O-type foot-and-mouth disease virus-like particle antigen and preparation method and application thereof

Abstract

The present application relates to the technical field of virology, more particularly to O-type foot-and-mouth disease virus-like particle antigen and its preparation method and application. The O-type foot-and-mouth disease virus structural protein is constructed, and the recombinant expression vector comprises a Sumo-VP1 sequence, a Sumo-VP3 sequence, a Sumo-VP0 sequence and a chaperone sequence; the recombinant expression vector is transformed into a genetically engineered bacterium, and Sumo-VP1 protein, Sumo-VP3 protein, Sumo-VP0 protein and chaperone protein are expressed; the initial extraction solution is obtained by crushing; the purified extraction solution is obtained by purification; the self-assembled O-type foot-and-mouth disease virus-like particle antigen is obtained by enzyme cutting and removal of Sumo enzyme. The present application improves the soluble expression proportion and overall expression amount of the target protein in the supernatant by optimizing the construction mode and transformation strategy of the foot-and-mouth disease structural protein, and overcomes the technical bottleneck that the supernatant expression amount of the existing E. coli expression system is low and the inclusion body is prone to be formed.

Description

Technical Field

[0001] This invention relates to the field of virology, and more specifically, to type O foot-and-mouth disease virus-like particle antigen, its preparation method, and its application. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, highly contagious infectious disease of cloven-hoofed animals caused by the foot-and-mouth disease virus (FMDV), posing a serious threat to livestock farming. FMDV belongs to the genus FMDV in the family Picornaviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus with a genome approximately 8500 nt in length, containing a 5'UTR, ORF, 3'UTR, and Poly A. The ORF encodes proteins such as Lpro, P1, P2, and P3. The P1 gene further encodes structural proteins VP4, VP2, VP3, and VP1. These proteins assemble into sixty protomers, forming an icosahedral capsid. There are seven serotypes of the virus: O, A, C, Asia-1, SAT1, SAT2, and SAT3. Cross-protection between serotypes is weak, with serotypes O and A being the most prevalent.

