Preparation method and application of pig foot-and-mouth disease O-type and A-type bivalent virus-like particles

Abstract

The invention discloses a preparation method and application of pig foot-and-mouth disease type O and type A divalent virus-like particles, and belongs to the field of biological products. The technical problem to be solved by the invention is how to better realize the prevention effect of the foot-and-mouth disease of pigs. In order to solve the technical problem, the invention provides DNA (deoxyribonucleic acid), the DNA is a gene cluster OVP1-2A-AVP1-3C, and the gene cluster OVP1-2A-AVP1-3C comprises three expression cassettes, namely an expression cassette of OVP1-2A, an expression cassette of A type foot and mouth disease virus VP1 and an expression cassette of 3C protease. The invention also provides a foot-and-mouth disease bivalent antigen prepared by taking the DNA as a biological material. The foot-and-mouth disease bivalent antigen provided by the invention has high immunogenicity in pigs, and can induce a strong foot-and-mouth disease antibody reaction. Research results prove that the immune antigen provided by the invention is effective and has great potential and value of industrial transformation.

Description

Technical Field

[0001] The present application relates to the field of biological products, and specifically to a method for preparing and applying bivalent virus-like particles of swine foot-and-mouth disease type O and type A.

[0002] Priority application: This application claims the priority of the Chinese patent application (application number 202411967136.X) with an application date of December 30, 2024. The entire contents of the patent application are hereby incorporated by reference into this document. Background Art

[0003] Foot-and-mouth disease (FMD) is a highly contagious disease caused by infection with the FMD virus (FMDV), which causes significant economic losses to the pig industry worldwide. FMDV belongs to the Picornaviridae family and mainly infects even-toed ungulates. The genome of the virus is a stranded RNA (about 8.5 kb) surrounded by a protein coat of four structural proteins, namely VP1, VP2, VP3 and VP4. FMDV is divided into seven immunotypes: A, O, C, AsiaI and South African Territory (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 complete, assembled FMDV capsids to induce protective immune responses. Traditional FMD antigens are produced by chemical inactivation of purified, highly virulent FMDV virions, but this production method carries inherent risks for the accidental release of FMDV. Recombinant protein antigens are safer alternatives to traditional FMDV antigens because they express only the FMDV proteins or virus-like particles (VLPs) required to assemble empty FMDV capsids. Recombinant protein antigens all require the expression of FMDV3C protease in the cytoplasm to process the FMDVP1-2A polyprotein into individual viral capsid proteins (VP0, VP3, VP1). However, the wild-type FMDV3C protease is toxic and has low expression in host cells and host bacteria. Summary of the invention

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

[0006] In order to solve the above technical problems, this application provides the following technical solutions:

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

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

[0009] g11) The coding sequence is SEQ ID NO: 1, positions 5071-7284;

[0010] g12) a sequence having a homology of more than 75% with the sequence shown in g11);

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

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

[0013] g22) a sequence having a homology of more than 75% with the sequence shown in g21);

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

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

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

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

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

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

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

[0021] B3) a recombinant cell, the recombinant cell containing the DNA or the recombinant vector described in B1);

[0022] B4) RNA molecules obtained by transcription from said DNA.

[0023] As used herein, the term "vector" means any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell, such as a plasmid, cosmid, virus, phage, or linear or circular DNA.

[0024] The present application also provides a method for preparing a protein, which comprises the steps of expressing the DNA in a biological cell to obtain the protein.

[0025] In the above method, expressing the DNA in biological cells includes the step of introducing the gene cluster OVP1-2A-AVP1-3C into Escherichia coli through the recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C to obtain recombinant Escherichia 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 was performed with IPTG, tetracycline and arabinose.

[0028] The proteins include but are not limited to OVP1-2A, type A foot-and-mouth disease virus VP1 and 3C protease,

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

[0030] (a11) the amino acid sequence is the protein shown in SEQ ID NO: 2;

[0031] (a12) a sequence with 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 C-terminus of a11) or a12);

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

[0034] (a21) the amino acid sequence is the protein 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 C-terminus of a21) or a22);

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

[0038] (a31) the amino acid sequence is the protein 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 C-terminus of a31) or a32).

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

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

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

[0044] C1) Prokaryotic microorganisms;

[0045] C2) Gram-negative bacteria;

[0046] C3) Escherichia bacteria;

[0047] C4) Escherichia coli;

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

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

[0050] The protein can be used to prepare swine foot-and-mouth disease virus antigen.

