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

Abstract

The present invention provides a chimeric recombinant expression protein of African swine fever virus p30, p72 and p54, and its preparation method and application. The protein expressed by the chimeric recombinant expression of African swine fever virus comprises partial fragments of p30, p72 and p54, which play a major role in African swine fever virus infection and viral capsid formation. Each fragment is subjected to epitope analysis software to select a gene fragment with a relatively concentrated antigenic epitope. The recombinant protein provided by the present invention is a chimeric expression of the main antigenic epitope partial sequence of the three structural proteins p30, p72 and p54 of African swine fever virus, which is intended to make up for the shortcomings of insufficient protection when a single expressed protein is used as an antigen to prepare a subunit vaccine, and unsatisfactory sensitivity and specificity as a diagnostic reagent antigen for the entire process of viral infection. The p30, p54 and p72 proteins selected by the present invention run through the entire process of African swine fever virus infection and have broad market application value in the field of diagnosis and prevention of African swine fever.

Description

Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology, and specifically relates to African swine fever virus (ASFV) p30, p72 and p54 chimeric recombinant proteins, and a preparation method and application thereof. Background Art

[0002] African swine fever (ASF) is a highly contagious disease of pigs caused by infection with the African swine fever virus (ASFV). Clinical symptoms primarily include fever, respiratory and nervous system dysfunction, visceral organ lesions, and extensive bleeding. The disease course is short, with high morbidity and mortality rates, approaching 100% within ten days of infection in domestic pigs. Currently, there are no effective commercial vaccines or drugs to prevent or treat the disease. Once detected, large-scale culling is necessary, resulting in severe economic losses for the pig farming industry and pork trade. Therefore, identifying ASFV structural proteins with excellent immunogenicity and antigenicity is of great significance for the prevention and treatment of ASF, as well as for the development of highly sensitive and specific detection kits.

[0003] ASFV is a regular icosahedron that replicates within the cytoplasm of infected cells. ASFV encodes over 200 proteins, including over 50 structural proteins. Studies have reported that these 50 ASFV proteins are packaged into virions and play a role in viral infection. ASFV virions are complex, multilayered structures with a diameter of 170–190 nm (Salas et al., Virus Res 2013, 173, 29–41). ASFV-encoded structural proteins 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 ASFV entry. p54, encoded by the E183L gene, has a molecular weight of 25 kDa and is a key antigenic structural protein of ASFV. The p54 protein, located on the outer lipid envelope of the virion, plays a key role in host cell endoplasmic reticulum membrane aggregation and conversion to the viral envelope precursor (Rodriguez et al., J Virol 2004, 78, 4299–4313). It also plays a crucial role in virus growth and the induction of specific antibodies in pigs immunized with attenuated strains (Rodriguez et al., Virus Res 1996, 40, 161–167). Antibodies against p54 can inhibit viral attachment, the first step in the ASFV infection cycle. Furthermore, p54 can induce apoptosis in infected cells (Gómez-Puertas et al., Virus Res 1997, 49, 115–122). Like p54, p30 is an early viral protein encoded by the CP204L gene. With a relative molecular mass of 30 kDa, it is one of the major antigenic structural proteins involved in ASFV entry into cells. (Sánchez et al., Virus Res 2013, 173, 58–75.) ASFV begins expressing the p30 protein 2–4 hours after entering cells and continues throughout the viral infection cycle. Therefore, p30 protein expression marks the onset of viral entry and early viral gene expression. (Lithgow et al., Vet Microbiol 2014, 168, 413–419.) p72, encoded by the B646L (VP72) gene, is a key antigenic protein with a molecular weight of 73.2 kDa and is one of the major structural proteins of ASFV. p72 has high antigenicity and immunogenicity and is a major component of the ASFV icosahedron. P72 is located on the surface of the viral capsid and plays a key role in nucleocapsid formation during late viral infection (Neilan et al., Virology 2004, 319, 337–342.).

[0004] In view of the above-mentioned characteristics, the innovation of the present invention lies in the simultaneous recombinant expression of the three main structural proteins, resulting in a recombinant antigen containing the three proteins. Since the three antigens p72, p30, and p54 are present throughout the entire infection process of the African swine fever virus, from invasion to late expression, they have broad application prospects in the diagnosis and prevention of African swine fever. However, currently, no diagnostic kits or vaccines using the recombinant proteins of the three ASF virus proteins p72, p30, and p54 as chimeric antigens have been found on the market, as the simultaneous expression of the three proteins using existing technologies is difficult. Summary of the Invention

[0005] The present invention aims to provide a chimeric recombinant protein expressing p30, p72, and p54 of African swine fever virus (ASFV), as well as its preparation method and application. The recombinant protein tandemly expresses the structural proteins of ASFV p30, p72, and p54. The present invention clones and expresses the protective antigen protein, a major structural protein of ASFV, and constructs an expression strain that expresses the recombinant protein at a high level.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A chimeric recombinant expression protein of African swine fever virus p30, p72 and p54, which is a protein (named p307254) that chimerally expresses structural protein fragments of African swine fever virus p30, p72 and p54, wherein the amino acid sequence of p30 is shown in SEQ ID NO.3, the amino acid sequence of p72 is shown in SEQ ID NO.4, and the amino acid sequence of p54 is shown in SEQ ID NO.5.

[0008] The chimeric recombinant expression proteins of African swine fever virus p30, p72 and p54 as described above, preferably, have an amino acid sequence as shown in SEQ ID NO.1, or are proteins derived from SEQ ID NO.1, which have undergone one or more amino acid residue substitutions, deletions or additions with the amino acid sequence shown in SEQ ID NO.1 and have the same immunogenicity and neutralizing activity as the amino acid residue sequence shown in SEQ ID NO.1.

