A method for preparing and use of a porcine foot-and-mouth disease O and A bivalent virus-like particle

Abstract

The application discloses a preparation method and application of porcine foot-and-mouth disease O type and A type bivalent virus-like particles, and belongs to the field of biological products. The technical problem to be solved by the application is how to better realize the prevention effect of porcine foot-and-mouth disease. In order to solve the above technical problem, the application provides DNA, wherein the DNA is a gene cluster OVP1-2A-AVP1-3C, and the gene cluster OVP1-2A-AVP1-3C comprises three expression boxes of an expression box of OVP1-2A, an expression box of foot-and-mouth disease virus VP1 of type A and an expression box of 3C protease. The application also provides foot-and-mouth disease bivalent antigens prepared by taking the DNA as a biological material. The foot-and-mouth disease bivalent antigens provided by the application have high immunogenicity in pigs and can induce a strong foot-and-mouth disease antibody response. The research results prove that the immunization antigens provided by the application are effective, and have great industrialization transformation potential and value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological products, in particular to a preparation method and application of a bivalent virus-like particle of swine foot-and-mouth disease O and A types.

[0002] Priority application: This application claims priority to Chinese patent application (application number 202411967136.X) with a filing date of December 30, 2024, the entire contents of which are hereby incorporated by reference. BACKGROUND

[0003] Foot-and-mouth disease (FMD) is a highly contagious disease caused by infection with FMD virus (FMDV), which has caused significant economic losses to the swine industry worldwide. FMDV belongs to the Picornaviridae family and mainly infects odd-toed animals. The genome of the virus is a linear RNA (about 8.5 kb) surrounded by a protein shell of four structural proteins, namely VP1, VP2, VP3, and VP4. FMDV is divided into seven immunotypes: A, O, C, AsiaI, and South African territories (SAT1, SAT2, and SAT3). These seven serotypes have no cross-immunity, and animals infected with one serotype are still susceptible to the other six serotypes. Among them, the O type FMDV serotype is the most prevalent of the seven serotypes.

[0004] Effective FMD antigens require the production of intact, assembled FMDV capsids to induce a protective immune response. Traditional FMD antigens are produced by chemical inactivation of purified, virulent FMDV virions, but this production method has inherent risks for accidental release of FMDV. Recombinant protein antigens are safer alternatives to traditional FMDV antigens because they only express FMDV proteins or virus-like particles (VLPs) required for assembling empty FMDV capsids. Recombinant protein antigens all require the expression of FMDV 3C protease in the cytoplasm to process FMDV P1-2A polyprotein into individual viral capsid proteins (VP0, VP3, VP1). However, wild-type FMDV 3C protease is toxic and has low expression in host cells and host bacteria. SUMMARY

[0005] The technical problem to be solved by the present application is how to better achieve the preventive effect of swine foot-and-mouth disease.

[0006] To solve the above technical problems, the present application provides the following technical solutions:

[0007] The present application provides DNA, the DNA is gene cluster OVP1-2A-AVP1-3C, the gene cluster OVP1-2A-AVP1-3C includes the coding sequence (CDS) of OVP1-2A, the coding sequence (CDS) of type A foot-and-mouth disease virus VP1 and the coding sequence of 3C protease;

[0008] The coding sequence of OVP1-2A is any one of the following:

[0009] g11) the coding sequence is 5071-7284 of SEQ ID NO:1;

[0010] g12) the sequence with more than 75% identity to the sequence shown in g11);

[0011] The coding sequence of type A foot-and-mouth disease virus VP1 is any one of the following:

[0012] g21) the coding sequence is 8253-8894 of SEQ ID NO:1;

[0013] g22) the sequence with more than 75% identity to the sequence shown in g21);

[0014] The coding sequence of 3C protease is any one of the following:

[0015] g31) the coding sequence is 9094-9741 of SEQ ID NO:1;

[0016] g32) the sequence with more than 75% identity to the sequence shown in g31).

[0017] The gene cluster OVP1-2A-AVP1-3C comprises three expression cassettes of an expression cassette of OVP1-2A, an expression cassette of VP1 of type A foot-and-mouth disease virus and an expression cassette of 3C protease. The nucleotide sequence of the expression cassette of OVP1-2A is SEQ ID NO: 1 from position 4984 to 7281, wherein SEQ ID NO: 1 from position 4984 to 5002 is a T7 promoter, SEQ ID NO: 1 from position 5003 to 5027 is a lac operator, and SEQ ID NO: 1 from position 5071 to 7284 is a coding sequence of OVP1-2A. The nucleotide sequence of the expression cassette of VP1 of type A foot-and-mouth disease virus is SEQ ID NO: 1 from position 7285 to 8894, wherein SEQ ID NO: 1 from position 7285 to 8252 is a TETR promoter, and SEQ ID NO: 1 from position 8253 to 8894 is a coding sequence of VP1 of type A foot-and-mouth disease virus. The nucleotide sequence of the expression cassette of 3C protease of type O foot-and-mouth disease virus is SEQ ID NO: 1 from position 8895 to 9741, wherein SEQ ID NO: 1 from position 8895 to 9093 is an araBAD promoter, and SEQ ID NO: 1 from position 9094 to 9741 is a coding sequence of 3C protease of type O foot-and-mouth disease virus.