[0003] Currently, the main vaccines for foot-and-mouth disease (FMD) are traditional attenuated live vaccines and inactivated vaccines. However, these two types of vaccines carry the risk of virus escape and cannot effectively distinguish between naturally infected animals and vaccinated animals, while also having high production costs. Therefore, the development of novel vaccines has become a research hotspot. Among them, virus-like particles (VLPs) vaccines, due to their nanoscale structure similar to natural virus particles and composed of structural proteins without viral genetic material, can effectively induce immune responses and distinguish between infected and immunized animals, showing promising application prospects. Currently, FMD virus-like particles are mostly produced using prokaryotic expression systems (such as E. coli), which have advantages such as simple operation, low cost, and rapid processing. However, in practical applications, the viral structural proteins expressed in prokaryotic systems often exist in the form of inclusion bodies, making it difficult to correctly fold into the spatial conformation required to form virus-like particles, resulting in low yields. Therefore, how to achieve high-yield production of FMD virus-like particles has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] The nucleotide sequence of the P1 gene of the O-type foot-and-mouth disease strain MYA98 / BY2010 used in this invention is as follows: GGAGCCGGAC AATCCAGTCC GGCTACTGGG TCACAGAACC AATCAGGCAA CACTGGGAGC ATCATCAACA ACTACTACAT GCAGCAGTAC CAGAACTCCA TGGACACCCA ACTTGGTGAC AACGCCATCA GCGGAGGCTC CAATGAGGGA TCCACGGACA CAACTTCCAC CCACACAACC AACACTCAGA ACAATGACTG GTTTTCAAAG TTGGCCAGCT CTGCTTTCAG CGGTCTTTTC GGCGCTCTTC TCGCCGACAA GAAAACCGAG GAGACCACTC TTCTCGAGGA CCGCATCCTC ACCACCCGAA ACGGACACAC CACTTCGACG ACCCAGTCGA GCGTTGGTGT AACGCACGGG TACGCAACAG CTGAGGACTT TGTGAGCGGG CCAAACACCT CTGGTCTCGA GACCAGAGTT GTCCAGGCGG AACGATTCTT CAAAACCCAC CTGTTCGACT GGGTCACCAC TGATCCGTTC GGACGGTGCC ACTTGTTGGA GCTCCCGACT GACCACAAAG GTGTCTACGG CGGCCTGATC GACTCATATG CCTATATGAG AAACGGTTGG GACGTTGAAG TCACCGCTGT GGGGAACCAG TTCAACGGAG GCTGCTTACT TGTGGCCATG GTACCTGAGC TTTGTTCCAT CGGGCAGAGA GAGCTGTTTC AACTCACACT CTTTCCCCAC CAGTTCATCA ACCCCCGGAC GAACATGACA GCCCACATCA AGGTGCCCTT TGTTGGCGTC AACCGCTACG ACCAGTACAA GGTACACAAG CCGTGGACCC TTGTGGTTAT GGTCGTAGCC CCACTGACCG TCAATACCGA GGGCGCCCCC CAAATCAAGG TGTACGCCAA CATCGCGCCC ACCAACGTGC ACGTCGCGGG TGAGTTCCCT TCCAAAGAGG GGATTTTCCC CGTGGCCTGT AGCGATGGTT ACGGCGGCTT GGTGACAACT GACCCAAAGA CGGCTGATCC CGTTTATGGC AAAGTGTTCA ACCCCCCCCG CAACATGTTG CCGGGGCGGT TCACCAACCT CCTGGATGTG GCCGAGGCTT GTCCTACGTT TCTGCACTTT GAAGGTGGCG TGCCATACGT GACCACAAAG ACGGACTCGG ACAGGATGCT CACACGATTT GACCTGTCTC TGGCAGCAAA ACATATGTCA AACACCTTCC TTGCAGGTCT TGCCCAGTAC TACACACAGT ACAGCGGAAC TATCAACCTG CATTTCATGT TCACAGGTCC CACTGACGCG AAAGCGCGTT ACATGATTGC ATATGCCCCC CCCGGCATGG AGCCGCCCAA AACACCTGAG GCTGCTGCTC ACTGCATTCA TGCAGAGTGG GACACGGGTC TGAACTCAAA GTTCACTTTT TCCATCCCCT ACCTTTCGGC GGCTGATTAC GCGTACACCG CGTCTGATAC CGCTGAGACC ACAAATGTTC AGGGGTGGGT CTGCCTGTTT CAAATAACAC ACGGGAAAGC TGATGGTGAC GCTCTTGTTG TGCTGGCCAG TGCCGGCAAA GACTTTGAGC TGCGCCTGCC TGTGGACGCC CGTCGAGAGA CCACGTCGAC AGGTGAATCG GCTGACCCCG TGACTGCCAC CGTTGAGAAC TACGGTGGTG AAACACAGGT TCAGAGACGC CACCACACAG ACGTCTCATT CATACTGGAT AGGTTTGTGA AAGTCACACC ACAAGACTCA ACAAACGTAT TGGACCTGAT GCAGACCCCT TCCCACTCCC TGGTGGGGGC CCTCCTCCGC ACTGCCACCT ACTACTTCGC CGATTTAGAG GTGGCGGTGA AACACAAGGG GGACCTCACC TGGGTGCCAA ACGGAGCACC TGAAGCAGCT CTGGATAACA CCACCAACCC AACGGCGTAC CACAAGCGC CGCTTACCCG GCTTGCACTG CCTTACACGG CACCACACCG TGTTATGGCC ACCGTTTACA ACGGGAACTG CAAATACGCC GGAGGCTCAC TGACCAACGT GAGAGGTGAT CTCCAAGTGC TGGCTCAGAA GGCGGCGCGG CCGCTGCCTA CTTCTTCAA CTACGGTGCC ATTAAAGCCA CCCGGGTGAC AGAACTGTTG TATCGCATGA AGAGGGCCGA GACGTACTGT CCCCGGCCCC TCTTAGCTGT CCACCCGAGT GAGGCTAGAC ACAAACAGAA AATAGTGGCA CCTGCAAAGC AGTCCTTG.