[0009] The gene encoding the recombinant expression protein described above contains the nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

[0010] The gene encoding the recombinant expression protein as described above has a nucleotide sequence of one of the following sequences:

[0011] 1) the DNA sequence shown in SEQ ID NO. 2;

[0012] 2) A DNA sequence that has more than 90% homology with the DNA sequence defined by SEQ ID NO. 2 and encodes a protein with the same function.

[0013] Among them, the sequence shown in SEQ ID NO.2 encodes the chimeric gene sequence of African swine fever virus p30, p72 and p54 proteins (p307254 gene) consisting of 1182 nucleotides, of which the bases 1 to 366 from the 5' to 3' end are the nucleotide sequence of African swine fever virus structural protein p30, the bases 367 to 786 are the nucleotide sequence of African swine fever virus structural protein p72, and the bases 787 to 1182 are the nucleotide sequence of African swine fever virus structural protein p54.

[0014] An expression vector contains the gene sequence shown in SEQ ID NO.2.

[0015] A cell line comprising the gene sequence shown in SEQ ID NO.2.

[0016] A method for preparing chimeric recombinant P30, p72 and p54 proteins of African swine fever virus comprises the following steps:

[0017] Amplify the target fragment shown in SEQ ID NO.2;

[0018] The target fragment was connected to the expression vector and transformed into Escherichia coli;

[0019] Screening of positive clones;

[0020] The target protein was obtained by inducing expression.

[0021] In the preparation method described above, preferably, the method for amplifying the target fragment is:

[0022] First, three pairs of primers, SEQ ID NO.11 and SEQ ID NO.12; SEQ ID NO.13 and SEQ ID NO.14; and SEQ ID NO.15 and SEQ ID NO.16, were used to amplify SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.8 in sequence;

[0023] In the second step, primers SEQ ID NO.11 and SEQ ID NO.14 were used to amplify the gene fragments of SEQ ID NO.9 and SEQ ID NO.10 as templates to obtain the p3072 chimeric gene;

[0024] In the third step, primers SEQ ID NO.11 and SEQ ID NO.16 were used to further chimerically amplify the chimeric gene p3072 and the gene fragment of SEQ ID NO.8 as templates to obtain the target gene fragment of the present invention, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0025] In the preparation method described above, preferably, the PCR amplification product of the target fragment is purified and recovered, and then digested with BamHI and SalI, the digested fragment is purified and recovered, connected to the pET28a(+) vector digested with the same enzymes, and transformed into the expression type Escherichia coli BL21(DE3); the positive is picked, cultured, induced by IPTG to express the target protein and purified.

[0026] By utilizing the above method provided by the present invention, a high yield of expression products can be obtained, and 15 mg of recombinant protein can be harvested per 100 ml of culture medium. The recombinant protein has good antigenicity and immunogenicity.

[0027] The present invention also provides vaccine antigens that can effectively neutralize African swine fever virus and antigens for detecting African swine fever virus infection.

[0028] In order to achieve this object, the present invention adopts the following technical solutions:

[0029] A vaccine for preventing African swine fever virus infection, the active ingredient of which is the chimeric recombinant expression protein of African swine fever virus p30, p72 and p54 as described above, which contains chimeric recombinant protein protective antigens of ASFv p30, p72 and p54.

[0030] Specifically, it is a protein having the amino acid sequence as shown in SEQ ID NO.1, or a protein derived from SEQ ID NO.1, which has the same activity as the amino acid sequence shown in SEQ ID NO.1 by replacing, deleting or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1.

[0031] In practical applications, a subunit vaccine with the protein shown in SEQ ID NO. 1 as the antigenic active ingredient is preferred.

[0032] To enhance the efficacy of this subunit vaccine, an adjuvant may be added. The adjuvant may be No. 10 mineral oil adjuvant, Freund's adjuvant, or Vibrio cholerae ghost adjuvant. No. 10 mineral oil adjuvant is preferred in the present invention, with the antigen and adjuvant mixed in a 1:3 (v / v) ratio.

[0033] The immunization dose of the vaccine of the present invention is generally 0.5-1 mg / kg body weight.

[0034] Rabbits immunized with the vaccine containing the chimeric recombinant structural protein p307254 of the African swine fever virus of the present invention as an active ingredient produced specific antibodies against the African swine fever virus, indicating that the vaccine has good immunogenicity and has broad market application prospects.

[0035] The present invention also provides an antigen for detecting serum antibodies infected with African swine fever virus, the active ingredient of which is the chimeric recombinant expression protein of African swine fever virus p30, p72 and p54 as described above, with the p307254 antigen having the amino acid sequence shown in SEQ ID NO.1.

[0036] The beneficial effects of the present invention are:

[0037] The recombinantly expressed African swine fever virus protein p307254 provided by the present invention is a chimeric amino acid fragment that expresses concentrated antigenic epitopes of the three structural proteins of African swine fever virus, p30, p72 and p54, and is intended to make up for the shortcomings of insufficient or no protection when a single expressed recombinant protein is used as a vaccine antigen, and insufficient sensitivity or low specificity when used as a diagnostic detection antigen.

[0038] The chimeric recombinant expression protein prepared by the present invention is obtained by expressing the three proteins p30, p72 and p54 on a single vector. Firstly, it has the functional characteristics of the three proteins, has very good immunogenicity and antigenic activity, and has broad market application value in the field of detection and prevention of African swine fever virus.

