protein concentration is 0.24mg/mL.

4. Cell localization analysis of recombinant protein gp64-Fp30

To verify that recombinant protein Fp30 was displayed on the cell surface, fluorescent microscopy was started 48h (moi=5) after infection with recombinant virus-infected Sf9 cells, and after a large amount of green fluorescence was observed, the infected cells were immobilized, blocked, primary antibody-bound, cy3 (red) -labeled secondary antibody-bound, DAPI (blue) -nuclear-stained, blocked, and the like, and samples 63x/1.40 (oil-mirror) were analyzed under zeiss LSM510 confocal microscope, and it was clearly observed that recombinant protein gp64-Fp30 expression was anchored on Sf9 cell surfaces (see fig. 7).

2. Antigen P30 protein antigenicity detection

1. Western-blot detection of immune serum

To evaluate the immunogenicity of gp64-Fp30, antigen was mixed with Freund's adjuvant in equal volumes to prepare an antigen solution for immunization studies in BALB/c mice. After boosting twice with 100mg/kg dose, 1000-fold dilutions of serum samples are collected at week 2 as primary antibodies, wild-type immunized mouse serum mixed with an adjuvant is used as a negative control, AP-labeled goat anti-mouse IgG is used as a secondary antibody for Western-blot detection (as shown in figure 8), the experimental groups all have a darker specific band, and the control groups do not have the specific band. This indicates that the surface-displayed gp64-Fp30 has excellent immunogenicity, and can induce the generation of a murine antibody to strongly and specifically bind with the gp64-Fp30 protein expressed in vitro.

2. Indirect ELISA determination of immune serum antibody titers

The antibody titer of serum of immunized mice is determined by adopting an indirect ELISA method (as shown in figure 9), tail-broken blood collection serum is positive to be detected, blank mouse serum is negative control, goat anti-mouse IgG marked by HRP is used as secondary antibody, and the antibody titer of positive serum is detected by adopting a "matrix method" titration. The surface-displayed fusion protein gp64-Fp30 was diluted 1:100 and then immobilized on a 96-well ELISA plate. Antisera were prepared according to 1:200,1:800,1:1600,1:3200,1:6400,1:128000,1:25600,1:51200, negative serum 1:200 is diluted as primary antibody, then added into an ELISA plate in sequence, 4-hole repetition is carried out on each gradient, and incubation is carried out for 2 hours at 37 ℃; goat anti-mouse IgG (1:500 dilution) secondary antibody labeled with HRP, incubated at 37℃for 1h; TMB was added to the reaction mixture to carry out a dark color reaction for 5min, followed by 2M H ₂ SO ₄ The reaction was terminated by the reaction termination liquid. The ELISA plate is placed in a multifunctional microplate detector to detect OD ₄₅₀ Values. Blank Kong Diaoling, if the test group OD ₄₅₀ The value (P) is more than or equal to 2.1 times of the negative control (N), namely, the P/N is more than or equal to 2.1, and the positive can be judged. The ratio of the OD values (P/N) of the positive serum to the negative control serum at a dilution of 1:12800 was over 2.1 for immunized mice serum, and furthermore the OD of the positive serum at 1:25600 was higher than that of the positive serum ₄₅₀ =1.828, OD 1:12800 ₄₅₀ The difference is obvious, the variation coefficient of the OD value corresponding to each gradient coated serum hole among 6 repeated experiment groups is within 5%, which indicates that the stability of the data result is good and the experimental data has better repeatability. The positive serum titer of the immunized mice is 1:12800, and the p30 protein displayed on the surface of the constructed baculovirus has excellent antigen sensitivity and can detect diluted antibodies with high gradient.

3. Indirect Immunofluorescence (IFA) analysis of immune serum

After infection of Sf9 cells with recombinant baculovirus for 4d, the cells were collected, washed with PBS, and fixed with 4% paraformaldehyde for 20min at 37 ℃; adding 0.2% Triton-X-100 blocking solution, and blocking at room temperature for 30min; adding serum antibody subjected to dilution sedimentation treatment by using a blocking solution 1:1000, and allowing the mixture to act at 37 ℃ for 1h; adding secondary anti-dilution liquid which is diluted by secondary anti-dilution liquid 1:500 and contains Cy3 (red) marked goat anti-mouse antibody into the mixture after washing in a dark place, and enabling the mixture to act for 1h at room temperature; after washing, a drop of anti-fluorescence quenching liquid was dropped on the slide glass and the cover glass was back-fastened to the slide glass, and fluorescence image analysis was performed under an inverted fluorescence microscope after sealing the slide glass with nail oil (see fig. 10). The results of indirect Immunofluorescence (IFA) of all the test wells showed bright red color, and the control wells showed black background, which indicates that serum antibodies generated after immunization reacted strongly with the p30 protein displayed on the surface, and the baculovirus surface displayed p30 protein had excellent immunogenicity.

