CN115850501A - African swine fever virus p30, p72 and p54 chimeric recombinant expression protein, preparation method and application thereof - Google Patents

Abstract

The invention provides an African swine fever virus p30, p72 and p54 chimeric recombinant expression protein, a preparation method and application thereof. The protein of the African swine fever virus chimeric recombinant expression is a partial fragment containing p30, p72 and p54 which play a main role in African swine fever virus infection and virus capsid formation, and each fragment selects a gene fragment with more concentrated antigen epitopes through epitope analysis software. The recombinant protein provided by the invention is a main antigen epitope partial sequence of three structural proteins p30, p72 and p54 of the chimeric expression African swine fever virus, and aims to make up for the defects that the protection power is insufficient when a single expression protein is used as an antigen to prepare a subunit vaccine, and the sensitivity and specificity of the antigen used as a diagnostic reagent to the whole virus infection process are not ideal. The p30, p54 and p72 proteins selected by the invention penetrate through the whole process of African swine fever virus infection, and have wide market application value in the field of diagnosis and prevention of African swine fever.

Description

African swine fever virus p30, p72 and p54 chimeric recombinant expression protein, preparation method and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a chimeric recombinant protein of African Swine Fever Virus (ASFV) p30, p72 and p54, a preparation method and application thereof.

Background

African Swine Fever (ASF) is a highly contagious disease of pigs caused by infection with African Swine Fever Virus (ASFV). The clinical symptoms mainly comprise fever, respiratory and nervous system dysfunction, visceral organ pathological changes and extensive bleeding. The disease course is short, the morbidity and the mortality are high, and the mortality rate of the domestic pig is close to 100% within ten days after the infection. At present, no effective commercial vaccine or medicine can prevent and treat the disease, and once the disease is discovered, the vaccine or medicine can only kill the disease on a large scale, so that serious economic loss is caused to the fields of pig breeding industry and pork trade. Therefore, the ASFV structural protein with good immunogenicity and antigenicity is screened out, which has great significance for the prevention, treatment and development of high-sensitivity and specificity detection kits of ASF.

ASFV is a positive 20-sided body, a virus that replicates within the cytoplasm of infected cells. The number of ASFV-encoded proteins is more than 200, and there are more than 50 structural proteins. Studies have reported that over 50 proteins of ASFV are packaged into virions and play a role in viral infection. The virion of ASFV is a complex multi-layered structure with a diameter of 170-190 nm (Salas et al, virus Res 2013,173,29-41.). Structural proteins encoded by ASFV are involved in genome replication and viral infection (Dixon et al, virus Res 2013,173,3-14). p54 and p30 are structural proteins involved in the invasion process of ASFV. p54 is encoded by E183L gene, has relative molecular weight of 25KDa, and is ASFV important antigen structural protein. The p54 protein is localized on virion exolipid and plays a key role in the aggregation and conversion of the plasma membrane of the omentum in host cells to virion membrane precursors (rodri guez et al, J Virol 2004, 78, 4299-4313.) and plays an important role in the growth of viruses and the induction of specific antibodies in the body of low virulent strain immunized pigs (Rodriguez et al,. Virus Res 1996, 40, 161-167). The p54 antibody is capable of inhibiting the first step of the ASFV infection cycle, virus attachment. In addition, p54 protein can induce apoptosis of infected cells (G Lo mez-Puertas et al, virus Res1997, 49, 115-122). p30 is also an early viral protein like p54, is encoded by CP204L gene, has a relative molecular weight of 30kDa, and one of the main antigen structural proteins, and participates in the cell invasion process of ASFV. (S, nchez et al, virus Res 2013,173, 58-75.). Expression of p30 protein begins 2-4 hours after invasion of cells by ASFV and throughout the entire cycle of viral infection. Thus, expression of the p30 protein marks viral entry and the onset of early viral gene expression. (Lithgow et al, vet Microbiol 2014, 168, 413-419.). The p72 is an important antigen protein coded by a B646L (VP 72) gene, the molecular weight is 73.2KDa, and the antigen protein is one of ASFV main structural proteins. p72 has high antigenicity and immunogenicity, and is the main component of ASFV 20-hedron. P72 is located on the surface of the viral capsid and plays a key role in the formation of the nucleocapsid during late expression in viral infection (Neilan et al, virology 2004, 319, 337-342.).

In view of the characteristics, the innovation point of the invention is that three main structural proteins are embedded together for one-time recombinant expression to obtain a recombinant expression antigen containing three proteins, and the three antigens p72, p30 and p54 penetrate through the whole infection process of African swine fever virus invasion and later expression, so the invention has wide application prospect in the aspects of diagnosis and prevention of African swine fever. However, no diagnostic kit and vaccine which take three proteins of African swine fever virus p72, p30 and p54 as an antigen through chimeric expression of recombinant proteins are found in the market, and the simultaneous expression of the three proteins is difficult by adopting the prior art.

Disclosure of Invention

The invention aims to provide an African Swine Fever Virus (ASFV) p30, p72 and p54 chimeric recombinant expression protein, a preparation method and application thereof, wherein the recombinant expression protein is used for carrying out tandem chimeric expression on structural proteins of the African swine fever virus p30, p72 and p 54. The invention clones and expresses the protective antigen protein of the main structural protein of the African swine fever virus and constructs an expression strain of recombinant protein with higher expression.

In order to achieve the purpose, the invention adopts the following technical scheme that:

a chimeric recombinant expression protein of African swine fever viruses p30, p72 and p54 is a protein (named as p 307254) of a structural protein fragment of the African swine fever viruses p30, p72 and p54, wherein an amino acid sequence of p30 is shown as SEQ ID No.3, an amino acid sequence of p72 is shown as SEQ ID No.4, and an amino acid sequence of p54 is shown as SEQ ID No. 5.

The chimeric recombinant expression protein of the African swine fever viruses p30, p72 and p54 is preferably a protein of which the amino acid sequence is shown in SEQ ID NO.1, or a protein which is derived from the SEQ ID NO.1 and has the same immunogenicity and the neutralizing activity with the amino acid residue sequence shown in the sequence SEQ ID NO.1 after the substitution, deletion or addition of one or more amino acid residues with the amino acid sequence shown in the SEQ ID NO. 1.

The gene of the recombinant expression protein contains nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.

A gene encoding the recombinant expression protein as described above, the nucleotide sequence of the gene being one of the following sequences:

1) A DNA sequence shown as SEQ ID NO. 2;

2) DNA sequence with over 90% homology with the DNA sequence defined by SEQ ID NO.2 and encoding the same functional protein.