[0051] The present application also provides a virus-like particle, wherein the virus-like particle contains the above-mentioned protein.

[0052] The present application also provides an application, wherein the application is any of the following:

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

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

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

[0056] A4) Use of the above virus-like particles in the preparation of products for preventing swine foot-and-mouth disease virus infection.

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

[0058] Furthermore, the antigen also includes an adjuvant.

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

[0060] The type of adjuvant is mineral oil.

[0061] The adjuvants include but are 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 this application, consistency refers to the consistency of an amino acid sequence or a nucleotide sequence. The consistency of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for a pair of amino acid sequences to calculate the consistency, and then the consistency value (%) can be obtained.

[0064] Herein, the identity may specifically be 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% identity.

[0065] In the above biological material, the expression cassette refers to a DNA capable of expressing the protein in a recombinant cell, which may include not only a promoter for initiating transcription of the protein encoding gene, but also a terminator for terminating transcription of the protein encoding gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0066] The term "recombinant vector" means a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide, and the polynucleotide is operably connected to a regulatory sequence provided for its expression. The recombinant vector comprises a polynucleotide of the present application, which is connected to one or more regulatory sequences, such as a promoter and transcription and translation termination signals, and the regulatory sequence directs the polypeptide to be produced in an expression host. Various nucleotides and regulatory sequences can be combined together to produce a recombinant vector, and the recombinant vector can include one or more convenient restriction sites to allow the insertion or replacement of the polynucleotide encoding the polypeptide at these sites. Alternatively, the polynucleotide can be expressed by inserting a nucleic acid construct or polynucleotide comprising the polynucleotide in a suitable vector for expression. In the process of preparing the expression vector, the coding sequence is placed in a vector, so that the coding sequence is operably connected to a suitable regulatory sequence for expression. The recombinant vector can be any vector (e.g., a plasmid or a virus) that can conveniently carry out a recombinant DNA step and can produce expression of the polynucleotide. The choice of the vector will generally depend on the compatibility of the vector with the recombinant cell into which the vector will be introduced. The vector can be a linear or closed circular plasmid. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring self-replication; alternatively, the vector may be a vector that, when introduced into a recombinant cell, is integrated into the genome and replicates together with the chromosome into which the vector is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids may be used that together contain the complete DNA to be introduced into the genome of the recombinant cell, or a transposon may be used.

[0067] The vector preferably contains one or more selective markers, which allow simple selection of cells transformed, transfected, transduced, etc. The vector preferably contains elements that allow the vector to be integrated into the recombinant cell genome or the vector to replicate independently of the genome in the cell. In order to be integrated into the recombinant cell genome, the vector can rely on the sequence of the polynucleotide encoding the polypeptide or any other vector element for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for guiding the precise position of integration into the host cell genome chromosome by homologous recombination. In order to increase the possibility of integration at the precise position, the integration element should contain a sufficient number of nucleic acids, such as 100 to 10000 base pairs, 400 to 10000 base pairs, and 800 to 10000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integration element may be any sequence, which is homologous to the target sequence in the host cell genome. In addition, the integration element may be a non-coding or encoding polynucleotide. On the other hand, the vector may be integrated into the genome of the recombinant cell by non-homologous recombination. For autonomous replication, the vector may further comprise an origin of replication, which enables the vector to replicate autonomously in the recombinant cell. The origin of replication may be any plasmid replicon that mediates autonomous replication and functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0068] More than one copy of the polynucleotide of the present application can be inserted into a recombinant cell to increase the production of the polypeptide. The increase in the number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome, or including an amplifiable selective marker gene in the polynucleotide, wherein cells containing an amplified copy of the selective marker gene and thus containing additional copies of the polynucleotide can be selected by culturing the cells in the presence of a suitable selection agent. Methods for connecting the above elements to construct the recombinant expression vector of the present application are well known to those skilled in the art.

[0069] The recombinant microorganisms can be bacteria such as Escherichia coli, mammalian cells, insect cells, or other expression systems such as yeast, algae, and plants. Mammalian cells can be 293, CHO, BHK, MDCK, etc., and insect cells can be SF9 and Hifv5.