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide a method for preparing type O foot-and-mouth disease virus-like particle antigen, the specific steps of which are as follows: S1. Construct the structural protein of type O foot-and-mouth disease virus. The recombinant expression vector includes the Sumo-VP1 sequence, Sumo-VP3 sequence, Sumo-VP0 sequence, and molecular chaperone sequence. S2. The S1 recombinant expression vector was transformed into genetically engineered bacteria and expressed Sumo-VP1 protein, Sumo-VP3 protein, Sumo-VP0 protein and molecular chaperone protein; S3. The S2 genetically engineered bacteria were disrupted to obtain the initial extract; S4. Purify the initial extract from S3 to obtain a purified extract; S5. The purified extract from S4 was digested with Sumo enzyme and the Sumo enzyme was removed to obtain self-assembled type O foot-and-mouth disease virus-like particle antigen.

[0006] Preferably, the amino acid sequence corresponding to the Sumo-VP0 sequence is as shown in SEQ ID NO.2 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.2; and / or, The SEQ ID NO.2: GAGQSSPATG SQNQSGNTGS IINNYYMQQY QNSMDTQLGD NAISGGSNEG STDTTSTHTT NTQNNDWFSK LASSAFSGLF GALLADKKTE ETTLLEDRIL TTRNGHTTST TQSSVGVTYG YATAEDFVSG PNTSGLETRV VQAERFFKTH LFDWVTSDPF GRCHQLELPT DHKGVYGSLT DSYAYMRNGW DVEVTAVGNQ FNGGCLLVAM VPELCSIDKR GLYQLTLFPH QFINPRTNMT AHITVPFVGV NRYDQYKVHK PWTLVVMVVA PLIVNTEGAP QIKVYANIAP TNVHVAGEFP SKE; The amino acid sequence corresponding to the Sumo-VP1 sequence is as shown in SEQ ID NO.3 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.3; and / or, The SEQ ID NO.3: GIFPVACSDG YGGLVTTDDPK TADPAYGKVF NPPRNMLPGR FTNFLDVAEA CPTFLHFEGD VPYVTTKTDS DRVLAQFDLS LAAKHMSNTF LAGLAQYYTQ YSGTINLHFM FTGPTDAKAR YMIAYAPPGM EPPKTPEAAA HCIHAEWDTG LNSKFTFSIP YLSAADYAYT ASDAAETTNV QGWVCLFQIT HGKADGDALV VLASAGKDFE LRLPVDARTQ; The amino acid sequence corresponding to the Sumo-VP3 sequence is as shown in SEQ ID NO.4 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.4; The SEQ ID NO.4: TTSTGESADP VTATVENYGV ETQVQRRQHT DVSFILDRFV KVTPKDQINV LDLMQTPAHT LVGALLRTAT YYFADLEVAV KHEGNLTWVP NGAPETALDN TTNPTAYHKA PLTRLALPYT APHRVLATVY NGNCKYGESP VTNARGDLQV LAQKAARALP TSFNYGAIKA TRVTELLYRM KRAETYCPRP LLAIHPSAAR HKQKIVAPVK Q.

[0007] Preferably, the recombinant expression vector comprises a first shell recombinant vector, a second shell recombinant vector, and a molecular chaperone recombinant expression vector, wherein the first shell recombinant vector contains a Sumo-VP1-Sumo-VP3 sequence, and the second shell recombinant vector contains a Sumo-VP0 sequence; and / or, The recombinant expression vector includes a first recombinant vector and a second recombinant vector, wherein the first recombinant vector contains the Sumo-VP1-Sumo-VP3 sequence, and the second shell recombinant vector contains the Sumo-VP0 sequence and a molecular chaperone sequence.

[0008] Preferably, the recombinant expression vector further includes a molecular chaperone recombinant expression vector, wherein the molecular chaperone protein is DsbA protein, pG-KJE8 protein, pGro7 protein, pKJE7 protein, or pTf16 protein.