[0039] The chimeric expression recombinant protein prepared by the present invention is used as an antigen in an African swine fever diagnosis kit, and the sensitivity and specificity are greatly improved compared with the single antigen preparation kit. If it is used as a subunit vaccine antigen, compared with the single protein preparation vaccine, only the chimeric recombinant antigen prepared by the present invention is immunized, and specific antibodies against the three proteins p72, p30 and p54 are produced in the immunized animal, which effectively improves the protection against African swine fever virus. Secondly, the present invention uses the PCR method to chimerize the three protein genes together, and then constructs a recombinant expression plasmid with a prokaryotic expression vector, which is transformed into Escherichia coli BL21 (DE3) to induce expression to obtain the chimeric expression p307254 protein, which greatly eliminates the tedious and repetitive work required for the separate expression of the three proteins, saves manpower, material resources and time costs, and is conducive to large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The p30, p72 and p54 gene fragments are PCR amplified products in Example 1;

[0041] Figure 2 The p30+p72 chimeric gene PCR amplification product in Example 1 is p3072;

[0042] Figure 3The PCR amplification product of the p3072+p54 chimeric gene in Example 1 is p307254;

[0043] Figure 4 This is a diagram for identifying the inducible expression of the recombinant p307254-pet28a / BL21 (DE3) in Example 1;

[0044] Figure 5 This is a diagram of the purification of the recombinant p307254-pet28a / BL21 (DE3) expressed protein in Example 1;

[0045] Figure 6 This is the antigenicity identification result of the recombinant p307254 in Example 3. DETAILED DESCRIPTION

[0046] The following examples are used to further illustrate the present invention but should not be construed as limiting the present invention. Any modifications or substitutions made to the present invention without departing from the spirit and substance of the present invention are considered within the scope of the present invention. The genes and amino acid sequences of the recombinant proteins p30, p72, and p54 used in the comparative experiments are identical to those used for the p307254 protein. Furthermore, the vectors, host bacteria, and purification methods used for the individual expression of the p30, p72, and p54 proteins are the same as those used for the p307254 protein and will not be further described in detail herein.

[0047] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents used in the present invention are of analytical grade or above.

[0048] Example 1 Preparation of African swine fever virus chimeric recombinant expression protein p307254

[0049] The present invention utilizes the PCR method to chimera the highly concentrated gene fragments of the antigenic epitopes of the African swine fever virus structural proteins p30, p72 and p54 in the order of p30-p72-p54 to obtain the p307254 connecting gene.

[0050] The specific amplification method is as follows:

[0051] 1.1 ASFv P30, p72 template synthesis and p54 template genome extraction

[0052] Based on the Anhui XCGQ strain sequence provided by GenBank (accession number: MK128995.1), the p30, p72 and p54 protein gene sequences were selected. After the p30 and p72 protein gene sequences were optimized for Escherichia coli preferred codons, they were synthesized by BGI and cloned into the pLB vector respectively. The p54 protein is a transmembrane protein. The present invention selected an intramembrane protein fragment, and the gene sequence was not optimized. The p54 gene was amplified using genomic DNA of liver samples from sick pigs in a private household in Henan as a template. The genomic DNA extraction method of the diseased sample tissue was carried out according to the instructions of the tissue DNA extraction kit (DP304) of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0053] 1.2 Amplification of selected fragments of ASFv p30, p72, and p54:

[0054] After analyzing the three proteins using online epitope prediction software (Bepipred 1.0 Server), fragments with highly concentrated epitopes were selected and primers were designed. The optimized nucleotide sequence encoding the ASFv p30 protein is shown in SEQ ID NO. 6; the optimized nucleotide sequence encoding the ASFv p72 protein is shown in SEQ ID NO. 7. These served as PCR templates for amplifying the p30 and p72 target fragments, respectively. The nucleotide sequence encoding the ASFv p54 intramembrane protein is shown in SEQ ID NO. 8.

[0055] SEQ ID NO.6:ATGGACTTCATCCTGAACATCAGCATGAAGATGGAAGTGATCTTCAAGACCGACCTGAGGAGCTCCAGCCAGGTGGTGTTCCACGCAGGATCCCTGTACAACTGGTTCAGCGTGGAGATCATCAACTCCGGCAGGATCGTGACCACCGCCATCAAGACCCTGCTGAGCACCGTGAAGTACGACATCGTGAAGTCCGCCAGGATCTACGCAGGACAGGGATACACCGAGCACCAGGCCCAGGAGGAGTGGAACATGATCCTGCACGTGCTGTTCGAGGAGGAGACCGAGTCCAGCGCCTCCAGCGAGAACATCCACGAGAAGAACGACAACGAGACCAACGAGTGCACCTCCAGCTTCGAGACCCTGTTCGAGCAGGAGCCCTCCAGCGAGGTGCCTAAGGACTCCAAGCTGTACATGCTGGCCCAGAAGACCGTGCAGCACATCGAGCAGTACGGCAAGGCCCCAGACTTCAACAAAGTGATCAGAGCCCACAACTTCATCCAGACCATCTACGGCACCCCCCTGAAGGAGGAGGAGAAGGAGGTGGTGCGCCTGATGGTCATCAAGCTGCTGAAGAAGAAG

[0056]

[0057] SEQ ID NO.8: TCAAGAAAGAAAAAAGCTGCTGCTATTGAGGAGGAAGATATACAGTTTATAAATCCTTATCAAGATCAGCAGTGGGTAGAAGTCACTCCACAACCAGGTACCTCTAAACCAGCTGGAGCGACTACAGCAAGTGTAGGCAAGCCAGTCACGGGCAGACCGGCAACAAACAGACCAGCAACAAACAAACCAGTTACG GACAACCCAGTTACGGACAGACTAGTCATGGCAACTGGCGGCCGGCGGCCGCACCTGCGGCCGCGAGTGCTCCTGCTCATCCGGCTGAGCCTTACACGACAGTCACTACTCAGAACACTGCTTCACAAACAATGTCGGCTATTGAAAATTTACGACAAAGAAACACCTATACGCATAAAGACCTAGAAAACTCCTTGTAA

[0058] The viral genomes extracted from SEQ ID NO.6, SEQ ID NO.7 and 1.1 were used as templates, and specific primers were used to amplify the P30, p72 and p54 target fragments (the corresponding gene sequences are SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.8).