Wherein, the sequence shown in SEQ ID NO.2 codes the chimeric gene sequence of the African swine fever virus p30, p72 and p54 proteins (p 307254 gene) and consists of 1182 nucleotides, wherein the bases from the 1 st to the 366 th from the 5 'end to the 3' end are the nucleotide sequence of the African swine fever virus structural protein p30, the bases from the 367 th to the 786 th are the nucleotide sequence of the African swine fever virus structural protein p72, and the bases from the 787 th to the 1182 nd are the nucleotide sequence of the African swine fever virus structural protein p 54.

An expression vector, which contains a gene sequence shown as SEQ ID NO. 2.

A cell line, which contains a gene sequence shown as SEQ ID NO. 2.

A preparation method of chimeric recombinant P30, P72 and P54 proteins of African swine fever virus comprises the following steps:

amplifying a target segment shown as SEQ ID NO. 2;

connecting the target fragment with an expression vector and transferring into escherichia coli;

screening positive clones;

inducing expression to obtain the target protein.

The preparation method as described above, preferably, the method for amplifying the target fragment is:

firstly, primers SEQ ID NO.11 and SEQ ID NO.12 are used; three pairs of primers of SEQ ID NO.13 and SEQ ID NO.14 and SEQ ID NO.15 and SEQ ID NO.16 amplify SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.8 in sequence;

secondly, amplifying by using primers SEQ ID NO.11 and SEQ ID NO.14 and using the amplified gene segments of SEQ ID NO.9 and SEQ ID NO.10 as templates to obtain a p3072 chimeric gene;

thirdly, the primers SEQ ID NO.11 and SEQ ID NO.16 are used, the amplified chimeric gene p3072 and SEQ ID NO.8 gene fragments are used as templates for further chimeric amplification to obtain the target gene fragment of the invention, and the nucleotide sequence is shown as SEQ ID NO. 2.

In the preparation method as described above, preferably, the PCR amplification product of the target fragment is purified and recovered, then digested with BamHI and SalI, and the digested fragment is purified and recovered, linked with the pET28a (+) vector digested with the same, and transferred into expression type Escherichia coli BL21 (DE 3); selecting positive, culturing, inducing expression with IPTG to obtain target protein, and purifying.

By using the method provided by the invention, higher yield of the expression product can be obtained, 15mg of recombinant protein can be obtained in each 100ml of culture solution, and the recombinant protein has better antigenicity and immunogenicity.

The invention also provides a vaccine antigen capable of effectively neutralizing African swine fever virus and an antigen for detecting African swine fever virus infection.

In order to realize the purpose, the invention adopts the following technical scheme:

a vaccine for preventing African swine fever virus infection, the active component of which is the chimeric recombinant expression protein of the African swine fever virus p30, p72 and p54, which comprises the protective antigen of the chimeric recombinant protein of ASFv p30, p72 and p 54.

In particular to a protein with an amino acid sequence shown in SEQ ID NO.1, or a protein which is obtained by substituting, deleting or adding one or more amino acid residues on the amino acid sequence shown in SEQ ID NO.1, has the same activity with the amino acid sequence shown in the sequence SEQ ID NO.1 and is derived from the SEQ ID NO. 1.

In practical application, the subunit vaccine taking the protein shown in SEQ ID NO.1 as an antigen active component is preferred.

In order to make the subunit vaccine more effective, the vaccine can also be added with an adjuvant. The adjuvant can be No.10 mineral oil adjuvant or Fowler's adjuvant or Vibrio cholerae ghost adjuvant. The invention preferably selects No.10 mineral oil adjuvant, and the antigen and the adjuvant are mixed according to the proportion of 1:3 (v/v).

The immunizing dose of the vaccine of the present invention is generally 0.5-1mg/kg body weight.

The vaccine immunized rabbit using the African swine fever virus chimeric recombinant structural protein p307254 as an active component generates a specific antibody of the African swine fever virus, so that the African swine fever virus chimeric recombinant structural protein has better immunogenicity and very wide market application prospect.

The invention also provides an antigen for detecting the African swine fever virus infection serum antibody, the active components of the antigen are the chimeric recombinant expression proteins of the African swine fever virus p30, p72 and p54, and the p307254 antigen of the amino acid sequence shown in SEQ ID NO. 1.

The invention has the beneficial effects that:

the African swine fever virus recombinant expression protein p307254 provided by the invention is an amino acid fragment with concentrated antigen epitopes for chimeric expression of three structural proteins p30, p72 and p54 of the African swine fever virus, and aims to overcome the defects that when a single expression recombinant protein is used as a vaccine antigen, the protection is insufficient or does not generate protection, and when the single expression recombinant protein is used as a diagnostic detection antigen, the sensitivity is insufficient or the specificity is low, and the like.

The chimeric recombinant expression protein prepared by the invention is obtained by integrating three proteins, namely p30, p72 and p54, on one carrier for expression, firstly has the functional characteristics of the three proteins, has very good immunogenicity and antigen activity, and has wide market application value in the field of detection and prevention of African swine fever viruses.

Compared with a single antigen preparation kit, the chimeric expression recombinant protein prepared by the invention is used as an antigen of the African swine fever diagnostic kit, so that the sensitivity and specificity are greatly improved; if the recombinant protein is used as a subunit vaccine antigen, compared with a vaccine prepared from a single protein, the chimeric recombinant antigen prepared by the invention is only immunized, and the specific antibodies aiming at the three proteins of p72, p30 and p54 generated in an immune animal body effectively improve the protection effect on the African swine fever virus; the invention uses PCR method to embed three protein genes together and then constructs recombinant expression plasmid with prokaryotic expression vector to be transformed into escherichia coli BL21 (DE 3) to obtain the embedded expression p307254 protein by induction expression, thus greatly saving the tedious repeated work required by independently expressing the three expressions, saving manpower, material resources and time cost, and being beneficial to large-scale production and application.

Drawings

FIG. 1 shows PCR amplification products of three gene fragments, p30, p72 and p54, in example 1;

FIG. 2 shows the PCR amplification product of the p30+ p72 chimeric gene of example 1, i.e., p3072;

FIG. 3 shows the PCR amplification product of the p3072+ p54 chimeric gene of example 1, i.e., p307254;

FIG. 4 is the identification chart of induced expression of recombinant p307254-pet28a/BL21 (DE 3) in example 1;

FIG. 5 shows the purification scheme of the recombinant p307254-pet28a/BL21 (DE 3) expression protein in example 1;

FIG. 6 shows the results of the antigenic characterization of recombinant p307254 in example 3.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Modifications and alterations of this invention are within the scope of this invention without departing from the spirit and nature of this invention. The genes and amino acid sequences of the recombinant proteins p30, p72 and p54 which are used for independently expressing the recombinant proteins in the comparative test of the invention are completely the same as the fragments used for the p307254 protein. In addition, the vector, host bacteria and purification method for independently expressing p30, p72 and p54 proteins are all the same as those of p307254 protein, and are not described in detail in the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and unless otherwise specified, the reagents used in the present invention are analytically pure or above.