[0009] Preferably, the DsbA protein sequence is as shown in SEQ ID NO.5 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.5; The SEQ ID NO.5: MAQYEDGKQY TTLEKPVAGA PQVLEFFSFF CPHCYQFEEV LHISDNVKKK LPEGVKMTKY HVNFMGGDLG KDLTQAWAVA MALGVEDKVT VPLFEGVQKT QTIRSASDIR DVFINAGIKG EEYDAAWNSF VVKSLVAQQE KAAADVQLRG VPAMFVNGKY QLNPQGMDTS NMDVFVQQYA DTVKYLSEKK.

[0010] Preferably, a tag sequence is included before the Sumo fragment of the first recombinant expression vector; the purification of the initial extract to obtain a purified extract includes: Based on the tag sequence, the initial extract is purified to obtain a purified extract.

[0011] Another aspect of the present invention is to provide an application of type O foot-and-mouth disease virus-like particle antigen, said type O foot-and-mouth disease virus-like particle antigen for the preparation of vaccine compositions.

[0012] Another aspect of the present invention aims to provide a vaccine composition comprising an immunologic amount of the type O foot-and-mouth disease virus-like particle antigen and a pharmaceutically acceptable carrier.

[0013] Preferably, the pharmaceutically acceptable carrier is a biphasic adjuvant.

[0014] Preferably, the vaccine composition is used in the preparation of a medicament for the prevention and / or treatment of foot-and-mouth disease.

[0015] The beneficial effects of this invention are as follows: This invention significantly improves the soluble expression ratio and overall expression level of the target protein in the supernatant by optimizing the construction method and transformation strategy of foot-and-mouth disease structural proteins, overcoming the technical bottlenecks of low supernatant expression levels and easy inclusion body formation in existing E. coli expression systems. Verification showed that the expression supernatant obtained using the method described in this invention had the highest target protein content, providing sufficient and high-quality raw materials for subsequent assembly of virus-like particles (VLPs). Further in vitro assembly, verified by electrophoresis and transmission electron microscopy, confirmed the successful formation of morphologically uniform and structurally complete VLPs, indicating that the method described in this invention can effectively support the correct spatial conformational assembly of VLPs.

[0016] Furthermore, immunization experiments on rabbits showed that the VLP assembled in this invention could induce high levels of specific antibodies and neutralizing antibodies, exhibiting good immunogenicity. In summary, this invention provides a new technical means for developing efficient, safe, and mass-producible foot-and-mouth disease VLP vaccines, with promising industrial application prospects and economic benefits.

[0017] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is the verification of foot-and-mouth disease recombinant protein expression in Example 2 of the present invention; Figure 2 This refers to the purification effect detection of foot-and-mouth disease recombinant protein in Example 2 of the present invention; Figure 3 This is an SDS-PAGE electrophoresis image of the foot-and-mouth disease recombinant protease digestion assembly in Example 2 of the present invention; Figure 4 These are the electron microscopic observation results of the foot-and-mouth disease VLPs assembled in vitro in Example 2 of this invention; Figure 5 This is an SDS-PAGE electrophoresis image of DsbA fusion expression verification in Comparative Example 1 of this invention; Figure 6 This is an SDS-PAGE electrophoresis image of DsbA fusion expression affinity purification in Comparative Example 1 of this invention; Figure 7 This is an SDS-PAGE electrophoresis image of the DsbA fusion expression purified by ion exchange in Comparative Example 1 of this invention; Figure 8 This is an electrophoresis image of DsbA fusion expression verified by enzyme digestion in Comparative Example 1 of this invention; Figure 9 This is an electrophoresis diagram verifying the Sumo fusion expression in Comparative Example 2 of this invention. Detailed Implementation

[0020] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0022] Foot-and-mouth disease virus (FMDV) belongs to the Picornaviridae family and the Oral genus. This virus has seven serotypes: O, A, C, SAT1, SAT2, SAT3 (i.e., South African FMDV types 1, 2, and 3), and Asia1 (Asian type 1). There is no cross-protection between these serotypes, and each serotype contains multiple subtypes. At the center of the virus is a single-stranded positive-sense RNA, composed of approximately 8,000 bases, which is the basis for infection and heredity. The surrounding proteins determine the virus's antigenicity, immunogenicity, and serological reactivity. The viral capsid is a symmetrical icosahedron.