[0059] The target gene sequence obtained by amplification was amplified by PCR in the order of p30-p72-p54, and the chimeric junction was amplified. The restriction sites were analyzed by DANMAN. The restriction endonuclease sites of BamHI and SalI were added to the first and sixth primers respectively. The primer sequences used are as follows:

[0060] SEQ ID NO.11:5'-ATAATTGGATCCGACATCGTGAAGTCCGC-3';

[0061] SEQ ID NO.12:5'-TTCTCGTACAGTCTCTC cacctccttctcctcct -3';

[0062] SEQ ID NO.13:5'-aggaggagaaggaggtgGAGAGACTGTACGAGAA-3';

[0063] SEQ ID NO.14:5'-GCTTTTTTTCTTTCTTGActggtagtacttagggg-3';

[0064] SEQ ID NO.15:5'-cccctaagtactaccagTCAAGAAAGAAAAAAGC-3';

[0065] SEQ ID NO.16:5'-TTAGTCGACTTACAAGGAGTTTCCA-3';

[0066] Step 1: Using the optimized synthetic p30 / plB plasmid as a template, two primers, SEQ ID NO.11 and SEQ ID NO.12, were used to amplify the selected p30 gene fragment. The total gene length was 366 bp. The target fragment was recovered by gel excision for future use. The gene sequence is shown in SEQ ID NO.9; the amino acid sequence of the corresponding encoded protein is shown in SEQ ID NO.3.

[0067] SEQ ID NO.9: GACATCGTGAAGTCCGCCAGGATCTACGCAGGACAGGGATACACCGAGCACCAGGCCCAGGAGGAGTGGAACATGATCCTGCACGTGCTGTTCGAGGAGGAGACCGAGTCCAGCGCCTCCAGCGAGAACATCCACGAGAAGAACGACAACGAGACCAACGAGTGCACCTCCAGCTTCGAGAC CCTGTTCGAGCAGGAGCCCTCCAGCGAGGTGCCTAAGGACTCCAAGCTGTACATGCTGGCCCAGAAGACCGTGCAGCACATCGAGCAGTACGGCAAGGCCCCAGACTTCAACAAAGTGATCAGAGCCCACAACTTCATCCAGACCATCTACGGCACCCCCCTGAAGGAGGAGGAGAAGGAGGTGSEQ ID NO.3: DIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEV

[0068] Step 2: Using the optimized synthetic p72 / pLB as a template, the selected p72 gene fragment was amplified using primers SEQ ID NO.13 and SEQ ID NO.14. The total gene length was 420 bp. The target fragment was recovered by gel cutting for future use. The gene sequence is shown in SEQ ID NO.10; the amino acid sequence of the corresponding encoded protein is shown in SEQ ID NO.4.

[0069] SEQ ID NO.10: GAGAGACTGTACGAGAACGTGCGCTTCGACGTGAACGGCAACTCCCTGGACGAGTACAGCTCCGACGTGACCACCCTGGTGAGAAAGTTCTGTATCCCCGGCGACAAGATGACCGGCTACAAGCACCTGGTGGGACAGGAGGTGAGCGTGGAGGGCACCTCCGGACCTCTGCTGTGCAACATCCACGACCTGCACAAGCCACACCAGAG CAAGCCCATCCTGACCGACGAGAACGACACCCAGCGCACCTGTTCCCACACCAACCCAAAGTTCCTGAGCCAGCACTTCCCCGAGAACTCCCACAACATCCAGACCGCCGGCAAGCAGGACATCACCCCCATCACCGACGCCACCTACCTGGACATCAGGCGGAACGTGCACTACAGCTGCAACGGCCCCCAGACCCCTAAGTACTACCAG; SEQ ID NO.4: ERLYENVRFDVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPILTDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYYQ

[0070] Step 3: Using the genomic DNA extracted in 1.1 as a template, amplify the selected p54 intramembrane protein fragment using primers SEQ ID NO.15 and SEQ ID NO.16. The total gene length is 396 bp. The target fragment is recovered by gel excision for future use. The gene sequence is shown in SEQ ID NO.8; the amino acid sequence of the corresponding encoded protein is shown in SEQ ID NO.5.

[0071] SEQ ID NO.5: SRKKKAAAIEEEDIQFINPYQDQQWVEVTPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPAHPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSL

[0072] Step 4: Using 1 μl each of p30 and p72 (sequences shown in SEQ ID NOs. 9 and 10) recovered from gel amplification as templates, primers SEQ ID NOs. 11 and 14 were used to amplify the p3072 chimeric gene. The total gene length was 786 bp and the p3072 chimeric gene was recovered from the gel amplification for future use.

[0073] Step 5: Using 1 μl each of p3072 obtained by amplification in Step 4 and p54 obtained by amplification in Step 3 (sequence shown in SEQ ID NO. 8) as templates, and using primers SEQ ID NO. 11 and SEQ ID NO. 16, p3072 and p54 were chimerized to obtain a p307254 gene fragment with a total length of 1182 bp. The gene sequence is shown in SEQ ID NO. 2; the amino acid sequence of the corresponding encoded protein is shown in SEQ ID NO. 1.

[0074]

[0075] The PCR reaction system for each step was 50 μL, and the amplification was performed using the pfu high-fidelity enzyme from Sangon Biochemical (Shanghai) Co., Ltd. The PCR amplification was performed using a general method and will not be described in detail here. The PCR products obtained in each step were identified by 1% DNA gel. The results were as follows: Figure 1-3 As shown, Figure 1 These are PCR amplification products of the p30, p72, and p54 gene fragments. Band 1 is the p72 gene fragment, 420 bp in size; band 2 is the p30 gene fragment, 366 bp in size; and band 3 is the p54 gene fragment, 396 bp in size. M: DNA molecular weight standard (100-2000 bp). Figure 2 It is the PCR amplification product of the p30+p72 chimeric gene, namely p3072; band 1: p3072 gene, size 786 bp; M: DNA molecular weight standard (100-2000 bp). Figure 3 The PCR amplification product of the p3072+p54 chimeric gene (SEQ ID NO. 2) is p307254; band 1 in the figure: p307254 gene, size 1182 bp; M: DNA molecular weight standard (100-2000 bp).