[0018] The present application also provides a biological material related to the DNA, which is any one of the following:

[0019] B1) a recombinant vector containing the DNA;

[0020] B2) a recombinant microorganism containing the DNA or containing the recombinant vector of B1);

[0021] B3) a recombinant cell containing the DNA or containing the recombinant vector of B1);

[0022] B4) an RNA molecule obtained by transcription of the DNA.

[0023] As used herein, the term "vector" means any construct useful for the purpose of transformation, i.e. the introduction of heterologous DNA into a host cell. Such as a plasmid, a cosmid, a virus, a bacteriophage, or linear or circular DNA.

[0024] The present application also provides a method for producing a protein, comprising the step of expressing the DNA in a biological cell, to obtain the protein.

[0025] In the above method, the expression of the DNA in the biological cell comprises the step of introducing the gene cluster OVP1-2A-AVP1-3C into E. coli through a recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C to obtain a recombinant E. coli; the nucleotide sequence of pET-28a(+)-OVP1-2A-AVP1-3C is SEQ ID NO: 1.

[0026] The expression is induced expression.

[0027] The induced expression is performed using IPTG, tetracycline and arabinose.

[0028] The protein includes, but is not limited to, OVP1-2A, type A foot-and-mouth disease virus VP1 and 3C protease,

[0029] The OP1-2A is any one of the following:

[0030] (a11) a protein with an amino acid sequence shown in SEQ ID NO: 2;

[0031] (a12) a sequence having more than 75% identity with a11);

[0032] (a13) a fusion protein having the same function obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of a11) or a12);

[0033] The type A foot-and-mouth disease virus VP1 is any one of the following:

[0034] (a21) a protein with an amino acid sequence shown in SEQ ID NO: 3;

[0035] (a22) a sequence having more than 75% identity with a21);

[0036] (a23) a fusion protein having the same function obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of a21) or a22);

[0037] The 3C protease is any one of the following:

[0038] (a31) a protein with an amino acid sequence shown in SEQ ID NO: 4;

[0039] (a32) a sequence having more than 75% identity with a31);

[0040] (a33) a fusion protein having the same function obtained by connecting a tag or a signal peptide to the N-terminus and / or the C-terminus of a31) or a32).

[0041] Under the action of the 3C protease, the OVP1-2A continues to be cleaved into VP0 with the amino acid sequence of SEQ ID NO: 2 from 1 to 304, VP3 with the amino acid sequence of SEQ ID NO: 2 from 305 to 524, and VP1 protein with the amino acid sequence of SEQ ID NO: 2 from 525 to 737, and assembled into a 5 -sided body; VP0 also continues to be cleaved and continues to be assembled into 146S.

[0042] Further, in the method, the organism is a microorganism, a plant, or an animal.

[0043] Further, in the method, the microorganism is any one of the following:

[0044] C1) a prokaryotic microorganism;

[0045] C2) a gram-negative bacterium;

[0046] C3) an Escherichia bacterium;

[0047] C4) Escherichia coli;

[0048] C5) Escherichia coli BL21 (DE3).

[0049] The application also provides a protein prepared by the above method.

[0050] The protein can be used for preparing a porcine foot-and-mouth disease virus antigen.

[0051] The application also provides a virus-like particle containing the above protein.

[0052] The application also provides an application, which is any one of the following:

[0053] A1) use of the above DNA in the preparation of a product for preventing porcine foot-and-mouth disease virus infection;

[0054] A2) use of the above biological material in the preparation of a product for preventing porcine foot-and-mouth disease virus infection;

[0055] A3) use of the above protein in the preparation of a product for preventing porcine foot-and-mouth disease virus infection;

[0056] A4) use of the above virus-like particle in the preparation of a product for preventing porcine foot-and-mouth disease virus infection.

[0057] The application also provides an antigen for preventing porcine foot-and-mouth disease virus, which comprises the protein and / or the virus-like particle.

[0058] Further, the antigen further comprises an adjuvant.

[0059] The present application also provides a method for preparing the antigen, the method comprising the step of mixing the protein and / or the virus-like particle with an adjuvant.

[0060] The adjuvant is a mineral oil.

[0061] The adjuvant includes, but is not limited to, Monanide TM ISA206 VG adjuvant.

[0062] In some embodiments of the present application, the adjuvant is Monanide TM ISA206 VG adjuvant.

[0063] In the present application, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the identity of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and conducting a search in Advanced BLAST 2.1, and then the value of identity (%) can be obtained.

[0064] In the present application, the identity can be specifically 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0065] In the above biological material, the expression cassette refers to a DNA capable of expressing the protein in a recombinant cell, which can include not only a promoter that initiates the transcription of the protein-encoding gene, but also a terminator that terminates the transcription of the protein-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0066] The term "recombinant vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to regulatory sequences providing for its expression. The recombinant vector comprises a polynucleotide of the application linked to one or more regulatory sequences, such as a promoter and transcription and translation termination signals, that direct the production of the polypeptide in an expression host. A variety of nucleotide and regulatory sequences can be joined together to produce a recombinant vector, which can include one or more convenient restriction sites for the insertion or substitution of a polynucleotide encoding a polypeptide. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide into an appropriate vector in the art for expression. In making the expression vector, the coding sequence is operably linked to the appropriate regulatory sequences, such as promoters and transcription and translation terminators, for expression. The recombinant vector can be any vector (e.g., a plasmid or virus) that can conveniently be subjected to recombinant DNA procedures and that can bring about the expression of the polynucleotide. The choice of vector will often depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be linear or closed circular. The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a non-chromosomal element, a mini-chromosome or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into a host cell, is integrated into the DNA of the cell, and replicated together with the chromosome into which it has been integrated. Furthermore, a purpose-built vector or plasmid, or two or more vectors or plasmids, can be used which together contain the complete DNA to be introduced into the genome of the recombinant cell, or a transposon can be used.