[0023] An antigen is a substance that can induce an immune response in the body. It is a substance that can be specifically recognized and bound by antigen receptors (TCR / BCR) on the surface of T / B lymphocytes, activate T / B cells, cause them to proliferate and differentiate, produce immune response products (sensitized lymphocytes or antibodies), and can specifically bind to the corresponding products in vivo and in vitro.

[0024] "Virus-like particles (VLPs)" are particles assembled from one or more viral structural proteins. They have an external structure and antigenicity similar to virus particles, but do not contain viral genes.

[0025] "Foot-and-mouth disease virus VP0, VP3, and VP1 antigen proteins": The FMDV structural protein precursor protein P1 is processed by protease 3C into VP0, VP3, and VP1. These three proteins can self-assemble into an icosahedral viral capsid. VP0 protein is an intermediate product of P1 after cleavage by protease 3C. In the final stage of virus particle formation, VP0 matures and cleaves into VP4 and VP2.

[0026] As used in this invention, the terms "vaccine" and "vaccine composition" refer to a pharmaceutical composition containing foot-and-mouth disease virus-like particle antigen, which can induce, stimulate, or enhance an immune response in pigs against foot-and-mouth disease.

[0027] The term "immune dose" should be understood as "immunely effective dose," also known as immune protective dose or effective dose to elicit an immune response. It refers to the amount of antigen that can effectively induce an immune response in a recipient, sufficient to prevent or improve the signs or symptoms of disease, including adverse health effects or complications thereof. This immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of disease, including adverse health outcomes or complications of infection caused by a pathogen. Humoral immunity or cell-mediated immunity, or both, can be induced. An animal's immune response to an immunogenic composition can be indirectly assessed, for example, by measuring antibody titers, lymphocyte proliferation analysis, or directly assessed by monitoring signs or symptoms after challenge with a wild-type strain. The protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs in the subject such as mortality, reduction in morbidity, temperature values, overall physiological status, and overall health and performance. The immune response may include, but is not limited to, the induction of cellular and / or humoral immunity.

[0028] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent in the vaccine composition of the present invention that does not irritate the body or hinder the biological activity and properties of the compound, except for the foot-and-mouth disease virus antigen. The term "adjuvant" may include aluminum gel adjuvants; water-in-oil emulsions; oil-in-water emulsions; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid, etc. The term "emulsion" may be particularly based on light liquid paraffin oil; isoprene-like oils produced by olefin oligomerization, etc.

[0029] "Transformation" refers to the process by which cells or cultured recipient cells acquire a new genetic phenotype through the automatic acquisition or artificial supply of exogenous DNA.

[0030] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called a recombinant expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, and cosmids.

[0031] The term "assembly" refers to the process by which structural proteins of a virus (such as capsid proteins) interact with each other or with nucleic acids to form regular granular structures. This includes the assembly of natural viral particles and the assembly of virus-like particles.

[0032] According to the present invention, the disruption of host cells can be achieved by various methods known to those skilled in the art, including but not limited to homogenization, homogenizer disruption, ultrasonic treatment, grinding, high-pressure extrusion, lysozyme treatment, etc.

[0033] The term "prevention and / or treatment" in the context of foot-and-mouth disease virus (FMD) infection refers to inhibiting FMD virus replication, inhibiting FMD virus transmission, or preventing FMD virus colonization in its host, as well as alleviating the symptoms of disease or symptom of FMD virus infection. Treatment is considered effective if viral load decreases, symptoms lessen, and / or food intake and / or growth increases.

[0034] The term "SUMO (small ubiquitin-associated modifier)" refers to SUMO-tagged proteins, which are small ubiquitin-associated modifiers. These are large proteins highly conserved in eukaryotes and involved in protein microubiquitination-related modifications. Compared to GST, MBP, or NusA, SUMO not only serves as a fusion tag for recombinant protein expression but also functions as a molecular chaperone, promoting proper protein folding, exhibiting heat and protease resistance, and contributing to the stability of the target protein. Furthermore, the SUMO tag has a specific protease that recognizes the SUMO tertiary structure, exhibiting highly specific cleavage without any amino acid residue, thus making it suitable for recombinant protein expression.