[0076] Then, the DNA was recovered by gel excision using the agarose gel DNA recovery kit (DP209) from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0077] 1.3 Construction of p307254-pet28a recombinant expression plasmid:

[0078] 32 μL of the recovered product of the p307254 gene fragment obtained in step 5 of 1.2 was added with 2 μL each of BamHI and SalI enzymes (Takara restriction endonucleases), 4 μL of 10× HO Buffer, a total volume of 40 μL, placed at 37 ° C enzyme digestion reaction for 5 hours, the enzyme digestion product was purified and recovered, the digested p307254 gene fragment was mixed with the pET28a (+) vector digested with the same restriction endonucleases BamHI and SalI at a ratio of 4.3:1 (V / V), and the reaction was ligated at 25 ° C for 30 minutes using the T4 enzyme rapid ligation method of Tiangen Biochemical Technology (Beijing Co., Ltd.), 10 μL of the ligation product was transferred into Escherichia coli BL21 (DE3) competent cells, placed at 37 ° C, 130 rpm shaking incubator for 1 hour, and 100 μL of the bacterial solution was aseptically drawn and plated on solid LB medium containing 25 μg / mL kanamycin, placed at 37 ° C for 14 to 18 hours, and several single colonies were picked and cleaved with primers SEQ ID NO.11 and SEQ ID NO. No. 16 was first identified by PCR, and plasmids were extracted from PCR-positive colonies for sequencing identification. If both PCR and sequencing results were positive, it was a positive bacterium containing the p307254 / pet28 recombinant expression plasmid.

[0079] 1.4 Induced expression, purification and quantitative packaging of recombinant plasmid p307254 / pet28a:

[0080] Step 1: Determine whether the positive E. coli BL21(DE3) containing the recombinant expression plasmid p307254 / pet28 obtained in 1.3 can express the target protein. The method is as follows: p307254 / pet28a / BL21(DE3), empty BL21(DE3) bacteria, and BL21(DE3) / pet28a transformed with the empty vector pet28a were induced with 1 ml of IPTG (final concentration 0.5 mM). Empty BL21(DE3) bacteria and BL21(DE3) / pet28a transformed with the empty vector pet28a served as controls. The results are shown in the figure below. Figure 4As shown in the figure, 1: BL21 (DE3) empty bacteria induced expression; 2: pet28a / BL21 (DE3) empty vector induced expression; 3: p307254-pet28a / BL21 (DE3) induced expression, theoretical size 47.5KDa; M: protein molecular weight standard (14.4, 18.4, 25, 35, 45, 66.2, 116KDa). The results showed that the induced bacteria of 307254 / pet28a / BL21(DE3) had a thicker expression band between 45 and 66.2 KDa, which was consistent with the size of the target recombinant protein p307254 / pet28a. Neither the empty vector pet28a / BL21(DE3) nor the empty bacteria BL21(DE3) had an expression band consistent with the size of the target protein, indicating that the target gene recombinant expression plasmid p307254 / pet28a was successfully expressed in Escherichia coli BL21(DE3), that is, p307254 / pet28a / BL21(DE3) positive expression bacteria were successfully obtained.

[0081] Step 2: Add the positive p307254 / pet28a / BL21 (DE3) bacterial solution at a ratio of 1:100 to LB liquid medium containing 25 g / mL kanamycin. Incubate at 37°C, 250 rpm, and shake for 2 hours until the OD600 of the bacterial solution reaches 0.5-0.7. Add IPTG to a final concentration of 0.5 mM and continue incubation for another 4 hours. Centrifuge, discard the supernatant, and collect the bacterial pellet.

[0082] Step 3: Resuspend the bacterial precipitate with 1 / 10 volume of phosphate buffer (0.01M, pH 7.2-7.4 PBS) of the total induced expression bacterial solution (i.e., the LB liquid culture medium in the first step, which refers to the volume of the induced expression bacterial solution. For example, if 200 ml is induced, the bacterial precipitate obtained by centrifuging 200 ml of bacterial solution is resuspended with 20 ml of PBS. Only the volume of the resuspended precipitate is calculated without using LB) and ultrasonically disrupt the bacterial cells. The ultrasonication is performed for 5 seconds with a rest period of 10 seconds, 40 times with a power of 300-400w. The disrupted bacterial solution is centrifuged at 6000rpm for 15min, the supernatant is discarded, and the precipitate is collected.

[0083] Step 4: Repeat step 2 twice and collect the precipitate, which is the inclusion body. (The supernatant after two ultrasonic centrifugations must be confirmed by SDS-PAGE to be free of the target protein before being discarded.)

[0084] Step 5: The obtained inclusion bodies were fully dissolved with 10 mL of 8 M urea buffer (NaH2PO4 0.1 M, Tris-HCl pH 8.0, 0.01 M). Ultrasound was performed 20 times at a power of 150-200 W with a 5-second interval and a 10-second interval. The mixture was centrifuged at 10,000 rpm for 15 minutes and the supernatant, which was the denatured p307254 protein, was collected.

[0085] Step 5: The denatured and cleaved p307254 protein solution was centrifuged at 10,000 rpm for 15 minutes. The supernatant, containing the recombinant p307254 protein, was gently pipetted into a dialysis bag (3 kDa molecular weight cutoff) and dialyzed for 6 hours against a refolding buffer (100 mM Tris-HCl, 0.004 M oxidized glutathione, 0.002 M reduced glutathione) containing 4 M urea. The solution was then dialyzed against a refolding buffer containing 2 M urea for 16 hours, a refolding buffer containing 1 M urea for 10 hours, and finally against 0.1 M PBS for 12-16 hours, changing the buffer twice. After dialysis, the solution in the dialysis bag was transferred to a clean centrifuge tube for use in the next purification step.