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

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

[0072] The foot-and-mouth disease O-A bivalent candidate antigen provided in this application is highly immunogenic in pigs and can induce a strong foot-and-mouth disease antibody response. Our research results prove that the immune antigen provided in this application is effective and has great potential and value for industrial transformation. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0074] Figure 2 The expression results of the recombinant strain ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C; the sources of the products represented by each lane are as follows: Lane 1: supernatant of bacterial lysis of the pET-28a(+) empty vector-transformed strain ArcticExpress(DE3) induced by tetracycline; Lane 2: supernatant of bacterial lysis of the pET-28a(+) empty vector-transformed strain ArcticExpress(DE3) induced by arabinose; Lane 3: supernatant of bacterial lysis of the pET-28a(+) empty vector-transformed strain ArcticExpress(DE3) induced by 1mM IPTG; Lane 4: supernatant of bacterial lysis induced by tetracycline and arabinose; Lane 5: supernatant of bacterial lysis induced by 1mM IPTG; Lane 6: precipitate of bacterial lysis induced by 1mM IPTG;

[0075] Figure 3The expression results of the recombinant strain ArcticExpress (DE3) / pET-28a (+) -OVP1-2A-AVP1-3C under low temperature and low inducer are shown in Figure 2. The sources of the products represented by each lane are as follows: Lane 1: precipitation induced by IPTG, tetracycline and arabinose at a culture temperature of 16°C; Lane 2: precipitation induced by IPTG at a culture temperature of 16°C; Lane 3: supernatant induced by low IPTG, tetracycline and arabinose at 16°C; Lane 4: supernatant induced by IPTG at a culture temperature of 16°C;

[0076] Figure 4 Electron microscopic results of supernatant protein induced by ArcticExpress (DE3) expressing pET-28a(+)-OVP1-2A-AVP1-3C at 16°C;

[0077] Figure 5 The antigen content was quantified by agar diffusion method. DETAILED DESCRIPTION

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

[0079] The bivalent FMD O-A candidate antigen provided in this application is highly immunogenic in mice and pigs and can induce a strong FMD antibody response. Our results demonstrate the potential of the pET-28a(+)-OVP1-2A-AVP1-3C candidate antigen as an effective and affordable immunization antigen.

[0080] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0081] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0082] The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0083] The following examples used GraphPad Prism statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. The t-test was used for the test. P < 0.05 (*) indicated a significant difference, and P < 0.001 (***) indicated a very significant difference.

[0084] The present application is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present application, not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present application in any way.

[0085] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[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 were respectively implanted into different promoters (the T7 promoter that promotes the expression of O-type foot-and-mouth disease VP1-2A is provided by the backbone vector), and the coding sequence of the recombinant protein pET-28a(+)-OVP1-2A-AVP1-3C and the corresponding expression cassette were obtained.

[0089] Shanghai Bioengineering was commissioned to synthesize the coding gene of the recombinant protein OVP1-2A-AVP1-3C, and its nucleotide sequence is shown in SEQ ID NO: 1, positions 5071-9741. Among them, positions 5071 to 7284 of SEQ ID NO: 1 are the coding sequence of O-type foot-and-mouth disease VP1-2A (also known as OVP1-2A) (this gene encodes VP0, VP3 and VP1 core proteins), positions 7285-8252 are TETR promoters, positions 8253-8894 are the coding sequence of A-type foot-and-mouth disease VP1 (also known as AVP1), positions 8895 to 9093 are araBAD promoters, and positions 9094 to 9741 are the coding sequence of O-type foot-and-mouth disease 3C protease (also known as 3C protease). The sequence between NcoI and XhoI of pET28a(+) vector was replaced by the DNA molecule shown in positions 5071 to 9741 of SEQ ID NO: 1, and the obtained recombinant expression vector was named as recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C, and the nucleotide sequence of the recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C was SEQ ID NO: 1. Positions 4984 to 9741 of SEQ ID NO: 1 were named as gene cluster OVP1-2A-AVP1-3C, and the gene cluster OVP1-2A-AVP1-3C included three expression cassettes: the expression cassette of OVP1-2A, the expression cassette of type A 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, positions 4984 to 7281, wherein SEQ ID NO: 1, positions 4984 to 5002 are T7 promoters, SEQ ID NO: 1, positions 5003 to 5027 are lac operators, and SEQ ID NO: 1, positions 5071 to 7284 are the coding sequence of OVP1-2A. The nucleotide sequence of the expression cassette of type A foot-and-mouth disease VP1 is SEQ ID NO: 1, positions 7285 to 8894, wherein SEQ ID NO: 1, positions 7285 to 8252 are TETR promoters, and positions 8253 to -8894 are the coding sequence of type A 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, positions 8895 to 9741, wherein SEQ ID NO: 1, positions 8895 to 9093 are araBAD promoter, and positions 9094 to 9741 are the coding 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 as follows Figure 1As shown. The recombinant expression vector pET-28a (+) -OVP1-2A-AVP1-3C expresses a recombinant protein (peptide chain) having an amino acid sequence of SEQ ID NO: 2, an A-type foot-and-mouth disease VP1 (AVP1) having an amino acid sequence of SEQ ID NO: 3, and an O-type foot-and-mouth disease 3C enzyme (referred to as 3C protease) having an amino acid sequence of SEQ ID NO: 4. Among them, positions 1 to 737 of SEQ ID NO: 2 are the amino acid sequences of the O-type foot-and-mouth disease P1-2A core protein. In the middle and late stages of fermentation, under the action of 3C protease, P1-2A continues to be cleaved into VP0 having an amino acid sequence of SEQ ID NO: 2 positions 1 to 304, VP3 having an amino acid sequence of SEQ ID NO: 2 positions 305 to 524, and VP1 proteins having an amino acid sequence of SEQ ID NO: 2 positions 525 to 737, and assembles into a 5-hedron; VP0 continues to be cleaved and continues to be assembled into 146S.