Example 1 preparation of African swine fever Virus chimeric recombinant expression protein p307254

The invention uses a PCR method to carry out mosaic on the antigen epitope highly concentrated gene segments of African swine fever virus structural proteins p30, p72 and p54 in the sequence of p30-p72-p54 to obtain the p307254 connecting gene.

The specific amplification method is as follows:

1.1 ASFv P30, P72 template synthesis and P54 template genome extraction

According to an Anhui XCGQ strain sequence (accession number: MK 128995.1) provided by GenBank, selecting p30, p72 and p54 protein gene sequences, carrying out escherichia coli preferred codon optimization on the p30 and p72 protein gene sequences, synthesizing by using Huada gene, and cloning with a pLB carrier respectively; the p54 protein is a transmembrane protein, an intramembrane protein fragment is selected, a gene sequence is not optimized, the p54 gene amplification is carried out by taking the liver disease material genome DNA of a sick pig of a certain individual family in Henan as a template, and the DNA extraction kit (DP 304) is extracted by a disease material tissue genome DN A extraction method according to the instruction of the tissue extraction DNA kit of Tiangen Biochemical technology (Beijing) Co.

1.2 Amplification of selected fragments of the ASFv p30, p72 and p 54:

after the three proteins are analyzed by online antigen epitope prediction software (Bepipred 1.0 Server), fragments with highly concentrated antigen epitopes are selected, synthetic primers are designed, wherein the nucleotide sequence of the optimized ASFv p30 protein is shown as SEQ ID NO. 6; the excellent nucleotide sequence of the coded ASFv p72 protein is shown in SEQ ID NO.7 and is respectively used as a PCR amplification template of a p30 and a p72 target fragment. The nucleotide sequence of the protein in the membrane of the coding ASFv p54 is shown as SEQ ID NO. 8.

SEQ ID NO.6：ATGGACTTCATCCTGAACATCAGCATGAAGATGGAAGTGATCTTCAAGACCGACCTGAGGAGCTCCAGCCAGGTGGTGTTCCACGCAGGATCCCTGTACAACTGGTTCAGCGTGGAGATCATCAACTCCGGCAGGATCGTGACCACCGCCATCAAGACCCTGCTGAGCACCGTGAAGTACGACATCGTGAAGTCCGCCAGGATCTACGCAGGACAGGGATACACCGAGCACCAGGCCCAGGAGGAGTGGAACATGATCCTGCACGTGCTGTTCGAGGAGGAGACCGAGTCCAGCGCCTCCAGCGAGAACATCCACGAGAAGAACGACAACGAGACCAACGAGTGCACCTCCAGCTTCGAGACCCTGTTCGAGCAGGAGCCCTCCAGCGAGGTGCCTAAGGACTCCAAGCTGTACATGCTGGCCCAGAAGACCGTGCAGCACATCGAGCAGTACGGCAAGGCCCCAGACTTCAACAAAGTGATCAGAGCCCACAACTTCATCCAGACCATCTACGGCACCCCCCTGAAGGAGGAGGAGAAGGAGGTGGTGCGCCTGATGGTCATCAAGCTGCTGAAGAAGAAG

SEQ ID NO.7：ATGGCAAGCGGAGGAGCATTCTGCCTGATCGCAAACGACGGCAAGGCCGACAAGATCATCCTGGCCCAGGACCTGCTGAACAGCAGGATCTCCAACATCAAGAACGTGAACAAGAGCTACGGCAAGCCAGACCCCGAGCCTACCCTGTCCCAGATCGAGGAGACCCACCTGGTGCACTTCAACGCCCACTTCAAGCCATACGTGCCCGTGGGCTTCGAGTACAACAAGGTGCGGCCACACACCGGAACCCCTACCCTGGGCAACAAGCTGACCTTCGGCATCCCACAGTACGGCGACTTCTTCCACGACATGGTGGGCCACCACATCCTGGGAGCATGTCACAGCTCCTGGCAGGACGCACCAATCCAGGGCACCAGCCAGATGGGAGCACACGGACAGCTGCAGACCTTCCCAAGAAACGGCTACGACTGGGACAACCAGACCCCACTGGAGGGAGCCGTGTACACCCTGGTGGACCCTTTCGGCAGACCTATCGTGCCAGGCACCAAGAACGCCTACCGCAACCTGGTGTACTACTGCGAGTACCCTGGCGAGAGACTGTACGAGAACGTGCGCTTCGACGTGAACGGCAACTCCCTGGACGAGTACAGCTCCGACGTGACCACCCTGGTGAGAAAGTTCTGTATCCCCGGCGACAAGATGACCGGCTACAAGCACCTGGTGGGACAGGAGGTGAGCGTGGAGGGCACCTCCGGACCTCTGCTGTGCAACATCCACGACCTGCACAAGCCACACCAGAGCAAGCCCATCCTGACCGACGAGAACGACACCCAGCGCACCTGTTCCCACACCAACCCAAAGTTCCTGAGCCAGCACTTCCCCGAGAACTCCCACAACATCCAGACCGCCGGCAAGCAGGACATCACCCCCATCACCGACGCCACCTACCTGGACATCAGGCGGAACGTGCACTACAGCTGCAACGGCCCCCAGACCCCTAAGTACTACCAGCCCCCTCTGGCCCTGTGGATCAAGCTGAGGTTCTGGTTCAACGAGAACGTGAACCTGGCCATCCCTAGCGTGTCCATCCCATTCGGCGAGCGGTTCATCACCATCAAGCTGGCCTCCCAGAAGGACCTGGTGAACGAGTTCCCAGGCCTGTTCGTGAGGCAGAGCCGGTTCATCGCAGGCAGGCCCTCCAGAAGGAACATCCGGTTCAAGCCTTGGTTCATCCCAGGCGTGATCAACGAGATCAGCCTGACCAACAACGAGCTGTACATCAACAACCTGTTCGTGACCCCTGAGATCCACAACCTGTTCGTGAAGAGGGTGCGGTTCTCCCTGATCAGGGTGCACAAGACCCAGGTGACCCACACCAACAACAACCACCACGACGAGAAGCTGATGAGCGCCCTGAAGTGGCCAATCGAGTACATGTTCATCGGCCTGAAGCCTACCTGGAACATCTCCGACCAGAACCCACACCAGCACAGAGACTGGCACAAGTTCGGCCACGTGGTGAACGCCATCATGCAGCCTACCCACCACGCCGAGATCTCCTTCCAGGACAGGGACACCGCCCTGCCAGACGCCTGCAGCTCCATCAGCGACATCTCCCCCGTGACCTACCCTATCACCCTGCCAATCATCAAGAACATCAGCGTGACCGCCCACGGCATCAACCTGATCGACAAGTTCCCCTCCAAGTTCTGTAGCTCCTACATCCCTTTCCACTACGGCGGCAACGCCATCAAGACCCCAGACGACCCAGGAGCCATGATGATCACCTTCGCCCTGAAGCCCAGGGAGGAGTACCAGCCTAGCGGCCACATCAACGTGTCCAGAGCCCGCGAGTTCTACATCAGCTGGGACACCGACTACGTGGGCTCCATCACCACCGCAGACCTGGTGGTGAGCGCCTCCGCCATCAACTTCCTGCTGCTGCAGAACGGAAGCGCCGTGCTGCGGTACTCCACC