Sequence listing

<110> university of Yangzhou

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gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080

ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440

cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860

gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980

tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400

cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460

ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520

caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580

acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640

tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700

ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760

aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820

tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880

ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940

tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000

gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060

gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120

cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180

ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240

agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300

ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360

tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420

acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480

ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540

ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600

cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660

ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720

cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780

ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840

gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900

tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960

ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020

ccatcgggcg cggatcccgg tccgaagcgc gcggctaggt ctgtactatt ctatgtaact 4080

actcaaacct gtttggtgtt gatcttacgt cacttttttt acgaaataaa cactttaaac 4140

actacgataa cgaaataaac attggtaata ttcgacgtta tttgttcaaa gttgagcaac 4200

gcgtccaaac gacgtggcac aactaccgca ggttgttaac aagtcgatta aatagattgt 4260

acctgcagtt cagcccattg ctcagacatt agagccgttc gttacacaaa ttagaccagt 4320

tttattgtag aaatcgccaa aggttgcggc gctcctcgac gtgcaaaact aacgcgtgcg 4380

ggcagtagta caacctgtta agatttttaa ctaatcaaac caactctaaa taaaccaact 4440

gcaacgagta gttgtacagc aaaactactg cttagaaaag gtcgcgcagc ttataaacag 4500

ctgcgggagc ggaccctctg ctacttgcac tggcgctaga tctggccctg gctctagcgc 4560

ttgctccaga cgttactcct ctcgcgcttg ctccagacgt tactcctctt gcactagcgc 4620

tcgcactagc actcgcacta gcactagccg ctgctgccat tttcatcgtg tttattaaca 4680

aacgttattt acacgagttg ttgaccagcc gcttgctcgt tccgcaagcg ttttggtctt 4740

gccggcgagg catttacagg tggtacataa actggcacaa tactgtgcta ggcgggcgta 4800

gcaactagta aaaagataca aatctacctt accacttgtt ttcaacaact ttgcggacgt 4860

ttccgtaaaa atttaaaact atttacatca ggcacagcta ggtttcctgg ctgcgcagca 4920

gcttgtattg caataagcag ccgaagtaga attcaggcct gtcgacgagc tcgagctcac 4980

tagtgcggcc gctctagact gcaggcgcgg gctaccaccc tgccatactt attaggcctt 5040

ataaatatcc aaaaaaataa tgttttgaca atgcttttgt cattttatga ataaataaac 5100

gctctaccaa tagtaaaatt aatagaggta ctagatgtct ttgtgatgcg cgcgacattt 5160

ttgtaggtta ttgataaaat gaacggatac gttgcccgac attatcatta aatccttggc 5220

gtagaatttg tcgggtccat tgtccgtgtg cgctagcatg cccgtaacgg acctcgtact 5280

tttggcttca aaggttttgc gcacagacaa aatgtgccac acttgcagct ctgcatgtgt 5340

gcgcgttacc acaaatccca acggcgcagt gtacttgttg tatgcaaata aatctcgata 5400

aaggcgcggc gcgcgaatgc agctgatcac gtacgctcct cgtgttccgt tcaaggacgg 5460

tgttatcgac ctcagattaa tgtttatcgg ccgactgttt tcgtatccgc tcaccaaacg 5520

cgtttttgca ttaacattgt atgtcggcgg atgttctata tctaatttga ataaataaac 5580

gataaccgcg ttggttttag agggcataat aaaagaaata ttgttatcgt gttcgccatt 5640

agggcagtat aaattgacgt tcatgttgga tattgtttca gttgcaagtt gacactggcg 5700

gcgacaagat cgtgaacaac caagacggtg ggaggtctat ataagcagag ctggtttagt 5760

gaaccgtcag atccgctagc gctaccggtc gccaccatgg tgagcaaggg cgaggagctg 5820

ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 5880

agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 5940

tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 6000

gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 6060

atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 6120

acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 6180

atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 6240

cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 6300

cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 6360

atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 6420

agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 6480

gggatcactc tcggcatgga cgagctgtac aagaacttgt ttattgcagc ttataatggt 6540

tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 6600

agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctc gaggcatgcg 6660

gtaccaagct tgtcgagaag tactagagga tcataatcag ccataccaca tttgtagagg 6720

ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 6780

caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 6840

tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 6900

tcatcaatgt atcttatcat gtctggatct gatcactgct tgagcctagg agatccgaac 6960

cagataagtg aaatctagtt ccaaactatt ttgtcatttt taattttcgt attagcttac 7020

gacgctacac ccagttccca tctattttgt cactcttccc taaataatcc ttaaaaactc 7080

catttccacc cctcccagtt cccaactatt ttgtccgccc acagcggggc atttttcttc 7140

ctgttatgtt tttaatcaaa catcctgcca actccatgtg acaaaccgtc atcttcggct 7200

actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcgttatt aatgtttgta 7260

attgactgaa tatcaacgct tatttgcagc ctgaatggcg aatgg 7305

<210> 8

<211> 52

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cgtcgacgat tataaagacg atgacgataa gatgaaaatg gaggtcatct tc 52