[0091] 1.2. Expression of recombinant proteins

[0092] The recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C prepared in 1.1 was transformed into the host bacteria ArcticExpress(DE3)pRARE competent cells (Beijing Bomade Gene Technology Co., Ltd., catalog number: BC225-02). The specific steps include: adding 0.5 μL of the recombinant expression vector pET-28a(+)-OVP1-2A-AVP1-3C to an ArcticExpress (DE3) pRARE competent EP tube, placing it in an ice bath for 30 minutes, heat shocking at 42°C for 90S and placing it in an ice bath for 2 minutes, adding it to 1 mL of LB medium and culturing it at 37°C for 1 hour, centrifuging it at 5000 rpm for 5 minutes, discarding 700 μL of the supernatant, blowing the remaining precipitate evenly and coating it on a resistance plate (the plate contains 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, and 34 μg / μL chloramphenicol), culturing it at 37°C for 24 hours, picking a single bacterium and inoculating it in an LB medium (containing 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, 34 μg / μL chloramphenicol), cultured at 37°C, 180 rpm with shaking for 6 hours, and stored at -80°C with a ratio of 7:3 of bacterial solution: 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 ArcticExpress (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, and 34 μg / μL chloramphenicol) at 1%, and then grouped and treated as follows. The culture conditions were: 37°C + 180 rpm; after induction of expression, the bacterial solution was centrifuged at 12000 rpm for 10 min, ultrasonically broken the bacteria, and centrifuged at 12000 rpm for 10 min to take 20 μL of the centrifugal supernatant, of which the bacterial solution of group 1 was centrifuged again, and each group of samples was added with SDS-PAGE protein loading buffer (5× (Biyuntian Biotechnology; P0015L), boiled for 10 min, and then SDS PAGE detection was performed.

[0096] Group 1: IPTG was added to a final concentration of 1 mM and induced for 6 h;

[0097] Group 2: Tetracycline (stock 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 and cultured for 6 hours to induce type A VP1 and 3C protease.

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

[0099] The results are as follows Figure 2 As shown, Figure 2The middle lanes from left to right are the test results of samples 1-6; among them, lane 1: supernatant of bacterial lysis of pET-28a(+) empty vector transformed strain ArcticExpress(DE3) induced by tetracycline; lane 2: supernatant of bacterial lysis of pET-28a(+) empty vector transformed strain ArcticExpress(DE3) induced by arabinose, lane 3: supernatant of bacterial lysis of 1mM IPTGpET-28a(+) empty vector transformed strain ArcticExpress(DE3), lane 4: supernatant of bacterial lysis induced by tetracycline and arabinose; lane 5: supernatant of bacterial lysis induced by 1mM IPTG, lane 6: precipitate of bacterial lysis induced by 1mM IPTG. The results showed that two product bands (between 20KD and 30KD) appeared in the precipitate of lane 4, and it was speculated that both type A VP1 and 3C proteases were expressed; while there were no obvious products in lanes 1 to 3; lanes 5 and 6 had obvious product bands around 70-100KD, which were speculated to be OP1-2A proteins. This showed that the recombinant strain ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C could express the target protein at 37°C and 180rpm.