SEQ ID NO.8：TCAAGAAAGAAAAAAGCTGCTGCTATTGAGGAGGAAGATATACAGTTTATAAATCCTTATCAAGATCAGCAGTGGGTAGAAGTCACTCCACAACCAGGTACCTCTAAACCAGCTGGAGCGACTACAGCAAGTGTAGGCAAGCCAGTCACGGGCAGACCGGCAACAAACAGACCAGCAACAAACAAACCAGTTACGGACAACCCAGTTACGGACAGACTAGTCATGGCAACTGGCGGGCCGGCGGCCGCACCTGCGGCCGCGAGTGCTCCTGCTCATCCGGCTGAGCCTTACACGACAGTCACTACTCAGAACACTGCTTCACAAACAATGTCGGCTATTGAAAATTTACGACAAAGAAACACCTATACGCATAAAGACCTAGAAAACTCCTTGTAA

The virus genomes extracted from SEQ ID NO.6, SEQ ID NO.7 and 1.1 are taken as templates respectively, and specific primers are used for amplifying target fragments of P30, P72 and P54 (the corresponding gene sequences are SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO. 8).

Amplifying and embedding the amplified target gene sequence according to the sequence of p30-p72-p54 by using a PCR method, analyzing enzyme cutting sites by using DANMAN, and respectively adding BamHI and SalI restriction enzyme cutting sites into a first primer and a sixth primer, wherein the sequences of the primers are as follows:

SEQ ID NO.11:5’-ATAATTGGATCCGACATCGTGAAGTCCGC-3’；

SEQ ID NO.12:5’-TTCTCGTACAGTCTCTCcacctccttctcctcct-3’；

SEQ ID NO.13:5’-aggaggagaaggaggtgGAGAGACTGTACGAGAA-3’；

SEQ ID NO.14:5‘-GCTTTTTTCTTTCTTGActggtagtacttagggg-3’；

SEQ ID NO.15:5‘-cccctaagtactaccagTCAAGAAAGAAAAAAGC-3’；

SEQ ID NO.16:5’-TTAGTCGACTTACAAGGAGTTTTCCA-3’；

the first step is as follows: using an optimally synthesized p30/plB plasmid as a template, amplifying a gene fragment selected by p30 by using two primers of SEQ ID NO.11 and SEQ ID NO.12, wherein the total length of the gene is 366bp, cutting the gel and recovering a target fragment for later use, and the gene sequence is shown as a sequence SEQ ID NO. 9; the amino acid sequence of the corresponding coded protein is shown as SEQ ID NO.3,

SEQ ID NO.9：GACATCGTGAAGTCCGCCAGGATCTACGCAGGACAGGGATACACCGAGCACCAGGCCCAGGAGGAGTGGAACATGATCCTGCACGTGCTGTTCGAGGAGGAGACCGAGTCCAGCGCCTCCAGCGAGAACATCCACGAGAAGAACGACAACGAGACCAACGAGTGCACCTCCAGCTTCGAGACCCTGTTCGAGCAGGAGCCCTCCAGCGAGGTGCCTAAGGACTCCAAGCTGTACATGCTGGCCCAGAAGACCGTGCAGCACATCGAGCAGTACGGCAAGGCCCCAGACTTCAACAAAGTGATCAGAGCCCACAACTTCATCCAGACCATCTACGGCACCCCCCTGAAGGAGGAGGAGAAGGAGGTG

SEQ ID NO.3：DIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEV

the second step is that: using optimized synthesized p72/pLB as a template, amplifying a gene fragment selected from p72 by using two primers of SEQ ID NO.13 and SEQ ID NO.14, wherein the total length of the gene is 420bp, cutting gel and recovering a target fragment for later use, and the gene sequence is shown as a sequence SEQ ID NO. 10; the amino acid sequence of the corresponding coded protein is shown as SEQ ID NO.4,

SEQ ID NO.10：GAGAGACTGTACGAGAACGTGCGCTTCGACGTGAACGGCAACTCCCTGGACGAGTACAGCTCCGACGTGACCACCCTGGTGAGAAAGTTCTGTATCCCCGGCGACAAGATGACCGGCTACAAGCACCTGGTGGGACAGGAGGTGAGCGTGGAGGGCACCTCCGGACCTCTGCTGTGCAACATCCACGACCTGCACAAGCCACACCAGAGCAAGCCCATCCTGACCGACGAGAACGACACCCAGCGCACCTGTTCCCACACCAACCCAAAGTTCCTGAGCCAGCACTTCCCCGAGAACTCCCACAACATCCAGACCGCCGGCAAGCAGGACATCACCCCCATCACCGACGCCACCTACCTGGACATCAGGCGGAACGTGCACTACAGCTGCAACGGCCCCCAGACCCCTAAGTACTACCAG；SEQ ID NO.4：ERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQ

the third step: using the genome DNA extracted from 1.1 as a template, amplifying an intramembrane protein fragment selected by p54 by using two primers of SEQ ID NO.15 and SEQ ID NO.16, wherein the total length of the gene is 396bp, cutting the gel, and recovering a target fragment for later use, wherein the gene sequence is shown as SEQ ID NO.8; the amino acid sequence of the corresponding coded protein is shown as SEQ ID NO.5,

SEQ ID NO.5：SRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSL

the fourth step: using 1 microliter of each of p30 and p72 (shown in SEQ ID NO.9 and SEQ ID NO. 10) recovered by amplified gel cutting as a template, and using two primers of SEQ ID NO.11 and SEQ ID NO.14 to amplify to obtain a p3072 chimeric gene, wherein the total length of the gene is 786bp, and the gel cutting is recovered for later use;

the fifth step: taking 1 microliter of each of p3072 obtained by the fourth step of amplification and p54 (with a sequence shown as SEQ ID NO. 8) obtained by the third step of amplification as a template, and using two primers of SEQ ID NO.11 and SEQ ID NO.16 to embed the p3072 and the p54 to obtain a p307254 gene fragment with the total length of 1182bp, wherein the gene sequence is shown as a sequence SEQ ID NO. 2; the amino acid sequence of the corresponding encoded protein is shown in SEQ ID NO. 1.