[0067] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene that encodes a protein necessary for the survival or growth of cells harboring the gene but not for untransformed, untransfected, or the like cells. The vector preferably contains an element that permits integration of the vector into the recombinant cell genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector can rely on a sequence encoding a polypeptide, or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides that encode a protein necessary for the integration of the vector into the host cell genome at a precise location. To increase the likelihood of integration at a precise location, the integrational element would comprise a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which are homologous with the corresponding sequence to enhance the probability of recombination between the vector and a target sequence. The integrational element can be any sequence that is homologous with the corresponding sequence in the genome of the host cell. Furthermore, the integrational element can be non-encoding or encoding a polynucleotide. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination. For autonomous replication, the vector can further comprise an origin of replication enabling the vector to replicate autonomously in the host cell. The origin of replication can be any plasmid origin of replication or any chromosomal origin of replication that functions in the host cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide capable of eliciting in vivo autonomous replication of a plasmid or vector.

[0068] More than one copy of a polynucleotide of the present application can be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome, or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene are selectable with a suitable selection agent. Methods for amplifying the copy number of a polynucleotide include the co-introduction of a selectable marker gene with the polynucleotide, where cells containing amplified copies of the selectable marker gene are selectable with a suitable selection agent, and thereby contain amplified copies of the polynucleotide. Methods for ligating the above-described elements to construct recombinant expression vectors of the present application are well known to one skilled in the art.

[0069] The recombinant microorganism can be bacteria such as E. coli, mammalian cells, insect cells, and other expression systems such as yeast, algae, and plants. The mammalian cells can be 293, CHO, BHK, MDCK, and the like, and the insect cells can be SF9 and Hifv5.

[0070] The beneficial technical effects achieved by the present application are as follows:

[0071] The application constructs a recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C. In the recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C, the T7 promoter is used to initiate expression of the fusion protein P1-2A, the tetracycline promoter is used to induce expression of the VP1 type A, and the arabinose promoter is used to induce expression of the 3C protease which is optimized and modified. The O-type foot-and-mouth disease P1-2A and VP1 type A antigens are induced to express under the conditions of IPTG and tetracycline. The arabinose is added in the late fermentation stage to induce expression of the 3C protease which is initiated by the araBAD promoter. The 3C protease cuts the P1-2A protein to form VP0, VP3 and VP1 proteins in the prokaryotic package and assembles the 76S virus-like particles. The cut proteins can be directly used to immunize animals, and the pure antigens can be obtained by using the foot-and-mouth disease antibodies for purification. The antigen candidate proteins with the O-type and A-type two antigens are assembled in a specific buffer to form bivalent antigens, which are used to immunize animals to detect the effectiveness of the antigens.

[0072] The bivalent candidate antigen of foot-and-mouth disease O-type-A-type provided in the application has high immunogenicity in pigs and can induce strong foot-and-mouth disease antibody response. The research results prove that the immunizing antigen provided in the application is effective, and has great potential and value for industrialization transformation. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 It is a physical map of pET-28a(+)-OVP1-2A-AVP1-3C;

[0074] Figure 2 It is an expression result of the recombinant strain ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C. The product sources represented by each lane are as follows: lane 1, tetracycline-induced pET-28a(+) empty vector-transferred strain ArcticExpress(DE3) broken bacteria supernatant; lane 2, arabinose-induced pET-28a(+) empty vector-transferred strain ArcticExpress(DE3) broken bacteria supernatant, lane 3, 1 mM IPTG pET-28a(+) empty vector-transferred strain ArcticExpress(DE3) broken bacteria supernatant, lane 4, tetracycline and arabinose-induced broken bacteria supernatant; lane 5, 1 mM IPTG-induced broken bacteria supernatant, lane 6, 1 mM IPTG-induced broken bacteria precipitate;

[0075] Figure 3For the expression results of recombinant strain Arctic Express (DE3) / pET-28a (+) -OVP1-2A-AVP1-3C at low temperature and low inducer, the product sources represented by each lane are as follows, lane 1: IPTG, tetracycline and arabinose induction precipitate at 16℃ culture temperature; lane 2: IPTG induction precipitate at 16℃ culture temperature, lane 3: low IPTG, tetracycline and arabinose induction supernatant at 16℃; lane 4: IPTG induction supernatant at 16℃ culture temperature;

[0076] Figure 4 For the electron microscope results of the induced expression of pET-28a (+) -OVP1-2A-AVP1-3C in Arctic Express (DE3) at 16℃ supernatant;

[0077] Figure 5 For the simultaneous quantification of antigen content by ELISA method. DETAILED DESCRIPTION

[0078] The present application constructs a recombinant expression vector pET-28a (+) -OVP1-2A-AVP1-3C, in which the T7 promoter is used to express the fusion protein P1-2A, the tetracycline promoter is used to induce the expression of type A VP1, and the arabinose promoter is used to induce the expression of the optimized 3C protease. First, the O-type foot-and-mouth disease P1-2A and type A VP1 antigens are induced to express under the conditions of IPTG and tetracycline, and arabinose is added in the late fermentation to induce the expression of 3C protease activated by araBAD promoter. The 3C protease cuts the P1-2A protein to form VP0, VP3 and VP1 proteins in the prokaryotic package and assembles 76S virus-like particles. The cut proteins can be directly immunized to animals, or the pure antigens can be obtained by using foot-and-mouth disease antibodies to purify, assembled into antigen candidate proteins with O-type and A-type two antigens in a specific buffer, and made into bivalent antigens.