[0100] (2) Low temperature induction verification

[0101] ArcticExpress (DE3) / pET-28a (+)-OVP1-2A-AVP1-3C was cultured at 37°C and 180 rpm for 8 h, cooled to 16°C, and treated as follows. The inducer was added and induced at 16°C for 12 h to achieve soluble protein expression. After ultrasonic disruption, the cells were centrifuged at 12,000 rpm for 10 min, and the supernatant and precipitate were taken for SDS-PAGE detection.

[0102] Group 1: IPTG with a final concentration of 0.05 mM, tetracycline at 5 μL / L (stock solution concentration 10 g / L), and arabinose at 0.1 g / L were added to the culture medium to induce co-expression of the target protein;

[0103] Group 2: IPTG was added to the culture medium at a final concentration of 0.05 mM to induce co-expression of the target protein.

[0104] Results Figure 3 , Figure 3In the figure, lane 1: IPTG, tetracycline and arabinose induced Escherichia coli lysis precipitate; lane 2: IPTG Escherichia coli lysis precipitate; lane 3: IPTG, tetracycline and arabinose induced Escherichia coli lysis supernatant; lane 4: IPTG induced Escherichia coli lysis supernatant; Marker: product of Yishen Biotechnology (Shanghai) Co., Ltd., product number: 20350ES72; 140kDa, 115kDa, 80kDa, 70kDa, 50kDa, 40kDa, 30kDa, 25kDa. Figure 3 The results showed that the target protein could be successfully induced to express, and 3C protease cleaved P1-2A into VP0, VP3 and VP1, as shown in lane 3. Some of the cleavage products were unstable and were detected in inclusion bodies. P1-2A without induced 3C protease also showed some inclusion bodies at low temperature, as shown in lane 2, and most of it was soluble, as shown in lane 4.

[0105] The bacterial supernatant obtained from Group 1 was diluted into PBS buffer and sent to the Institute of Biophysics for electron microscopy observation. The results showed that ArcticExpress (DE3) expressed pET-28a(+)-OVP1-2A-AVP1-3C at 16°C, low IPTG, tetracycline and arabinose induced P1-2A to cleave the recombinant protein at 3C protease to form O-type 146S virus-like particles (VLPS). The electron microscopy results are shown in Figure 4 As shown, Figure 4 The legend in the middle figure is 100nm, and the electron microscope observation conditions are 98000×, 100kV.

[0106] Example 2, Fermentation of ArcticExpress (DE3) / pET-28a (+) -OVP1-2A-AVP1-3C and Product Purification

[0107] 2.1. High-density fermentation of E. coli in a fermenter

[0108] 2.1.1. Seed solution preparation or plate culture

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

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

[0111] 2.1.2. Secondary seed preparation

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

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

[0114] 2.1.3 Fermentation production

[0115] Fermentation culture is divided into three stages:

[0116] 2.1.3.1. Bacteria culture stage

[0117] After sterilization, 3 L of initial fermentation medium was placed in a 5 L fermenter, and antibiotics were added according to the amount of 50 μg / μL Kana, 34 μg / μL gentamicin, 34 μg / μL tetracycline, and 34 μg / μL chloramphenicol in the medium. The prepared seed solution was inoculated at a 1% inoculum (volume ratio), and 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. When the carbon source was consumed, the bacteria stopped growing, and the dissolved oxygen (DO) returned to 10%. Ammonia water 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 in the previous step is consumed (with DO rising by 20% and pH rising without adding acid as the standard), start this step. Use feed medium to control DO between 10-20%. Use ammonia water to maintain the pH at 72. After this step is maintained for 3 hours, start inducing expression.

[0120] 2.1.3.3. Induced expression stage

[0121] When induction started after 3 hours of feeding, the temperature was lowered by 16°C, IPTG was added to a final concentration of 0.05mM, tetracycline was added to a mother liquor concentration of 10g / L to a final concentration of 5μL / L, and arabinose was added to a final concentration of 0.1g / L. Feed was used to control DO between 10-30%. NaOH was used to maintain the pH at 7.0 during the entire induction process, and the fermentation broth was collected after 12 hours of induction.