[0086] Step 6: Use NI-NTA agarose column to mix with the renatured p307254 protein, shake slowly on ice for 2 hours, and purify. The purity of the purified p307254 protein is greater than 90% by SDS-PAGE analysis. Figure 5 As shown in the figure, band 1: recombinant p307254-pet28a / BL21 (DE3) purified product, theoretical size 47.5KDa; band 2: protein molecular weight standard (14.4, 18.4, 25, 35, 45, 66.2, 116KDa).

[0087] Step 7: The purified p307254 protein was quantified, aliquoted at 100 μg / tube, freeze-dried, and stored at -20°C until use.

[0088] Example 2 Immunological evaluation of the vaccine with the recombinant p307254 protein of the present invention as the active ingredient in rabbits

[0089] (1) The recombinant p307254 protein of African swine fever virus obtained in Example 1 was diluted to 0.5 mg / ml and coated on an ELIAS plate, and the serum titer was detected by ELISA.

[0090] Six large-eared white rabbits with negative serum OD450 values ​​less than 0.1 were randomly divided into experimental groups (numbered 1 to 3) and control groups (numbered 4 to 6). The large-eared white rabbits were 30 days old, male, and weighed approximately 1.8 to 2 kg each. They were provided by the Experimental Animal Center of China Agricultural University. The experimental group was immunized with the African swine fever virus recombinant p307254 protein of the present invention. The freeze-dried powdered recombinant p307254 protein was dissolved in PBS (0.01M, pH 7.2 to 7.4) and thoroughly stirred and mixed with an equal volume of Freund's adjuvant to prepare a latex solution. Three rabbits were injected subcutaneously at multiple points in the neck at 200 μg / kg body weight per rabbit. An additional injection was given at the same dose and location 14 days apart, and another injection was given 28 days later. Three large-eared white rabbits in the control group were injected only with PBS and Freund's adjuvant latex solution (the injection volume was the same as that of the experimental group) for a total of three immunizations. During the whole immunization process, rabbit serum was collected on the 28th and 35th day to detect the serum antibody titer, and the titer of the serum of the big-eared white rabbits in the experimental group and the control group was compared.

[0091] (2) Prepare materials:

[0092] Coating antigen: recombinant p307254 protein prepared in Example 1.

[0093] Serum to be tested: 3 rabbit sera in the experimental group and 3 rabbit sera in the control group.

[0094] Blocking solution: PBST containing 5% skim milk powder.

[0095] Secondary antibody: horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibody.

[0096] (III) ELISA method for detecting African swine fever virus antibody titer using P307254 antigen:

[0097] 1. Prepare coating antigen: dilute to 5 μg / mL with carbonate buffer (pH 9.6) and set aside;

[0098] 2. Coating ELISA plate: Add the diluted recombinant p307254 protein solution at a concentration of 5 μg / mL to the ELISA plate, 100 μL / well, and incubate at 4-8°C for 12-16 hours.

[0099] 3. Wash the ELISA plate: Take out the antigen-coated plate from the refrigerator at 4-8°C, pour out the antigen solution in the wells, fill the wells with PBST (PBST is a 0.01M PBS solution containing 0.5‰ Tween-20, pH 7.2-7.4) and wash three times, 3 minutes each time, and pat the ELISA plate dry;

[0100] 4. Block the ELISA plate: Add blocking solution (PBST solution containing 0.5% skim milk powder) to each well of the ELISA plate, 200 μL / well, and incubate at 37°C for 2 hours;

[0101] 5. Wash the ELISA plate: Repeat step 3;

[0102] 6. Add primary antibody: Add 1:100 diluted serum to each well of the ELISA plate (diluted with PBST), 100 μL / well, and incubate at 37°C for 30 minutes;

[0103] 7. Wash the ELISA plate: Pour out the primary antibody in the well, wash with PBST 5 times, 3 minutes each time, and pat the ELISA plate dry;

[0104] 8. Add secondary antibody: Add 100 μL / well of HRP-labeled goat anti-rabbit serum diluted 1:4000 (diluted with 0.01M, pH 7.2-7.4 PBS) to the ELISA plate and incubate at 37°C for 30 minutes;

[0105] 9. Wash the ELISA plate: Repeat step 7;

[0106] 10. Add substrate: Add TMB solution to each well of the ELISA plate, 100 μL / well, incubate at 37°C in the dark for 13-15 minutes;

[0107] 11. Add stop solution: Add 2M hydrochloric acid solution to each well of the ELISA plate, 100 μL / well, and gently shake to mix the liquid in the well;

[0108] 12. Read the OD450 value: Place the ELISA plate on the microplate reader and read the value at a wavelength of 450, i.e. the OD450 value.

[0109] (IV) The results are shown in Table 1. The OD450 values ​​of the serum from the control rabbits 14 days after the second and third immunizations were both less than 0.1, while the OD450 values ​​of the serum from the experimental rabbits 14 days after the second and third immunizations were both greater than 1.5. This indicates that the recombinant p307254 protein of the present invention can induce the production of neutralizing antibodies in rabbits, i.e., can activate a humoral immune response.

[0110] Table 1 OD450 values

[0111]

[0112] Example 3 Dot ELISA to compare the antigenicity of African swine fever virus recombinant p307254 and individually expressed p30, p72, and p54 recombinant proteins

[0113] Protein samples: purified and quantified African swine fever virus recombinant p307254, p30, p72 and p54 recombinant proteins.