[0079] The foot-and-mouth disease O-type-A-type bivalent candidate antigen provided by the present application has high immunogenicity in mice and pigs and can induce strong foot-and-mouth disease antibody response. The research results prove the potential of the pET-28a (+) -OVP1-2A-AVP1-3C candidate antigen as an effective and affordable immunizing antigen.

[0080] The present application will be further described in detail below in conjunction with the specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0081] The experimental methods in the following examples are routine methods, which are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0082] The quantitative tests in the following examples are performed in triplicate, unless otherwise specified, and the results are averaged.

[0083] The data in the following examples are processed using GraphPad Prism statistical software, and the experimental results are expressed as mean ± standard deviation, t-test is used, P<0.05 (*) indicates significant difference, P<0.001 (*** ) indicates extremely significant difference.

[0084] The present application is further described in detail below with reference to the specific embodiments, and the examples given are only to illustrate the present application, not to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not in any way constitute a limitation on the present application.

[0085] The experimental methods in the following examples are routine methods, which are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0086] Example 1, preparation of antigen protein

[0087] 1.1, synthesis of target gene and construction of recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C

[0088] In order to improve the immunogenicity of wild-type foot-and-mouth disease, the coding sequence of O-type foot-and-mouth disease VP1-2A, the coding sequence of A-type foot-and-mouth disease VP1 and the coding sequence of O-type foot-and-mouth disease 3C protease are respectively implanted under different promoters (the T7 promoter for expressing O-type foot-and-mouth disease VP1-2A is the backbone vector itself), to obtain the coding sequence of recombinant protein pET-28a(+)-OVP1-2A-AVP1-3C and the corresponding expression cassette.

[0089] The encoding gene of recombinant protein OVP1-2A-AVP1-3C was synthesized by Shanghai Generay Biotech Co., Ltd. The nucleotide sequence is shown in SEQ ID NO: 1 from 5071 to 9741. The nucleotide sequence of SEQ ID NO: 1 from 5071 to 7284 is the encoding sequence of O type foot-and-mouth disease VP1-2A (also referred to as OVP1-2A) (the gene encodes VP0, VP3 and VP1 core protein), the nucleotide sequence of SEQ ID NO: 1 from 7285 to 8252 is TETR promoter, the nucleotide sequence of SEQ ID NO: 1 from 8253 to 8894 is the encoding sequence of A type foot-and-mouth disease VP1 (also referred to as AVP1), the nucleotide sequence of SEQ ID NO: 1 from 8895 to 9093 is araBAD promoter, and the nucleotide sequence of SEQ ID NO: 1 from 9094 to 9741 is the encoding sequence of O type foot-and-mouth disease 3C protease (also referred to as 3C protease). The DNA molecule shown in SEQ ID NO: 1 from 5071 to 9741 is used to replace the sequence between Ncol and Xhol of pET28a(+) vector, and the obtained recombinant expression vector is named as recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C. The nucleotide sequence of the recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C is SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 1 from 4984 to 9741 is named as gene cluster OVP1-2A-AVP1-3C, which comprises the expression cassette of OVP1-2A, the expression cassette of A type foot-and-mouth disease virus VP1 and the expression cassette of 3C protease. The nucleotide sequence of the expression cassette of OVP1-2A is SEQ ID NO: 1 from 4984 to 7281, wherein the nucleotide sequence of SEQ ID NO: 1 from 4984 to 5002 is T7 promoter, the nucleotide sequence of SEQ ID NO: 1 from 5003 to 5027 is lac operator, and the nucleotide sequence of SEQ ID NO: 1 from 5071 to 7284 is the encoding sequence of OVP1-2A. The nucleotide sequence of the expression cassette of A type foot-and-mouth disease VP1 is SEQ ID NO: 1 from 7285 to 8894, wherein the nucleotide sequence of SEQ ID NO: 1 from 7285 to 8252 is TETR promoter, and the nucleotide sequence of SEQ ID NO: 1 from 8253 to 8894 is the encoding sequence of A type foot-and-mouth disease VP1. The nucleotide sequence of the expression cassette of O type foot-and-mouth disease 3C protease is SEQ ID NO: 1 from 8895 to 9741, wherein the nucleotide sequence of SEQ ID NO: 1 from 8895 to 9093 is araBAD promoter, and the nucleotide sequence of SEQ ID NO: 1 from 9094 to 9741 is the encoding sequence of O type foot-and-mouth disease 3C protease.

[0090] The map of the recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C is shown in FIG. 1. Figure 1The recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C expresses a recombinant protein with an amino acid sequence of SEQ ID NO: 2, a type A foot-and-mouth disease VP1 (AVP1) with an amino acid sequence of SEQ ID NO: 3, and an O-type foot-and-mouth disease 3C enzyme (referred to as 3C protease) with an amino acid sequence of SEQ ID NO: 4. Among them, the 1st to 737th of SEQ ID NO: 2 is the amino acid sequence of the O-type foot-and-mouth disease P1-2A core protein, and in the middle and late stages of fermentation, P1-2A is further cleaved into VP0 with an amino acid sequence of SEQ ID NO: 2 1st to 304th, VP3 with an amino acid sequence of SEQ ID NO: 2 305th to 524th, and VP1 protein with an amino acid sequence of SEQ ID NO: 2 525th to 737th under the action of 3C protease, and assembled into a 5-sided body; VP0 is further cleaved and further assembled into 146S.