[0035] The term "biphasic adjuvant" refers to a water-in-oil-in-water adjuvant.

[0036] The term "molecular chaperones" refers to a class of proteins that play important roles in cells. They assist other macromolecular structures in a non-covalent manner to unfold and assemble into their biological functional states.

[0037] The term "conserved mutation" refers to mutations that occur in the amino acid sequence of a protein that do not significantly alter the protein's structure and function, or have only a minor effect on them.

[0038] Example 1: Construction of recombinant expression vector and screening of target clones The recombinant expression vector in this embodiment is an expression vector containing the Sumo-VP1 sequence, Sumo-VP3 sequence, Sumo-VP0 sequence, and the expression sequence of a molecular chaperone. The number of plasmids corresponding to this recombinant expression vector is unlimited, and can be 1, 2, 3, or 4. Based on factors such as the expression capacity of the vector promoter, this embodiment uses a first-shell recombinant vector, a second-shell recombinant vector, and a molecular chaperone recombinant expression vector to construct the corresponding plasmids. The first-shell recombinant vector is used to express the Sumo-VP1 and Sumo-VP3 proteins, the second-shell recombinant vector is used to express the Sumo-VP0 protein, and the molecular chaperone recombinant expression vector is used to express the molecular chaperone protein. Molecular chaperones have the function of assisting protein folding, which can improve the correct folding rate after viral capsid protein expression, thereby improving subsequent correct assembly, such as DsbA, pG-KJE8, pGro7, pKJE7, and pTf16.

[0039] First shell recombination carrier: pACYCDuet-1A-Sumo-VP1-Sumo-VP3 The O-type foot-and-mouth disease virus sequence used in this embodiment is derived from the O-type MYA98 / BY2010 foot-and-mouth disease virus strain. Based on this, and combining E. coli codon bias and artificial sequence optimization, the amino acid sequences SEQ ID NO.11 and SEQ ID NO.12 were designed. The corresponding gene sequences were synthesized using chemical synthesis methods.

[0040] In this embodiment, pACYCDuet-1A was used as the backbone vector. Sumo-VP1 was digested with NdeI / KpnI, and Sumo-VP3 was digested with NcoI / EcoRI. This resulted in a recombinant expression vector that simultaneously expresses both Sumo-VP1 and Sumo-VP3 proteins.

[0041] Second shell recombination carrier: pET21a-Sumo-VP0 Based on E. coli codon bias and artificial sequence optimization, the amino acid sequence SEQ ID NO. 10 was designed. The corresponding gene sequence was synthesized chemically. Then, the Sumo-VP0 gene sequence was inserted into the recombinant expression vector by enzyme digestion. In this embodiment, the recombinant expression backbone vector was pET21a, and the enzyme used for digestion was NdeⅠ / XhoⅠ to obtain the recombinant expression vector expressing the SUMO-VP0 protein.

[0042] Molecular chaperone recombinant expression vector: pET39b-DsbA Molecular chaperones can enhance the expression level of target proteins by assisting in the correct folding and maintaining stability of proteins. In this embodiment, in addition to the first and second shell recombinant vectors, molecular chaperone expression vectors are also constructed. When proteins on the three recombinant vectors are expressed simultaneously, the molecular chaperone proteins can prevent protein aggregation and promote correct folding by binding to the hydrophobic regions exposed by the newly synthesized polypeptide chains, thus providing the microenvironment required for folding. Examples include GroEL / GroES, Hsp70 / DnaK, and Hsp90. Alternatively, they can reduce misconformations by assisting folding, thereby reducing the proportion of target protein that is degraded.