SEQ ID NO.2：GACATCGTGAAGTCCGCCAGGATCTACGCAGGACAGGGATACACCGAGCACCAGGCCCAGGAGGAGTGGAACATGATCCTGCACGTGCTGTTCGAGGAGGAGACCGAGTCCAGCGCCTCCAGCGAGAACATCCACGAGAAGAACGACAACGAGACCAACGAGTGCACCTCCAGCTTCGAGACCCTGTTCGAGCAGGAGCCCTCCAGCGAGGTGCCTAAGGACTCCAAGCTGTACATGCTGGCCCAGAAGACCGTGCAGCACATCGAGCAGTACGGCAAGGCCCCAGACTTCAACAAAGTGATCAGAGCCCACAACTTCATCCAGACCATCTACGGCACCCCCCTGAAGGAGGAGGAGAAGGAGGTGGAGAGACTGTACGAGAACGTGCGCTTCGACGTGAACGGCAACTCCCTGGACGAGTACAGCTCCGACGTGACCACCCTGGTGAGAAAGTTCTGTATCCCCGGCGACAAGATGACCGGCTACAAGCACCTGGTGGGACAGGAGGTGAGCGTGGAGGGCACCTCCGGACCTCTGCTGTGCAACATCCACGACCTGCACAAGCCACACCAGAGCAAGCCCATCCTGACCGACGAGAACGACACCCAGCGCACCTGTTCCCACACCAACCCAAAGTTCCTGAGCCAGCACTTCCCCGAGAACTCCCACAACATCCAGACCGCCGGCAAGCAGGACATCACCCCCATCACCGACGCCACCTACCTGGACATCAGGCGGAACGTGCACTACAGCTGCAACGGCCCCCAGACCCCTAAGTACTACCAGTCAAGAAAGAAAAAAGCTGCTGCTATTGAGGAGGAAGATATACAGTTTATAAATCCTTATCAAGATCAGCAGTGGGTAGAAGTCACTCCACAACCAGGTACCTCTAAACCAGCTGGAGCGACTACAGCAAGTGTAGGCAAGCCAGTCACGGGCAGACCGGCAACAAACAGACCAGCAACAAACAAACCAGTTACGGACAACCCAGTTACGGACAGACTAGTCATGGCAACTGGCGGGCCGGCGGCCGCACCTGCGGCCGCGAGTGCTCCTGCTCATCCGGCTGAGCCTTACACGACAGTCACTACTCAGAACACTGCTTCACAAACAATGTCGGCTATTGAAAATTTACGACAAAGAAACACCTATACGCATAAAGACCTAGAAAACTCCTTGTAA

SEQ ID NO.1：DIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQSRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSL

The PCR reaction system of each step is 50 mu L, pfu high fidelity enzyme of Biochemical (Shanghai) Co., ltd is used for amplification, and the PCR amplification adopts a general method which is not described again. The PCR products obtained in each step are identified by 1 percent DNA gel, the result is shown in figure 1-3, figure 1 is PCR amplification products of three gene fragments of p30, p72 and p54, wherein, a band 1 is a p72 gene fragment with the size of 420bp; the band 2 is a p30 gene fragment with the size of 366bp; the band 3 is a p54 gene fragment with the size of 396bp; m: DNA molecular weight standard (100-2000 bp). FIG. 2 shows the PCR amplification product of p30+ p72 chimeric gene, i.e., p3072; wherein the band 1 is the gene p3072, and the size is 786bp; m is DNA molecular weight standard (100-2000 bp). FIG. 3 shows the PCR amplification product of p3072+ p54 chimeric gene (SEQ ID NO. 2), i.e., p307254; in the figure, the strip 1: p307254 gene, size 1182bp; m is DNA molecular weight standard (100-2000 bp).

Then, agarose gel DNA recovery kit (DP 209) from Tiangen Biochemical technology (Beijing) Ltd was used for gel cutting and recovery.

1.3 Construction of p307254-pet28a recombinant expression plasmid:

recovering 32 mu L of the p307254 gene fragment obtained in the fifth step in 1.2, adding 2 mu L of each of BamHI and SalI enzyme (Takara restriction enzyme), 4 mu L of 10 XHbuffer, the total volume of which is 40 mu L, placing the obtained mixture in a 37 ℃ enzyme digestion reaction for 5 hours, purifying and recovering the enzyme digestion product, mixing the enzyme-digested p307254 gene fragment with a pET28a (+) vector which is digested by BamHI and SalI according to a 4.3 (V/V) method, performing 25 ℃ reaction and ligation for 30 minutes by using a Beijing Limited company T4 enzyme quick ligation method, transferring 10 mu L of the ligation product into escherichia coli BL21 (DE 3) competent cells, placing the cells in a 37 ℃ shaker for 1 hour, culturing the cells at 130rpm for 1 hour, then sucking 100 mu L of the cells under aseptic conditions, coating a solid LB medium containing 25 mu g/mL of the ligation products, placing the cells in a 37 ℃ sequencing culture medium for 14-18 hours, picking out a plurality of single primers, extracting a plurality of SEQ ID and PCR, and identifying the colonies which are positive plasmids containing the kanamycin, wherein the PCR strains, the PCR is the positive plasmid 3763, and the bacterial colony which is the PCR is the strain is identified as the positive plasmid containing the kanamycin 3716.

1.4 inducible expression, purification and quantitative subpackaging of the recombinant plasmid p307254/pet28 a:

the first step is as follows: and (3) identifying whether the positive escherichia coli BL21 (DE 3) strain containing the recombinant expression plasmid p307254/pet28 obtained in the step 1.3 can express the target protein. The method comprises the following steps: IPTG (final concentration of 0.5 mM) induction was carried out on 1 ml of p307254/pet28a/BL21 (DE 3)/pet 28a bacteria and BL21 (DE 3)/pet 28a bacteria transformed with empty vector pet28a, respectively, wherein BL21 (DE 3) empty bacteria and BL21 (DE 3)/pet 28a transformed with empty vector pet28a were used as controls. The results are shown in FIG. 4, where 1: BL21 (DE 3) empty bacterium induced expression; 2: pet28a/BL21 (DE 3) empty vector induction expression; 3: p307254-pet28a/BL21 (DE 3) induced expression, theoretical size 47.5KDa; m: protein molecular weight standards (14.4, 18.4, 25, 35, 45, 66.2, 116 KDa). The result shows that 307254/pet28a/BL21 (DE 3) induced bacteria have a thicker expression band in the middle of 45-66.2 KDa which is consistent with the size of the target recombinant protein p307254/pet28a, and both the empty vector pet28a/BL21 (DE 3) and the empty vector BL21 (DE 3) have no expression band which is consistent with the size of the target protein, which indicates that the target gene recombinant expression plasmid p307254/pet28a is successfully expressed in the escherichia coli BL21 (DE 3), i.e. the p307254/pet28a/BL21 (DE 3) positive expression bacteria are successfully obtained.