[0091] 1.2, Expression of recombinant protein

[0092] The recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C prepared in 1.1 was transformed into host bacteria ArcticExpress(DE3)pRARE competent cells (Beijing Bomeide Gene Technology Co., Ltd., Catalog No.: BC225-02). The specific steps include: adding 0.5 μL of recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C to the ArcticExpress(DE3)pRARE competent EP tube, ice bath for 30 min, 42°C heat shock for 90 s, ice bath for 2 min, add 1 mL LB medium, incubate at 37°C for 1 hour, centrifuge at 5000 rpm for 5 min, discard 700 μL supernatant, blow evenly and spread on resistance plate (plate contains 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol), 37°C culture for 24 hours, pick single bacteria and inoculate in LB medium (containing 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol), 37°C, 180 rpm shaking culture for 6 hours, store the bacterial liquid at -80°C at a ratio of 7:3 of bacterial liquid: 50% glycerol to obtain the recombinant strain ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C.

[0093] 1.3, Product expression and determination of expression form

[0094] (1) Normal temperature induction verification

[0095] The glycerol preserved recombinant strain Arctic Express (DE3) / pET-28a (+) -OVP1-2A-AVP1-3C in 1.2 was inoculated into 100 mL LB medium (the medium contained 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol) at 1%, and then was treated according to the following grouping, respectively, and the culture conditions were as follows: 37°C + 180 rpm rotation speed; after induction, the bacterial solution was centrifuged at 12000 rpm for 10 min, ultrasonically broken, centrifuged at 12000 rpm for 10 min, and 20 μL of the centrifugal supernatant was taken; the bacterial solution of group 1 was run another centrifugal precipitation, and the samples of each group were added with SDS-PAGE protein loading buffer (5x (Biyun Tian Biological; P0015L), boiled for 10 min, and then subjected to SDS PAGE detection.

[0096] Group 1: IPTG was added to a final concentration of 1 mM to induce culture for 6 hours;

[0097] Group 2: tetracycline (stock solution concentration 10 g / L) was added to a final concentration of 50 μL / L, and arabinose was added to a final concentration of 1.5 g / L to induce A-type VP1 and 3C protease for 6 hours.

[0098] Control group: the strain Arctic Express (DE3) transformed with pET-28a (+) empty vector was used as a control bacteria, and the same IPTG, tetracycline and arabinose were used for induction as controls, respectively.

[0099] The results are shown in Figure 2 Figure 2 ​Lane 1: supernatant of the strain ArcticExpress(DE3) transformed with pET-28a(+) empty vector induced by tetracycline; Lane 2: supernatant of the strain ArcticExpress(DE3) transformed with pET-28a(+) empty vector induced by arabinose; Lane 3: supernatant of the strain ArcticExpress(DE3) transformed with pET-28a(+) empty vector induced by 1 mM IPTG; Lane 4: supernatant induced by tetracycline and arabinose; Lane 5: supernatant induced by 1 mM IPTG; Lane 6: precipitate induced by 1 mM IPTG. The results show that two product bands (between 20 KD and 30 KD) appear in the precipitate of Lane 4, which indicates that both A-type VP1 and 3C protease are expressed; no obvious product appears in Lanes 1 to 3; obvious product bands appear in Lanes 5 and 6, which are presumed to be OP1-2A protein. It is shown that the recombinant strain ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C can express the target protein under the condition of 37℃ and 180 rpm.

[0100] (2) Low-temperature induction verification

[0101] The ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C is cultured under the condition of 37℃ and 180 rpm for 8 h, and then cooled to 16℃. The target protein is induced and cultured for 12 h under the condition of 16℃ after adding the inducers according to the following grouping. After ultrasonic disruption, the supernatant and the precipitate are centrifuged at 12000 rpm for 10 min, and then subjected to SDS-PAGE detection.

[0102] Group 1: the target protein is co-expressed by adding 0.05 mM IPTG, 5 μL / L (10 g / L of mother liquor) tetracycline and 0.1 g / L arabinose in the culture medium.

[0103] Group 2: the target protein is co-expressed by adding 0.05 mM IPTG in the culture medium.

[0104] The results are shown in Figure 3 , Figure 3Lane 1: IPTG, tetracycline and arabinose induced E. coli broken cell pellet; Lane 2: IPTG induced E. coli broken cell pellet; Lane 3: IPTG, tetracycline and arabinose induced E. coli supernatant; Lane 4: IPTG induced E. coli supernatant; Marker: Shanghai Yingnian Biotech Co., Ltd. product, item number: 20350ES72; 140 kDa, 115 kDa, 80 kDa, 70 kDa, 50 kDa, 40 kDa, 30 kDa, 25 kDa. Figure 3 The results show that the target protein can be successfully induced for expression, and the 3C protease cuts P1-2A into VP0, VP3 and VP1, and the protein is shown in lane 3, and part of the cutting product is unstable, and the cutting product is detected in the inclusion body, and P1-2A without induction of 3C protease also appears part of the inclusion body at low temperature, shown in lane 2, and most of them are soluble, shown in lane 4.