<210> 9

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ctctagattt tttttttaaa agtttaatga ccatgag 37

<210> 10

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ggaattcgag cgtccgtgtt catgatc 27

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cgtcgaccat ttgcgcgttg cagtg 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

ctctagactc ataaccacca tggag 25

<210> 13

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

actgcagtta atattgtcta ttacggtttc taat 34

Claims (6)

1. The recombinant protein gp64-Fp30 containing the African swine fever virus antigen P30 protein is characterized by comprising an N-terminal signal peptide SP of the baculovirus envelope glycoprotein gp64 connected with the N-terminal of the African swine fever virus antigen P30 protein and a fusion domain TMD of the baculovirus envelope glycoprotein gp64 connected with the C-terminal; the amino acid sequence of the African swine fever virus antigen P30 protein is shown as SEQ ID No.2, the amino acid sequence of an N-terminal signal peptide SP of baculovirus envelope glycoprotein gp64 is shown as SEQ ID No.4, and the amino acid sequence of a fusion domain TMD of baculovirus envelope glycoprotein gp64 is shown as SEQ ID No. 6.

2. The gene encoding the recombinant protein gp64-Fp30 containing the african swine fever virus antigen P30 protein according to claim 1, wherein the gene encoding the N-terminal signal peptide SP of the baculovirus envelope glycoprotein gp64 shown in seq id No.3, the gene encoding the african swine fever virus antigen P30 protein shown in seq id No.1, and the gene encoding the fusion domain TMD of the baculovirus envelope glycoprotein gp64 shown in seq id No.5 are sequentially connected.

3. The recombinant baculovirus with the surface displaying the African swine fever virus antigen P30 protein is characterized in that a recombinant expression vector pFPG-gp64-Fp30 is constructed by inserting a coding gene of the recombinant protein gp64-Fp30 in a recombinant vector pFPG, the recombinant expression vector pFPG-gp64-Fp30 is transformed into DH10Bac to obtain a recombinant Bacmid plasmid, then the recombinant Bacmid plasmid is transfected into Sf9 insect cells, and the recombinant baculovirus with the surface displaying the African swine fever virus antigen P30 protein is obtained by packaging in the Sf9 insect cells.

4. The recombinant baculovirus with surface displaying African swine fever virus antigen P30 protein as defined in claim 3, wherein the insertion site of the encoding gene of the recombinant protein gp64-Fp30 in the recombinant vector pFPG is after baculovirus polyhedrin promoter Pph.

5. The method for preparing the recombinant baculovirus with the surface displaying the African swine fever virus antigen P30 protein as defined in claim 3 or 4, comprising the following steps:

(1) The coding gene of N-terminal signal peptide SP of baculovirus envelope glycoprotein gp64, the coding gene of African swine fever virus antigen P30 protein and the coding gene of fusion domain TMD of baculovirus envelope glycoprotein gp64 are respectively amplified by PCR, cloned into a pMD19-T vector to construct recombinant plasmids T-PSP, T-Fp30 and T-TMD, and then respectively subjected to double digestion by EcoRI/SaL I, saL I/XbaI and XbaI/PstI, and cloned into the pMD19-T vector to construct recombinant plasmid T-gp64-Fp30 by using T4 DNA ligase;

(2) The recombinant plasmid T-gp64-Fp30 and the recombinant vector pFPG are subjected to double enzyme digestion by EcoRI/PstI, and then the DH10Bac competent strain is subjected to enzyme ligation and transformation, and the successfully constructed recombinant expression vector pFPG-gp64-Fp30 is obtained through blue-white spot screening and PCR verification;

(3) Transforming a recombinant expression vector pFPG-gp64-Fp30 into DH10Bac to obtain a recombinant Bacmid plasmid;

(4) And (3) transfecting the Sf9 insect cells with the recombinant Bacmid plasmid, culturing, and collecting the supernatant to obtain the recombinant baculovirus with the surface displaying the African swine fever virus antigen P30 protein.

6. Use of a recombinant baculovirus surface displaying african swine fever virus antigen P30 protein as claimed in claim 3 or 4 in the preparation of ASFV diagnostic reagents or ASF vaccines.

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