[0122] Among them, the formulas of the initial fermentation medium and the feed medium are as follows:

[0123] The initial fermentation medium was a modified M9 synthetic medium, which consisted of 30 g / L glucose, NH4 Cl 1.0g / L, (NH 4 ) 2 SO 4 2.7g / L, Na 2 HPO 4 6.8g / L, KH 2 PO 4 3.0g / L, NaCl 0.6g / L, MgSO 4 7H 2 O 0.2g / L, vitamin B1 1.0μg / L, trace element solution 10mL / L; the trace element solution composition is: CaCl 2 ·2H 2 O 0.55g / L, FeCl 3 6H 2 O 1.67g / L, MnCl 2 ·4H 2 O 0.10g / L, ZnCl 2 0.17g / L,CuCl 2 ·2H 2 O 0.04g / L, CoCl 2 6H 2 O 0.06g / L, Na 2 MoO 4 ·4H 2 O 0.06g / L.

[0124] The composition of the feed medium is: 80 g / L glucose, 80 g / L peptone, 80 g / L yeast powder, distilled water is added to make up to 1 L, and sterilized at 105°C for 10 min before use.

[0125] 2.1.4. Preparation process of ArcticExpress(DE3) / pET-28a(+)-OVP1-2A-AVP1-3C antigen

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

[0127] The collected bacteria were resuspended in PBS buffer containing 10% glycerol, and 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 sterilized by ATS high-pressure homogenization in an ice bath, and 2% Triton 114 was added. The mixture was stirred overnight at 4°C, preheated at 37°C for 2h, and centrifuged at 12000rpm for 20min at 25°C to separate cell debris and Triton 114. The supernatant was carefully taken and centrifuged repeatedly at 12000rpm at 25°C to remove residual Triton 114. The supernatant was filtered through a 0.22μm filter membrane and the filtrate was collected as an antigen for later use. At the same time, the agar diffusion method (detection principle, 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) was used for agar diffusion reaction experiment. The preparation method of the O-type positive serum for foot-and-mouth disease is as follows: take the O-type positive standard in the O, A foot-and-mouth disease antibody ELISA test kit produced by Lanzhou Veterinary Research Institute and mix it with Freund's incomplete adjuvant, then inject it into rabbits (100 to 120 days old, 2 to 3.0 kg in weight, 2 mL / rabbit) intramuscularly for the first immunization, perform a booster immunization 21 days after the first immunization, and collect blood and separate serum 28 days after the second immunization to obtain rabbit positive serum. The results are shown in Figure 5 The results showed that rabbit positive sera prepared with type O standard flat antigen could form agar expansion lines and the quantitative antigen content could reach 1:128.

[0128] Example 3: Preparation of Antigen

[0129] 3.1 Sterility test

[0130] All protein solutions are sterile filtered and tested according to the current appendix of the Chinese Pharmacopoeia of Veterinary Medicine, and should be free of sterile growth.

[0131] 3.2. Preparation and emulsification of antigen products OA series

[0132] 3.2.1 Oil-water ratio

[0133] Emulsification was performed at a mass ratio of aqueous phase: adjuvant = 1:1.

[0134] Prepare antigen from batch 2.1.4. Name the prepared product OA1: Take 100 mL of antigen from batch 2.1.4 and add Monanide TM 100 mL of ISA206 VG adjuvant was mixed in an emulsifying tank, stirred at a low speed for 30 minutes, and the temperature was controlled at 4°C to obtain the water-in-oil-in-water formulation of OA1 antigen. The titer of O-type agar was not less than 1:64 per portion;

[0135] Prepare antigen from batch 2.1.4 and name the prepared product OA2: Take 50 mL of the antigen from batch 2.1.4 and add 50 ml of PBS and Monanide TM 100 mL of ISA206 VG adjuvant was mixed in an emulsifying tank, stirred at a low speed for 30 minutes, and the temperature was controlled at 4°C to obtain the water-in-oil-in-water formulation of OA2 antigen. The titer of O-type agar was not less than 1:32 / head portion;

[0136] Prepare antigen from batch 2.1.4 and name the prepared product OA3: Take 25 mL of antigen from batch 2.1.4 and 75 mL of PBS and Monanide TM 100 mL of ISA206 VG adjuvant was mixed in an emulsifying tank, stirred at a low speed for 30 minutes, and the temperature was controlled at 4°C to obtain the water-in-oil-in-water formulation of OA3 antigen, wherein the titer of O-type agar was not less than 1:16 / head portion.

[0137] 3.3. Finished product inspection

[0138] 3.3.1. Properties

[0139] Appearance: Visually observe that the OA series antigens should remain as a white, slightly viscous emulsion when packaged.

[0140] Dosage form: Take a clean pipette, suck a small amount of OA series antigen and drop it on the surface of clean cold water. It should spread in a cloud-like manner. A stable water-in-oil-in-water (W / O / W) type should be maintained.