[0105] The supernatant of the bacterial liquid obtained in the above group 1 is diluted in PBS buffer and sent to the biophysics for electron microscope observation, and the results show that ArcticExpress(DE3) expressing pET-28a(+)-OVP1-2A-AVP1-3C at 16℃ under low IPTG, tetracycline and arabinose induction P1-2A can form O type 146S virus-like particles (VLPS) after cutting by 3C protease, and the electron microscope results are shown in Figure 4 Figure 4 The legend in the figure is 100 nm, and the electron microscope observation condition is 98000x, 100kV.

[0106] Example 2, fermentation and product purification of ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C

[0107] 2.1, high-density fermentation of E. coli in a fermentation tank

[0108] 2.1.1, preparation of seed liquid or plate culture

[0109] The ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C strain was cultured at 37℃, 180rpm in modified LB medium containing kanamycin for 12 hours.

[0110] The modified LB medium containing kanamycin is to add kanamycin to the modified LB medium to a concentration of 25μg / mL, wherein the composition of the modified LB medium is: 10g / L glycerol, 10g / L proteose peptone, 5g / L NaCL, 10g / L yeast powder, and the rest is water.

[0111] 2.1.2, preparation of secondary seed ​

[0112] The strain was cultured in LB medium containing 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol at 37°C, 220 rpm for 12 hours.

[0113] The fermentation medium was LB medium containing 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol, wherein the composition of the LB medium was: 10 g peptone, 5 g NaCL, 5 g yeast powder, and water was added to 1 L.

[0114] 2.1.3, fermentation production

[0115] The fermentation culture was divided into three stages:

[0116] 2.1.3.1, cell culture stage

[0117] After sterilizing 3 L of initial fermentation medium in a 5 L fermenter, antibiotics were added according to the amount of 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol, and seed liquid prepared according to 1% inoculation amount (volume ratio) was inoculated, and the culture was cultured at 37°C with aeration and stirring for 7 hours. During the culture process, as the strain grew, the sugar in the medium was gradually consumed, and when the carbon source was consumed, the cells stopped growing, and the dissolved oxygen (DO) returned to 10%. Ammonia was used to maintain the pH value at 7.2 during the culture process.

[0118] 2.1.3.2, source feeding stage

[0119] After confirming that the sugar was consumed in the previous step (using the standard of DO returning to 20% and the pH rising without adding acid), this step was started. The DO was controlled between 10-20% using the feed medium. The pH value was maintained at 72 using ammonia, and after 3 hours of this step, the expression was induced.

[0120] 2.1.3.3, induction expression stage

[0121] After 3 hours of feeding, the induction was started, the temperature was reduced to 16°C, IPTG was added to a final concentration of 0.05 mM, tetracycline was added to a final concentration of 5 μL / L with a mother liquor concentration of 10 g / L, and arabinose was added to a final concentration of 0.1 g / L. The DO was controlled between 10-30% using the feed, the pH value was maintained at 7.0 using NaOH during the entire induction process, and the fermentation broth was collected after 12 hours of induction.

[0122] The formula of the initial fermentation medium and the feed medium is as follows:

[0123] The initial fermentation medium was a modified M9 synthetic medium, which was composed of glucose 30 g / L, NH4CI 1.0 g / L, (NH4)2SO42.7 g / L, Na2HPO46.8 g / L, KH2PO43.0 g / L, NaCI 0.6 g / L, MgSO4·7H2O 0.2 g / L, vitamin B1 1.0 μg / L, trace element solution 10 mL / L; the trace element solution was composed of CaCI2·2H2O 0.55 g / L, FeCI3·6H2O 1.67 g / L, MnCI2·4H2O 0.10 g / L, ZnCI2 0.17 g / L, CuCI2·2H2O 0.04 g / L, CoCI2·6H2O 0.06 g / L, Na2MoO4·4H2O 0.06 g / L.

[0124] The composition of the fed-batch medium was: glucose 80 g / L, peptone 80 g / L, yeast powder 80 g / L, distilled water to 1 L, sterilized at 105°C for 10 min, and then used.

[0125] 2.1.4, Arctic Express (DE3) / pET-28a (+) -OVP1-2A-AVP1-3C antigen preparation process

[0126] The fermentation broth obtained in 2.1.3 was centrifuged at 5000 rpm for 10 min, and the bacterial cells were collected and stored at -20°C;

[0127] The collected bacteria were resuspended with PBS buffer containing 10% glycerol, then centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was collected. The collected precipitate was resuspended in PBS at a weight ratio of 1:10 and broken by ATS high pressure homogenization under ice bath, 2% triton X114 was added, stirred at 4°C overnight, preheated at 37°C for 2h, centrifuged at 12000 rpm for 20 min at 25°C to separate cell debris and triton X114, carefully take the supernatant, repeat centrifugation at 12000 rpm at 25°C to remove residual triton X114, take the supernatant and filter it through a 0.22μm filter membrane, collect the filtrate as antigen for standby, at the same time, do agar diffusion reaction experiment by quian extension method (see https: / / baike.baidu.com / item / %E5%85%8D%E7%96%AB%E6%89%A9%E6%95%A3%E8%AF%95%E9%AA%8C / 292020 for detection principle). The preparation method of foot-and-mouth disease O type positive serum is as follows: take the O type positive standard in O, A foot-and-mouth disease antibody ELISA detection kit produced by Lanzhou Veterinary Research Institute and mix it with Freund's incomplete adjuvant, then muscle injection for the first immunization of rabbits (age 100 to 120 days, body weight 2 to 3.0 kg, immunization dose 2 mL per rabbit), 21 days after the first immunization, strengthen the immunization, 28 days after the second immunization, collect blood and separate serum, and the rabbit positive serum is obtained. The results are shown in Figure 5 The results show that the rabbit positive serum prepared by O type standard antigen can form quian extension line and the quantitative antigen content can reach 1:128.