[0043] In this embodiment, the molecular chaperone DsbA (Disulfide bond-forming protein A) is used as the molecular chaperone for subsequent co-expression. It is a disulfide bond oxidoreductase mainly found in the periplasmic space of *E. coli*, which significantly increases the expression level of soluble proteins by promoting the correct formation of disulfide bonds in exogenous proteins. After transformation into genetically engineered bacteria, DsbA catalyzes the correct pairing of cysteine ​​residues in the Sumo-VP0, Sumo-VP1, and Sumo-VP3 peptide chains through redox reactions, thus catalyzing the correct formation of disulfide bonds. It can also bind to the target protein, assisting in the formation of disulfide bonds during folding, reducing the exposure of hydrophobic regions caused by misfolding, thereby inhibiting protein aggregation, reducing inclusion body formation, and increasing protein solubility.

[0044] Based on the protein sequence shown in SEQ ID NO.5 and the gene sequence shown in SEQ ID NO.9, the gene sequence of DsbA protein was synthesized by chemical synthesis. The DsbA gene sequence was then inserted into an expression vector via enzyme digestion. In this embodiment, the expression vector used was pET39b, and the enzyme digestion was NdeI / XhoI.

[0045] The first capsid recombinant vector, the second capsid recombinant vector, and the molecular chaperone recombinant expression vector were co-transformed into competent Escherichia coli BL21(DE3), plated, and positive clones were selected.

[0046] Based on the resistance or marker proteins carried by the expression vector, a suitable screening method can be determined to screen for positive clones of the transformed strain. In this example, based on the selection of the vector, chloramphenicol, ampicillin, and kanamycin were used for screening. Through triple screening, clones with triple resistance were obtained and used as positive clones.

[0047] Positive clones were expanded through culture. They were inoculated into 25 mL of LB liquid medium and cultured at 37°C and 220 rpm / min. The LB medium contained 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl), 50 μg / mL kanamycin, 50 μg / mL ampicillin, and 34 μg / mL chloramphenicol.

[0048] Induced expression of Sumo-VP1, Sumo-VP3, Sumo-VP0 proteins and molecular chaperone proteins. When cultured to OD... 600 When the value reaches 0.6~0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1mM to induce the expression of recombinant protein, and adjust the temperature to 20℃ and culture at 220rpm / min for 6-8 hours.

[0049] Positive clones were screened based on expression levels to determine target clones for subsequent culture. After culture, absorbance was measured using OD600. The cells were collected by centrifugation at 8000 rpm / min for 10 min at 4°C. The cells were resuspended in PBS at a ratio of 1:10 (m:v) and disrupted using an ultrasonic homogenizer. After disruption, the cells were centrifuged at 8000 rpm / min for 10 min at 4°C and analyzed by SDS-PAGE. Figure 1 Based on the electrophoresis results, clones expressing the three proteins at the highest expression levels were selected as target clones for subsequent experiments. The target clones were kept incubated according to standard molecular biology methods.

[0050] If the corresponding number of plasmids is 2, the recombinant expression vector may contain a first recombinant vector containing the Sumo-VP1-Sumo-VP3 sequence and a second recombinant vector containing the Sumo-VP0 sequence and the DsbA sequence. For example, the first recombinant vector is pACYCDuet-1A-Sumo-VP1-Sumo-VP3 and the second recombinant vector is pACYCDuet-1K-Sumo-VP0-DsbA.

[0051] Example 2 Assembly of the viral capsid 2.1 Microbial culture Expand the target clone. Take 100 μl of the preserved target clone and inoculate it into a 10 ml LB / 100 ml Erlenmeyer flask. Add 10 μl of 50 mg / ml chloramphenicol solution and incubate at 37°C and 220 rpm until OD600 reaches 0.6–0.8 (approximately 3–4 h). Take 1 ml of the culture and inoculate it into a 1 L LB / 3 L Erlenmeyer flask. Add 1 ml of 50 mg / ml kanamycin solution, 1 mL of 50 mg / mL ampicillin, and 1 mL of 34 mg / mL CMR solution. Incubate overnight at 37°C and 220 rpm (approximately 14–16 h). Gram staining and microscopic examination confirm that the bacteria are Gram-negative and free from contamination.