The second step is that: adding the positive p307254/pet28a/BL21 (DE 3) bacterial liquid into LB liquid culture medium containing 25g/mL kanamycin according to the proportion of 1:100, shaking and culturing at 37 ℃ and 250rpm for 2 hours until the bacterial liquid OD600=0.5-0.7, adding IPTG to the final concentration of 0.5mM, continuing culturing for 4 hours, centrifuging, pouring off the supernatant, and collecting bacterial precipitates.

The third step: the thalli precipitation is suspended in 1/10 volume of phosphate buffer solution (0.01M, pH7.2-7.4 PBS) of which the thalli precipitation is obtained by centrifuging 200 ml of bacterial liquid, is suspended in 20ml of PBS, only the volume of the suspended bacterial solution is calculated, and LB is not used), the thalli precipitation is suspended in the phosphate buffer solution (0.01M, pH7.2-7.4 PBS), the thalli cells are ultrasonically broken, the working is performed for 5 seconds and is interrupted for 10 seconds, the ultrasonic frequency is 40 times, the power is 300-400 w, the broken thalli solution is centrifuged at 6000rpm for 15min, the supernatant is discarded, and the precipitate is collected.

The fourth step: repeating the second step for 2 times, and collecting the precipitate to obtain the inclusion body. (after two times of ultrasonic centrifugation, the supernatant is discarded after SDS-PAGE confirms that the supernatant does not contain the target protein).

The fifth step: the inclusion bodies obtained were washed with 10mL of a buffer containing 8M urea (NaH) ₂ PO ₄ 0.1M, tris-HCl pH8.0, 0.01M), working for 5 seconds with 10 seconds interval and power of 150-200 w, performing ultrasonic treatment for 20 times, centrifuging at 10000rpm for 15min, and collecting supernatant, i.e. denatured p307254 protein.

The fifth step: centrifuging the obtained denatured and cracked p307254 protein solution at 10000rpm for 15 minutes to obtain supernatant which is buffer solution containing recombinant protein p307254, gently sucking the collected supernatant solution, filling the supernatant solution into a dialysis bag (molecular weight cut-off of 3 KDa), dialyzing the supernatant solution for 6 hours in renaturation solution (100 mM Tris-HCl,0.004M oxidized glutathione and 0.002M reduced glutathione) containing 4M urea, sequentially transferring the supernatant solution into renaturation solution containing 2M urea for dialysis for 16 hours and dialyzing the renaturation solution containing 1M urea for 10 hours, finally transferring the supernatant solution into 0.1M PBS buffer solution for dialysis for 12 to 16 hours, and replacing the buffer solution for 2 times in the period. After dialysis, the solution in the dialysis bag was filled into a clean centrifuge tube for further purification.

And a sixth step: mixing the renatured p307254 protein with NI-NTA agarose column, slowly shaking the mixture on ice for 2 hours, purifying, and analyzing the purified p307254 protein with the purity of more than 90% by SDS-PAGE. The results are shown in FIG. 5, where lane 1: the recombinant p307254-pet28a/BL21 (DE 3) purified product has a theoretical size of 47.5KDa; strip 2: protein molecular weight standards (14.4, 18.4, 25, 35, 45, 66.2, 116 KDa).

The seventh step: the purified p307254 protein is quantified, packaged in 100 ug/tube, freeze-dried, and stored at-20 deg.C.

Example 2 immunization evaluation of rabbits with a vaccine comprising the recombinant p307254 protein of the present invention as an active ingredient

Firstly, the African swine fever virus recombinant p307254 protein obtained in example 1 is diluted to 0.5mg/ml and then coated with ELIAS plate, and the serum titer is detected by ELISA method.

6 negative big-ear white rabbits (No. 1-3) with serum OD450 value less than 0.1 were selected and randomly divided into experimental groups (No. 4-6) and control groups (No. 4-6). The big ear white rabbit is 30 days old and male, the weight is approximately 1.8-2 kg/rabbit, and the weight is provided by the experimental animal center of Chinese agriculture university. The experimental group is immunized by the African swine fever virus recombinant p307254 protein, the freeze-dried powder recombinant p307254 protein is dissolved by PBS (0.01M, pH7.2-7.4) and is fully stirred and uniformly mixed with equal volume of Freund's adjuvant to prepare latex solution, 3 rabbits are injected at multiple points on the neck under the skin, each rabbit is added with 1 needle at the same dosage and position every 200 mug/kg body weight at intervals of 14 days, 1 needle is added at 28 days, 3 rabbits in big ear white are injected in a control group, only PBS and the Freund's adjuvant latex solution are injected (the injection volume is the same as the injection volume of the experimental group), and the immunization is carried out for 3 times. Collecting rabbit serum at 28 days and 35 days in the whole immunization process, detecting the titer of serum antibody, and comparing the titer of the rabbit serum of the big ear white rabbit of the experimental group and the control group.

(II) preparing materials:

coating antigen: recombinant p307254 protein prepared in example 1.

Serum to be detected: experimental group 3 rabbit sera and control group 3 rabbit sera.

Sealing liquid: PBST with 5% skim milk powder.

Secondary antibody: horse radish peroxidase labeled goat anti-rabbit polyclonal antiserum.

(III) the ELISA method for detecting the antibody titer of the African swine fever virus by using the P307254 antigen:

1. preparation of coating antigen: diluting to 5 μ g/mL with carbonate buffer (pH9.6) for use;

2. coating ELISA plate: adding the diluted recombinant p307254 protein solution with the concentration of 5 mu g/mL into an ELISA plate, wherein the concentration is 100 mu L/hole, and the temperature is 4-8 ℃ for 12-16 hours;

3. washing the ELISA plate: taking out the plate coated with the antigen from a refrigerator at 4-8 ℃, pouring out the antigen solution coated in the hole, filling and washing the plate with PBST (PBST is 0.01M containing 0.5 per mill of Tween-20 and PBS solution with pH value of 7.2-7.4) for 3 times and 3 min/time, and patting the plate dry;

4. blocking the ELISA plate: adding a sealing solution (PBST solution containing 0.5% skimmed milk powder) into each hole of the ELISA plate, placing 200 mu L of the solution into each hole, and reacting at a constant temperature of 37 ℃ for 2 hours;

5. washing the ELISA plate: repeating the step 3;

6. adding a primary antibody: adding the diluted serum to be detected (diluted by PBST) with the proportion of 1;

7. washing the ELISA plate: pour off primary antibody in well, wash 5 times with PBST, 3 min/time, pat dry ELISA plate;

8. adding a secondary antibody: 100 mu L/hole of HRP-labeled goat anti-rabbit serum diluted by 1;

9. washing the ELISA plate: repeating the step 7;

10. adding a substrate: adding TMB solution into each hole of the ELISA plate, keeping the temperature constant at 37 ℃ at 100 mu L/hole, and developing in a dark place for 13-15 min;

11. adding a stop solution: adding 2M hydrochloric acid solution into each hole of the ELISA plate, and slightly shaking and uniformly mixing liquid in the holes, wherein each hole is 100 mu L/hole;

12. reading OD450 values: the ELISA plate was placed on a microplate reader and the value read at 450 wavelengths, i.e. OD450 values.