[0128] Example 3, preparation of antigen

[0129] 3.1, sterility test

[0130] All protein solutions were sterile filtered and tested according to the current "Chinese Veterinary Pharmacopoeia" appendix, which should be sterile growth.

[0131] 3.2, preparation and emulsification of antigen product OA series

[0132] 3.2.1, oil-water ratio

[0133] Emulsification was carried out according to the mass ratio of water phase: adjuvant = 1:1.

[0134] Take 2.1.4 batch antigen to prepare antigen, and the prepared product is named OA1: take 100 mL of 2.1.4 batch antigen and mix with Monanide TM ISA206 VG adjuvant 100 mL in emulsification cylinder, low speed stirring emulsification 30 minutes, control temperature 4℃, to prepare water-in-oil-in-water type OA1 antigen. The O type quian extension titer is not less than 1:64 head;

[0135] Take 2.1.4 batch antigen preparation antigen, prepared product named OA2: Take 2.1.4 batch antigen 50 mL plus 50 ml PBS and Monanide TM ISA206 VG adjuvant 100 mL mixed in emulsification cylinder, low speed stirring emulsification 30 minutes, control temperature 4℃, namely prepared water-in-oil-in-water type OA2 antigen. Among them, O type joint expansion titer is not less than 1:32 / head;

[0136] Take 2.1.4 batch antigen preparation antigen, prepared product named OA3: Take 2.1.4 batch antigen 25 mL and 75ml PBS and Monanide TM ISA206 VG adjuvant 100 mL mixed in emulsification cylinder, low speed stirring emulsification 30 minutes, control temperature 4℃, namely prepared water-in-oil-in-water type OA3 antigen. Among them, O type joint expansion titer is not less than 1:16 / head.

[0137] 3.3, product inspection

[0138] 3.3.1, nature

[0139] Appearance: visual observation test OA series antigen should maintain the white slightly sticky emulsion when dispensing.

[0140] Dosage form: take a clean pipette, take a little OA series antigen drops in clean cold water surface, should be cloud-like diffusion. Should maintain stable water-in-oil-in-water (W / O / W).

[0141] Stability: take OA series antigen 10 mL into centrifuge tube, centrifugation at 3000 r / min for 15 minutes, the water phase corresponding to the bottom of the tube should not be more than 0.5 mL, all meet the requirements.

[0142] 3.3.2, loading inspection according to the current "Chinese veterinary pharmacopoeia" appendix inspection, should meet the requirements.

[0143] 3.3.3, sterile test according to the current "Chinese veterinary pharmacopoeia" appendix test, should be sterile growth, results as shown in table 1.

[0144] Table 1. storage for different time 3 batch OA series antigen loading detection and sterile test results

[0145]

[0146] 3.4, safety inspection

[0147] 3.4.1, with small animals test

[0148] Fifteen guinea pigs (white guinea pigs, purchased from Vinton Li Hua breed, source) weighing 350 to 450 g were divided into 5 groups, and each group was subcutaneously injected with 2 mL of OA1, OA2, and OA3 series antigens, respectively. Fifteen mice (BALB / c breed, purchased from Vinton Li Hua source) weighing 18 to 22 g were divided into groups, and each group was subcutaneously injected with 0.5 mL of OA1, OA2, and OA3 series antigens, respectively. They were observed daily for 7 days, and no death, obvious local reaction, or systemic adverse reaction caused by injection of OA series antigens should occur. The results are shown in Table 2. No death occurred in experimental animals, and the prepared OA1, OA2, and OA3 series antigens were safe.

[0149] 3.4.2, Test with pigs

[0150] Six healthy susceptible piglets (Landrace three-way crossbred pigs, purchased from a pig farm in Baoding) aged 30 to 40 days were used, and each was injected with 4 mL (2 mL per pig) of OA series antigens in multiple sites of the muscle behind the ear root, and observed for 14 days. No abnormal reaction caused by OA series antigens should occur. The results are shown in Table 2. No death occurred in experimental animals, and the prepared OA1, OA2, and OA3 series antigens were safe.

[0151] Table 2. Safety test results of three batches of antigens stored for different periods of time

[0152]

[0153]

[0154] 4. Detection of antibody levels of three batches of OA1, OA2, and OA3 series antigens

[0155] 4.1, Experimental materials

[0156] Three groups were set up: high-dose OA1 group, medium-dose OA2 group, and low-dose OA3 group. Thirty healthy susceptible piglets (Landrace three-way crossbred pigs, purchased from a pig farm in Baoding) aged 30 to 40 days were selected, and each was injected with 2 mL of corresponding OA1, OA2, and OA3 series antigen products in the muscle behind the ear root. Booster immunization was performed once after 21 days, and each piglet was injected with 2 mL. There were 10 piglets in each group. O-type foot-and-mouth disease antibody was detected using O, A foot-and-mouth disease antibody ELISA detection kit produced by Lanzhou Veterinary Research Institute, and the test was performed under natural feeding conditions.