[0114] Antigen preparation: The purified p307254 recombinant protein and the purified p30, p72 and p54 individually expressed recombinant proteins in Example 1 were diluted to 10 μg / mL with carbonate buffer (0.05 mol / L, pH 9.6) and fully dissolved for later use. 1% BSA was prepared with carbonate buffer (0.05 mol / L, pH 9.6) for later use.

[0115] Primary antibody serum: African swine fever standard positive serum (purchased from China Veterinary Drug Administration); negative control serum used normal pig serum that has not been immunized or infected with African swine fever virus.

[0116] Dot ELISA experimental steps:

[0117] 1. Antigen Coating of Nitrocellulose (NC) Membrane: Remove a 0.45 μm pore size NC membrane and cut into pieces of appropriate size. Use a pencil to mark 10 squares on the cut NC membrane. Apply 2 μL of diluted p307254, p30, p72, or p54 antigens (10 μg / mL) to each of 8 squares, resulting in 20 ng of antigen per spot. Apply 2 μL of 1% BSA to each of the remaining 2 squares. Place the NC membrane in a damp gauze-lined box, cover, and incubate at 4-8°C for 12-16 hours.

[0118] 2. Washing the NC membrane: Remove the coated NC membrane and place it in 10 mL to 20 mL of PBST (0.01 M PBS buffer containing 0.5‰ Tween-20, pH 7.2-7.4). Wash on a shaker for 5 minutes, then replace the washing solution. Repeat three times.

[0119] 3. Blocking NC membrane: Place the washed NC membrane into pre-prepared blocking solution (PBST solution containing 5% skim milk powder) and block at 37°C for 2 hours.

[0120] 3. Wash the NC membrane: same as step 2.

[0121] 4. Add primary antibody: Cut the 10 antigen spots of the NC membrane into 2 strips (each containing 5 antigen spots), and then put the two NC membranes into the 1:100 diluted African swine fever standard positive serum and negative control (normal pig serum) serum solution (both diluted with PBST containing 5% skim milk powder), shake steadily at room temperature (22-25℃), and incubate at room temperature for 2 hours.

[0122] 5. Wash the NC membrane: discard the primary antibody and wash the membrane with PBST for 5 minutes x 4 times.

[0123] 6. Add secondary antibody: Add two NC membranes to horseradish peroxidase-conjugated rabbit anti-pig secondary antibody solution (diluted 1:4000 with 0.01M PBS pH 7.2-7.4) and shake steadily at room temperature for 1 hour.

[0124] 7. Wash the NC membrane: discard the secondary antibody and wash the membrane with PBST for 5 minutes x 4 times.

[0125] 8. DAB color development: Place the membrane in the pre-prepared DAB color development solution, protect from light and develop the color until light brown spots appear. Continue to develop the color for 2 minutes, then remove the NC membrane and transfer it to double-distilled water to terminate the reaction.

[0126] The results are as follows Figure 6 As shown in the figure, column A is the results of normal pig serum that has not been immunized and infected with African swine fever virus, and column B is the results of African swine fever standard positive serum, 1: p307254-pet28a / BL21(DE3) recombinant protein; 2: p30-pet28a / BL21(DE3) recombinant protein; 3: p72-pet28a / BL21(DE3) recombinant protein: 4: BSA; 5: p54-pet28a / BL21(DE3) recombinant protein.

[0127] The results showed that the p307254 antigen reacted with the standard positive serum of African swine fever to produce very dark brown spots, and no color was observed with the negative control (normal pig serum); the p30 and p72 antigens expressed alone did not produce visible color with either the positive serum or the negative serum of African swine fever virus; the p54 antigen expressed alone produced visible light brown spots after reacting with the positive serum of African swine fever, and no color was observed with the negative serum; 1% BSA had no color reaction with either the positive serum of African swine fever or the negative pig serum.

[0128] Conclusion: According to the dot ELISA test results, it can be seen that the individually expressed p30 and p72 proteins have no antigenic activity with African swine fever virus-positive serum. Although the individually expressed p54 reacts with African swine fever virus-positive serum, the antigenicity is very weak according to the color development results. The recombinant protein p307254 obtained by chimeric recombinant expression of p30, p72 and p54 in the present invention shows a strong color reaction after reaction with African swine fever-positive serum, indicating that the antigenicity of the chimeric expression recombinant protein p307254 is significantly better than that of the individually expressed recombinant proteins p30, p72 and p54. At the same time, the chimeric expression recombinant protein p307254 does not show color with African swine fever-negative serum, indicating that the chimeric expression recombinant protein p307254 also has good specificity.

[0129] Example 4 Indirect ELISA method for detecting African swine fever serum antibodies using the African swine fever virus chimeric recombinant protein p307254 prepared by the present invention and the recombinant protein antigens expressing p30, p72 and p54 separately

[0130] 1. Preparation of Coating Antigens: (The genes and amino acid sequences of the antigens expressing the p30, p72, and p54 proteins used in the present invention, as well as the expression vectors, are identical to those of p307254 and are homemade in our laboratory. The methods and steps are not described in detail here.) Purified African swine fever virus p307254 recombinant protein and individually expressed p30, p72, and p54 recombinant proteins were diluted to 5 μg / mL in coating carbonate buffer (0.05 M, pH 9.6).

[0131] 2. Coating the ELISA reaction plate: Add the diluted p307254, p30, p72, and p54 antigen solutions to the ELISA reaction plate at 100 μL / well. Incubate at 4-8°C for 14-16 hours until the entire plate is coated with each antigen.

[0132] 3. Wash the ELISA reaction plate: Take out the reaction plate and wash it with PBST (0.01 M PBS containing 0.5‰ Tween-20, pH 7.2-7.4) 5 times, 3 minutes each time.

[0133] 4. Block the ELISA reaction plate: add PBST containing 5% skim milk powder (200 μL / well) to each well of the reaction plate, incubate at 37° C., and block for 2 hours.