The results are shown in table 1, and the detection results show that the OD450 values of the control group rabbit sera after the second and third immunizations are less than 0.1 after 2 times, and the OD450 values of the experimental group rabbit sera after the second and third immunizations are more than 1.5 after 2 times. The recombinant p307254 protein can induce and generate a neutralizing antibody in a rabbit body, and can activate humoral immune response.

TABLE 1OD450 values

Example 3 detection of antigenicity comparing African swine fever Virus recombinant p307254 with p30, p72 and p54 recombinant proteins expressed separately by dot ELISA method

Protein sample: purified and quantified African swine fever virus recombinant p307254, p30, p72 and p54 recombinant proteins.

Antigen preparation: the purified p307254 recombinant protein of example 1 and the purified p30, p72 and p54 recombinant proteins expressed alone were diluted to 10 μ g/mL with carbonate buffer (0.05 mol/L, ph 9.6) and sufficiently dissolved for 1% bsa: prepared with carbonate buffer (0.05 mol/L, pH9.6) for use.

An antiserum: standard positive serum for african swine fever (purchased from chinese veterinary medicine institute); negative control sera were normal swine sera not immunized and not infected with African swine fever virus.

Spot ELISA protocol:

1. nitrocellulose membrane (NC) antigen coating: the NC membrane with a pore size of 0.45 μm was taken out, the NC membrane with an appropriate size was cut out, 10 squares were drawn with a pencil on the cut NC membrane, 2 μ L of each spot in 8 squares, and diluted p307254, p30, p72, and p54 antigen (10 μ g/mL), that is, 20ng of antigen content per spot, 2 μ L of each spot in 2 additional squares 1% BSA. Placing the NC membrane in a box paved with damp gauze, covering the box with a cover, and incubating for 12-16 hours at 4-8 ℃.

2. And (3) washing the NC membrane, namely taking out the coated NC membrane, putting 10 mL-20 mL of PBST (0.01M PBS buffer solution with 0.5 thousandth of Tween-20 and pH value of 7.2-7.4) into the coated NC membrane, putting the coated NC membrane on a shaking table, washing for 5min, and replacing the washing liquid. Repeat 3 times.

3. Sealing NC membrane, the washed NC membrane is put into a prepared sealing liquid (PBST solution containing 5% of skimmed milk powder) and sealed for 2 hours at 37 ℃.

3. Washing the NC film: the same as the step 2.

4. Adding a primary antibody: cutting 10 antigen points of an NC membrane into 2 (each containing 5 antigen points), respectively putting the two NC membranes into a standard positive serum and a negative control (normal pig serum) serum solution (both diluted by PBST containing 5% of skimmed milk powder) of African swine fever diluted by a ratio of 1.

5. Washing an NC membrane: discard the primary antibody, wash the membrane with PBST, 5min × 4 times.

6. Adding a secondary antibody: 2 NC membranes were added to a solution of horseradish peroxidase-conjugated rabbit anti-porcine secondary antibody (diluted 1:4000 with 0.01M PBS pH 7.2-7.4) and shaken gently for 1 hour at room temperature.

7. Washing an NC membrane: discard the secondary antibody, wash the membrane with PBST, 5min × 4 times.

8. DAB color development: and (3) putting the membrane into a prepared DAB color development solution, performing color development in a dark place until light brown spots appear, continuing to perform color development for 2min, taking out the NC membrane, and transferring the NC membrane into double distilled water to terminate the reaction.

The results are shown in FIG. 6, in which A is the result of normal swine serum without immunization and infection with African swine fever virus, B is the result of standard positive serum for African swine fever, 1: p307254-pet28a/BL21 (DE 3) recombinant protein; 2: p30-pet28a/BL21 (DE 3) recombinant protein; 3: p72-pet28a/BL21 (DE 3) recombinant protein 4 BSA;5: p54-pet28a/BL21 (DE 3) recombinant protein.

The result shows that the p307254 antigen has dark brown spots when reacting with the African swine fever standard positive serum, and does not have color development with the negative control (normal pig serum); the p30 and p72 independently express antigens and the African swine fever virus positive serum and negative serum have no macroscopic color development; the p54 independently expressed antigen reacts with African swine fever positive serum to generate light brown spots which can be seen by naked eyes, and the light brown spots and the negative serum do not generate color development; 1% BSA showed no color reaction with both African swine fever positive and negative swine sera.

And (4) conclusion: according to the results of the dot ELISA experiments, the p30 and p72 proteins expressed separately have no antigen activity with the African swine fever virus positive serum, and the p54 expressed separately has weak antigenicity from the color development result although reacting with the African swine fever virus positive serum. The recombinant protein p307254 obtained by chimeric recombinant expression of p30, p72 and p54 has strong chromogenic reaction after reacting with African swine fever positive serum, which shows that the antigenicity of the p307254 chimeric expression recombinant protein is obviously better than that of the individually expressed recombinant protein of p30, p72 and p54, and simultaneously, the p307254 chimeric expression recombinant protein has no chromogenic reaction with African swine fever negative serum, which shows that the specificity of the p307254 chimeric expression recombinant protein is also good.

Example 4 detection of African swine fever serum antibody by African swine fever virus chimeric recombinant protein p307254 prepared by the invention and indirect ELISA method of recombinant protein antigens independently expressing p30, p72 and p54

1. Preparation of coating antigen: (the independently expressed p30, p72 and p54 protein antigen genes, amino acid sequences and expression vectors used in the invention are completely the same as those of p307254, are made by the laboratory, and the method and the steps are not described in detail). The purified recombinant protein of African swine fever virus p307254, separately expressed p30, p72 and p54 recombinant proteins were diluted to 5. Mu.g/mL with coated carbonate buffer (0.05M, pH 9.6).

2. Coating ELISA reaction plate: the diluted p307254, p30, p72 and p54 antigen solutions are added into an ELISA reaction plate, 100 mu L/hole is placed at 4-8 ℃ for 14-16 hours, and each antigen is coated on the whole plate.