[0157] Foot-and-mouth disease antibody was detected by liquid phase blocking ELISA method recommended by OIE. O-type foot-and-mouth disease antibody titer was detected using O, A foot-and-mouth disease antibody ELISA detection kit produced by Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences (Chinese Academy of Agricultural Sciences, Lanzhou Veterinary Research Institute, batch number: REF: 99-43220, LOT SN: G131) according to the instructions of the kit.

[0158] According to the national major epidemic immune antibody detection standard, more than 70% of the immune pigs should have foot-and-mouth disease antibody not less than 1:64, and be judged as effective. The specific result judgment: taking 50% of the average OD492nm value of the virus antigen control as the critical value, the wells with OD492nm value of the detected serum greater than the critical value are negative wells, the wells less than or equal to the critical value are positive wells, and the corresponding dilution degree of the wells equal to the critical value is the antibody titer of the detected serum. The antibody titer higher than 1:40 is judged as foot-and-mouth disease type OO antibody positive; close to 1:40 is judged as suspicious and should be retested once. The titers of each type of antibody meet the national major epidemic immune antibody detection standard.

[0159] 4.2, Safety evaluation method and index

[0160] Safety evaluation method: within 14 days after injection of OA1, OA2 and OA3 series of antigen products, the local and systemic adverse reactions of the test pigs caused by the inoculation of the antigen products are observed regularly. Special attention is paid to the mental health status and feeding conditions of the pigs within 12 hours after the inoculation of the antigen products, and records are made, and corresponding emergency measures are taken.

[0161] Results: individual pigs in the high-dose group may have slight swelling at the injection site, mild body temperature rise, depression, and food reduction, but recover to normal after 3 days of vaccination, which is a normal reaction to antigen inoculation. No systemic reactions such as restlessness, convulsions, vomiting, rapid breathing, elevated body temperature, high depression, loss of appetite, lying down, etc. occur, and the test pigs cannot recover to normal within 7 days, or individual pigs die or abort due to injection of the antigen product, which is judged as unsafe antigen product. It can be judged as safe antigen product.

[0162] 4.3, Efficiency evaluation method and index

[0163] Serum antibody determination: the test pigs in each group were bled at 1, 2 and 3 months after immunization, and the serum antibody level was determined by ELISA method according to the method of 4.1, to detect the antibody induction level, time, antibody maintenance and growth rule of OA1, OA2 and OA3 series of antigens in clinical use, and to determine the effectiveness of the antigens. The results are shown in Table 3.

[0164] 4.4, Analysis of foot-and-mouth disease efficiency experiment results

[0165] Foot-and-mouth disease is listed as a mandatory report of the World Organization for Animal Health (OIE) as a highly contagious disease. At present, developing countries mainly adopt effective antigen immunization control strategy, and the foot-and-mouth disease inactivated antigen product widely used for immunization has the shortcomings of short immune duration and poor ability to induce cellular immunity. Therefore, we use the protein expression technology platform, especially after emulsification with ISA206 adjuvant, to conduct immunization experiments on pigs as experimental animals, the natural host of foot-and-mouth disease. The experimental animals of OA1, OA2, OA3 series antigen groups prepared by the application contain a higher content of antibody production at each time point, at the same time, this result also confirms that the foot-and-mouth disease core vlp antigen can also induce the immune system of animals. This shows that the antigen has the prospect of being applied to foot-and-mouth disease immunization, and the specific results are shown in Tables 3 and 4.

[0166] Table 3 O-type antibody detection results of primed pigs

[0167]

[0168]

[0169] Table 4 A-type antibody detection results of primed pigs

[0170]

[0171]

[0172] The application is described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In general, according to the principle of the application, the application is intended to include any changes, uses or improvements of the application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the application.

Claims

1. DNA, said DNA is a gene cluster OVP1-2A-AVP1-3C, said gene cluster OVP1-2A-AVP1-3C comprises an expression cassette of OVP1-2A, an expression cassette of VP1 of Type A foot-and-mouth disease virus and an expression cassette of 3C protease; the nucleotide sequence of said expression cassette of OVP1-2A is SEQ ID NO: 1 from 4984 to 7284, the nucleotide sequence of said expression cassette of VP1 of Type A foot-and-mouth disease virus is SEQ ID NO: 1 from 7285 to 8894, the nucleotide sequence of said expression cassette of 3C protease is SEQ ID NO: 1 from 8895 to 9741. said biological material is any one of the following:

2. Biological material associated with the DNA of claim 1, characterized in that, B1) a recombinant vector, said recombinant vector contains said DNA of claim 1; B2) a recombinant microorganism, said recombinant microorganism contains said DNA of claim 1 or contains said recombinant vector of B1); B3) a recombinant cell, said recombinant cell contains said DNA of claim 1 or contains said recombinant vector of B1); B4) an RNA molecule obtained by transcription of said DNA of claim 1. said method comprises the step of expressing said DNA of claim 1 in a biological cell to obtain said protein; said organism is E. coli.

3. A method of preparing a protein, characterized in that, said E. coli is E. coli BL21 (DE3).

4. The method of claim 3, wherein, 5. Use of said DNA of claim 1 in the preparation of a product for preventing infection of pigs by foot-and-mouth disease virus.

6. Use of said biological material of claim 2 in the preparation of a product for preventing infection of pigs by foot-and-mouth disease virus. ​

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