[0141] Stability: Pipette 10mL of OA series antigen into a centrifuge tube and centrifuge at 3000r / min for 15 minutes. The water precipitated at the bottom of the tube should not exceed 0.5mL, which meets the requirements.

[0142] 3.3.2. The filling quantity inspection shall be carried out in accordance with the appendix of the current "Chinese Veterinary Pharmacopoeia" and shall comply with the regulations.

[0143] 3.3.3 Sterility test: The test shall be carried out according to the appendix of the current "Chinese Veterinary Pharmacopoeia". There should be no sterile growth. The results are shown in Table 1.

[0144] Table 1. Results of antigen loading and sterility tests for three batches of OA series stored at different times

[0145]

[0146] 3.4 Safety Inspection

[0147] 3.4.1. Testing with small animals

[0148] 15 guinea pigs weighing 350 to 450g (white guinea pigs, purchased from Vital River), 5 in each group, each group was subcutaneously injected with 2mL of OA1, OA2, and OA3 series antigens; 15 mice weighing 18 to 22g (BALB / c, purchased from Vital River), each group was subcutaneously injected with 0.5mL of OA1, OA2, and OA3 series antigens. Daily observation for 7 days, no death or 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 the experimental animals, and the prepared OA1, OA2, and OA3 series antigens were safe.

[0149] 3.4.2. Testing with Pigs

[0150] Six healthy susceptible piglets (Landrace hybrid pigs, purchased from a pig farm in Baoding) aged 30 to 40 days were used, and 4 mL of OA series antigens (2 doses) were injected into the muscles behind the ear roots on both sides, 2 mL each time, and observed for 14 days. No abnormal reactions caused by OA series antigens occurred, and the results are shown in Table 2. No experimental animals died, and the prepared OA1, OA2, and OA3 series antigens were safe.

[0151] Table 2. Safety test results of three batches of antigens stored at different times

[0152]

[0153]

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

[0155] 4.1 Experimental Materials

[0156] Divided into 3 groups: high-dose OA1 group, medium-dose OA2 group, and low-dose OA3 group, the experimental pigs were selected from 30 healthy susceptible piglets (Landrace hybrid pigs, purchased from a pig farm in Baoding) aged 30 to 40 days, and injected with 2mL of OA1, OA2, and OA3 series antigen products respectively through intramuscular injection behind the ear roots. After 21 days, booster immunization was given once, 2mL per head, and each group had 10 heads. O-type foot-and-mouth disease antibody ELISA detection kit produced by Lanzhou Veterinary Research Institute was used to detect O-type foot-and-mouth disease antibody, and the experiment was carried out under natural feeding conditions.

[0157] The antibody test for foot-and-mouth disease was performed according to the liquid phase blocking ELISA method recommended by OIE. The O, A foot-and-mouth disease antibody ELISA test kit produced by Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences (Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences, batch number: REF: 99-43220, LOT SN: G131) was used to detect the titer of O-type foot-and-mouth disease antibody according to the instruction manual of the kit.

[0158] According to the national standards for detecting antibodies to major epidemic diseases, the foot-and-mouth disease antibodies of more than 70% of immune pigs should not be less than 1:64, which is considered effective. Specific result determination: 50% of the average OD492 nm value of the virus antigen control is the critical value, the wells with OD492nm values ​​of the tested serum greater than the critical value are negative wells, the wells with OD492nm values ​​less than or equal to the critical value are positive wells, and the dilution corresponding to the positive well equal to the critical value is the antibody titer of the tested serum. Antibody titers higher than 1:40 are judged as positive for foot-and-mouth disease OO type antibodies; those close to 1:40 are judged as suspicious and should be retested once. The titers of various types of antibodies meet the national standards for detecting antibodies to major epidemic diseases.

[0159] 4.2 Safety evaluation methods and indicators

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

[0161] Results: Some pigs in the high-dose group had slight swelling at the injection site, slight temperature rise and depression, and decreased food intake, but returned to normal 3 days after the injection. These were normal reactions to the antigen product. No systemic reactions such as restlessness, convulsions, vomiting, rapid breathing, temperature rise, high mental depression, loss of appetite, and inability to lie on the ground occurred in the test pigs after the injection of the antigen product. If the pigs could not return to normal within 7 days, or if some pigs died or had a miscarriage due to the injection of the antigen product, the antigen product was judged to be unsafe. The antigen product can be judged to be safe.