3. Washing the ELISA reaction plate: the reaction plate was removed and washed 5 times for 3 minutes each with PBST (0.01M, pH 7.2-7.4, PBS containing 0.5% o Tween-20).

4. Blocking the ELISA reaction plate: PBST (200. Mu.L/well) containing 5% skim milk powder was added to each well of the reaction plate, and the plate was sealed at 37 ℃ for 2 hours.

5. Washing the ELISA reaction plate: the same step 36, primary antibody reaction: PBST was used to dilute 1.

7. Washing the ELISA reaction plate: the same as step 3.

8. Secondary antibody reaction: the ELISA reaction plate was incubated with HRP-labeled rabbit anti-porcine secondary antibody diluted in advance at 1:5000 (0.01M, pH 7.2-7.4 PBS) per well at 100. Mu.L/well for 30 minutes at 37 ℃.

9. Washing the ELISA reaction plate: the same as step 3.

10. And (3) color development reaction: the ELISA reaction plate was incubated with 100. Mu.L/well of TMB substrate solution at 37 ℃ for 10-15 min.

11. And (4) terminating: A2N hydrochloric acid solution (100. Mu.L/well) was added to the ELISA plate, and the plate was gently shaken and mixed well.

11. Read OD ₄₅₀ The value: the ELISA reaction plate is placed in a plate groove of an enzyme-linked immunosorbent assay (ELISA), and each hole OD is read ₄₅₀ The value is obtained.

12. As a result: see table 2.

TABLE 2

As can be seen from the results in Table 2, when the separately expressed P30, P72 and P54 proteins are used as ELISA antigens to detect positive and negative sera respectively, the P/N value is lower than that of the recombinant protein P307254 which is expressed by three proteins which are combined together as an antigen (38.7), which indicates that the chimeric recombinant protein P307254 prepared by the invention has higher antigenicity than the singly expressed recombinant protein. The p30 and p54 selected by the invention play a crucial role in the invasion of the African swine fever virus into cells, and can generate corresponding neutralizing antibodies in the early stage of virus infection and throughout the whole infection process. P72 is the formation of the viral shell late in viral infection. The chimeric recombinant expression protein p307254 prepared by the invention integrates three protein characteristics, and effectively makes up for the immune failure caused by single antibody or even incapability of activating to generate the antibody when the single protein is used as a vaccine antigen. On the other hand, when the p307254 is used as an African swine fever virus diagnosis antigen, compared with the single antigens of p30, p72 and p54, the sensitivity and specificity of the African swine fever virus antibody are greatly improved, and the African swine fever virus diagnosis antigen has important value for African swine fever diagnosis, especially for early detection of virus infection.

The method selects three structural proteins p30, p72 and p54 epitope concentrated fragments of African swine fever virus, connects the three genes together by a PCR method, constructs a prokaryotic expression vector, and prepares the p307254 chimeric recombinant protein by induced expression. The prepared p307254 protein is shown in examples 3 and 4, and is compared with recombinant proteins which independently express p30, p72 and p54, so that the antigenicity of the p307254 protein to the African swine fever virus positive serum is obviously higher than that of the three proteins which independently express p30, p72 and p54, and the defects that the antigen sensitivity and specificity of the existing detection reagent in the market are not ideal and the like are overcome. In addition, the p307254 chimeric recombinant protein is an expression product obtained by linking together at the gene level, so that the three proteins have respective functional characteristics, even the immunogenicity and antigenicity are mutually enhanced to a certain extent, and the P307254 chimeric recombinant protein has an unexpected application prospect in developing African swine fever virus subunit vaccines and detection reagents.

Claims (10)

1. A chimeric recombinant expression protein of African swine fever viruses p30, p72 and p54 is characterized in that the protein is a protein of a structural protein fragment of the chimeric expression African swine fever viruses p30, p72 and p 54; wherein, the amino acid sequence of p30 is shown as SEQ ID NO.3, the amino acid sequence of p72 is shown as SEQ ID NO.4, and the amino acid sequence of p54 is shown as SEQ ID NO. 5.

2. The recombinant expression protein of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID NO.1,

or a protein which is derived from the SEQ ID NO.1 and has the same activity with the amino acid residue sequence shown in the sequence SEQ ID NO.1 after the substitution, deletion or addition of one or more amino acid residues with the amino acid sequence shown in the sequence SEQ ID NO. 1.

3. Gene coding for a recombinant expression protein according to claim 1, characterized in that it is capable of chimerizing the genes of recombinant p30, p72 and p54 proteins, which contain the nucleotide sequences shown in SEQ ID No.9, SEQ ID No.10 and SEQ ID No. 8.

4. The gene encoding the recombinantly expressed protein of claim 1, wherein the nucleotide sequence of the gene is one of the following:

1) A DNA sequence shown as SEQ ID NO. 2;

2) DNA sequence with over 90% homology with the DNA sequence defined by SEQ ID NO.2 and encoding the same functional protein.

5. An expression vector, which contains a sequence shown as SEQ ID NO. 2.

6. A cell line comprising the sequence shown in SEQ ID No. 2.

7. A preparation method of chimeric recombinant expression African swine fever virus p30, p72 and p54 proteins comprises the following steps:

amplifying a target segment shown as SEQ ID NO. 2;

connecting the target fragment with an expression vector and transferring into escherichia coli;

screening positive clones;

inducing expression to obtain the target protein.

8. The method of claim 7, wherein the target fragment is amplified by: firstly, primers SEQ ID NO.11 and SEQ ID NO.12 are used; three pairs of primers of SEQ ID NO.13 and SEQ ID NO.14 and SEQ ID NO.15 and SEQ ID NO.16 amplify SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.8 in sequence;

secondly, amplifying by using primers SEQ ID NO.11 and SEQ ID NO.14 and using the amplified gene segments of SEQ ID NO.9 and SEQ ID NO.10 as templates to obtain a p3072 chimeric gene;

thirdly, primers of SEQ ID NO.11 and SEQ ID NO.16 are used, and the amplified chimeric gene p3072 and the amplified gene fragment of SEQ ID NO.8 are used as templates for further chimeric amplification to obtain a target fragment with the nucleotide sequence shown in SEQ ID NO. 2.

9. A vaccine for preventing African swine fever virus infection, which is characterized in that the active component is the chimeric recombinant expression protein of the African swine fever virus p30, p72 and p54 of claim 1, and the chimeric recombinant expression protein is a protective antigen consisting of the p30, p72 and p54 proteins.

10. An antigen for detecting serum antibodies of African swine fever virus infection, which is characterized in that the antigen component is the chimeric recombinant expression protein of the African swine fever virus p30, p72 and p54 of claim 1.

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