[0134] 5. Wash the ELISA reaction plate: Same as step 36. Primary antibody reaction: Dilute the African swine fever standard positive serum (purchased from the China Food and Drug Administration) and negative control serum (normal pig serum) with PBST at a ratio of 1:200, add p307254, p30, p72 and p54 different antigens to the corresponding wells of the ELISA reaction plate (all in duplicate), and use PBST as a blank control in duplicate, 100 μL / well, and incubate at 37°C for 30 minutes.

[0135] 7. Wash the ELISA reaction plate: same as step 3.

[0136] 8. Secondary antibody reaction: Add 100 μL / well of HRP-labeled rabbit anti-pig secondary antibody diluted 1:5000 (in 0.01 M, pH 7.2-7.4 PBS) to each well of the ELISA reaction plate and incubate at 37°C for 30 minutes.

[0137] 9. Wash the ELISA reaction plate: Same as step 3.

[0138] 10. Color development reaction: Add TMB substrate solution to each well of the ELISA reaction plate, 100 μL / well, and incubate at 37°C for 10-15 minutes.

[0139] 11. Termination: Add 2N hydrochloric acid solution to the ELISA reaction plate, 100 μL / well, and gently shake the reaction plate to mix thoroughly.

[0140] 11. Read OD 450 Value: Place the ELISA reaction plate into the plate slot of the enzyme reader and read the OD value of each well. 450 value.

[0141] 12. Results: See Table 2.

[0142] Table 2

[0143]

[0144] As can be seen from the results in Table 2, when the individually expressed p30, p72 and p54 proteins are used as ELISA antigens to detect positive and negative sera, the P / N values ​​are all lower than the P / N (38.7) value of the recombinant protein p307254 expressed by the three proteins as a chimeric antigen receptor, indicating that the antigenicity of the chimeric recombinant protein p307254 prepared by the present invention is higher than that of the single expressed recombinant protein. The p30 and p54 selected by the present invention play a vital role in the invasion of African swine fever virus into cells, and can produce corresponding neutralizing antibodies in the early stage of viral infection and throughout the entire infection process. P72 is responsible for the formation of the viral capsid in the late stage of viral infection. The chimeric recombinant expression protein p307254 prepared and provided by the present invention combines the characteristics of three proteins in one, which effectively makes up for the immune failure caused by the single antibody produced when a single protein is used as a vaccine antigen, or even fails to activate the production of antibodies. On the other hand, when p307254 is used as an African swine fever virus diagnostic antigen, the sensitivity and specificity of African swine fever virus antibodies are greatly improved compared to the use of single antigens p30, p72 and p54 alone, which is of great value for the diagnosis of African swine fever, especially the early detection of viral infection.

[0145] This study selected fragments of antigenic epitopes concentrated from three structural proteins of African swine fever virus (ASFV), p30, p72, and p54. These three genes were linked together using PCR to construct a prokaryotic expression vector, which induced expression and produced the p307254 chimeric recombinant protein. The prepared p307254 protein, as described in Examples 3 and 4, was compared with individually expressed p30, p72, and p54 recombinant proteins. The p307254 protein exhibited significantly higher antigenicity against ASFV-positive serum than individual p30, p72, and p54 proteins, thus addressing the shortcomings of existing commercially available detection reagents, such as suboptimal antigen sensitivity and specificity. Furthermore, the expression product obtained by genetically linking the p307254 chimeric recombinant protein possesses the functional properties of each of the three proteins, even reinforcing their immunogenicity and antigenicity to some extent. This holds promise for the development of subunit vaccines and detection reagents for ASFV.

Claims

1. A chimeric recombinant expression protein of African swine fever virus p30, p72 and p54, characterized in that: The protein is a chimeric protein expressing the structural protein fragments of African swine fever virus p30, p72 and p54; 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; the amino acid sequence of the protein is shown as SEQ ID NO.

1.

2. The gene encoding the recombinant expression protein according to claim 1, characterized in that: It can chimera recombinant p30, p72 and p54 protein genes, and contains nucleotide sequences shown in SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 8; the nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. An expression vector comprising the sequence shown in SEQ ID NO.

2.

4. A cell line comprising the sequence shown in SEQ ID NO.

2.

5. A method for preparing chimeric recombinant expression of African swine fever virus p30, p72 and p54 proteins, comprising the following steps: Amplify the target fragment shown in SEQ ID NO. 2; Connect the target fragment to the expression vector and transform it into Escherichia coli; Screening of positive clones; The target protein was obtained by inducing expression.

6. The preparation method according to claim 5, wherein The method for amplifying the target fragment is as follows: first, three pairs of primers, SEQ ID NO. 11 and SEQ ID NO. 12; SEQ ID NO. 13 and SEQ ID NO. 14; and SEQ ID NO. 15 and SEQ ID NO. 16, are used to sequentially amplify SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 8; In the second step, primers SEQ ID NO. 11 and SEQ ID NO. 14 were used to amplify the gene fragments of SEQ ID NO. 9 and SEQ ID NO. 10 as templates to obtain the p3072 chimeric gene; In the third step, primers SEQ ID NO. 11 and SEQ ID NO. 16 were used to further chimeric amplify the amplified chimeric gene p3072 and the gene fragment of SEQ ID NO. 8 as templates to obtain the target fragment whose nucleotide sequence is shown in SEQ ID NO.

2.

7. A vaccine for preventing African swine fever virus infection, characterized in that: The active ingredient is the chimeric recombinant expression protein of African swine fever virus p30, p72 and p54 according to claim 1, and the chimeric recombinant expression p30, p72 and p54 proteins are composed of protective antigens.

8. Use of the chimeric recombinant expression proteins of African swine fever virus p30, p72 and p54 according to claim 1 in the preparation of serum antibodies for detecting African swine fever virus infection.

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