[0162] 4.3 Effectiveness evaluation methods and indicators

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

[0164] 4.4 Analysis of the results of the foot-and-mouth disease efficacy experiment

[0165] Foot-and-mouth disease is listed as a legally reportable severe infectious disease by the World Organization for Animal Health. At present, developing countries mainly adopt the prevention and control strategy of effective antigen immunization, and the inactivated foot-and-mouth disease antigen products widely used for immunization have the disadvantages of short immunity duration and poor ability to induce cellular immunity. Therefore, we used the protein expression technology platform, especially after emulsification with ISA206 adjuvant, to conduct immunization experiments with pigs, the natural host of foot-and-mouth disease, as experimental animals. The experimental animals of the OA1, OA2, and OA3 series antigen groups prepared by the present invention contained high levels of antibody production at each time point. At the same time, this result also confirmed that the foot-and-mouth disease core vlps antigen can also induce immunity in animal bodies. This shows that the antigen has the prospect of being applied to foot-and-mouth disease immunization. The specific results are shown in Tables 3 and 4.

[0166] Table 3 Results of primary swine O-type antibody detection

[0167]

[0168]

[0169] Table 4 Results of primary swine type A antibody detection

[0170]

[0171]

[0172] The present application has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present application, and without the need to carry out unnecessary experimental conditions, the present application can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present application provides specific embodiments, it should be understood that further improvements can be made to the present application. In a word, according to the principles of the present application, the present application is intended to include any changes, uses or improvements to the present application, including departure from the disclosed scope in the present application and changes made with conventional techniques known in the art.

Claims

1. DNA, wherein the DNA is a gene cluster OVP1-2A-AVP1-3C, wherein the gene cluster OVP1-2A-AVP1-3C includes a coding sequence of OVP1-2A, a coding sequence of type A foot-and-mouth disease virus VP1, and a coding sequence of 3C protease; The coding sequence of OVP1-2A is any one of the following: g11) The coding sequence is SEQ ID NO: 1, positions 5071-7284; g12) a sequence having a homology of more than 75% with the sequence shown in g11); The coding sequence of the type A foot-and-mouth disease virus VP1 is any one of the following: g21) coding sequence is SEQ ID NO: 1, positions 8253-8894; g22) a sequence having a homology of more than 75% with the sequence shown in g21); The coding sequence of the 3C protease is any one of the following: g31) coding sequence is SEQ ID NO: 1, positions 9094-9741; g32) is a sequence with a homology of more than 75% with the sequence shown in g31).

2. The biological material related to DNA according to claim 1, characterized in that: The biological material is any of the following: B1) a recombinant vector comprising the DNA according to claim 1; B2) a recombinant microorganism containing the DNA according to claim 1 or the recombinant vector according to B1); B3) a recombinant cell, the recombinant cell containing the DNA according to claim 1 or the recombinant vector according to B1); B4) RNA molecules obtained by transcription from the DNA according to claim 1.

3. A method for preparing a protein, characterized in that: The method comprises the steps of expressing the DNA of claim 1 in biological cells to obtain the protein.

4. The method according to claim 3, characterized in that The organism is a microorganism, a plant or an animal.

5. The method according to claim 4, characterized in that The microorganism is any of the following: C1) Prokaryotic microorganisms; C2) Gram-negative bacteria; C3) Escherichia bacteria; C4) Escherichia coli; C5) Escherichia coli BL21(DE3).

6. A protein prepared by the method according to any one of claims 3 to 5.

7. A virus-like particle, characterized in that The virus-like particle contains the protein according to claim 6.

8. Application, characterized in that, The application is any of the following: A1) Use of the DNA according to claim 1 in the preparation of a product for preventing infection by swine foot-and-mouth disease virus; A2) Use of the biological material according to claim 2 in the preparation of a product for preventing swine foot-and-mouth disease virus infection; A3) Use of the protein according to claim 6 in the preparation of a product for preventing infection by swine foot-and-mouth disease virus; A4) Use of the virus-like particles according to claim 7 in the preparation of a product for preventing swine foot-and-mouth disease virus infection.

9. An antigen for preventing swine foot-and-mouth disease virus, comprising the protein of claim 6 and / or the virus-like particle of claim 7.

10. A method for preparing the antigen according to claim 8, characterized in that: The method comprises the step of mixing the protein of claim 6 and / or the virus-like particle of claim 7 with an adjuvant.

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