[0052] The target clone was fermented. A primary fermentation was performed first. The culture temperature was 37℃, the stirring speed was 300 rpm, the air flow rate was 8 L / min, and the pH was 6.9. The pH of the fermentation medium was controlled using 2 M NaOH and 10% H2SO4, and the dissolved oxygen was set to 100%. The culture was continued until the OD reached... 600 ≈10~20, as the secondary seed culture. Then, the secondary seed culture was inoculated at a 6% inoculation rate into a 10 L fermenter containing 4 L of high-density fermentation basal medium, and fermentation culture began. When OD... 600 =15±2, cool to 20℃, add 1M IPTG to a final concentration of 1 mM, and induce culture for 6 hours. During the induction culture, when dissolved oxygen <10%, it is linked to the stirring speed (maximum stirring speed 600 rpm); when dissolved oxygen >10% and the stirring speed reaches 600 rpm, start uniformly adding nutrient solution at a flow rate of 0.27 mL / min·L, controlling dissolved oxygen =40%±5%. After the culture is completed, centrifuge at 4℃ and 8000 rpm for 30 minutes, and collect the bacterial cells.

[0053] During fermentation, the fermentation medium formulation includes 25-30 g / L glycerol, 35-45 g / L yeast extract, 1.5-2 g / L potassium dihydrogen phosphate, 1.5-2 g / L citric acid, 4.5-5.5 mL salt solution, and 0.8-1.2 mL trace metal salt solution. Specifically, in this example, the formulation consists of 26.7 g glycerol, 40.0 g yeast extract, 1.8 g potassium dihydrogen phosphate, 1.8 g citric acid, 5 mL salt solution, and 1 mL trace metal salt solution, with distilled water added to bring the volume to 1 L, and the pH value set to 7.2. Additionally, the fermentation medium may contain 1 mL of 10% antifoaming agent to prevent excessive foaming during fermentation. The salt solution b comprises 240-260 g / L MgCl2•6H2O, 90-110 g / L CaCl2•2H2O, and 90-110 g / L KCl, using 1.5-2.5 M citric acid as the solvent, with a preferred pH of 7.0-7.2. Specifically, in this embodiment, it consists of 250 g / L MgCl2•6H2O, 100 g / L CaCl2•2H2O, 100 g / L KCl, and 2 M citric acid. The trace metal salt solution comprises 6.5-7 g / L ZnCl2, 53-55 g / L FeCl3•6H2O, 16-16.5 g / L MnCl2•4H2O, 2-2.5 g / L CuSO4•5H2O, 4.5-5 g / L CoCl2•6H2O, and (NH4)6Mo7O. 24 • 4H₂O 0.023-0.025 g / L, KI 0.15-0.25 g / L, concentrated hydrochloric acid 118-120 mL. Specifically, this example contains 6.8 g ZnCl₂, 54.0 g FeCl₃•6H₂O, 16.2 g MnCl₂•4H₂O, 2.2 g CuSO₄•5H₂O, 4.8 g CoCl₂•6H₂O, and 0.024 g (NH₄)₆Mo₇O. 24 • Add 4H2O, 0.2 g KI, and 119 mL concentrated hydrochloric acid (37% by volume) to distilled water to bring the volume to 1 L.

[0054] In addition, during the fermentation process, a feed nutrient solution is added, which comprises the following components: 270-280 g / L glycerol and 220-230 g / L yeast extract. In this embodiment, the specific components are 275 g / L glycerol and 225 g / L yeast extract.

[0055] 2.2 Protein purification After collecting the bacterial culture, it was disrupted to obtain the initial extract. The culture was resuspended in PBS at a ratio of 1:10 (m:v), mixed thoroughly using a tissue disperser, and then homogenized using a high-pressure homogenizer at 800 bar for two consecutive cycles. The homogenate was then centrifuged at 8000 rpm for 30 minutes at 4°C to obtain the supernatant, which was used as the homogenate. To further clarify the homogenate, chitosan was added to the homogenate at a ratio of 100 ml:1 ml (homogeneous solution:1% chitosan), stirred for 15 minutes, allowed to stand at 4°C for 30 minutes, and then centrifuged at 7500 rpm for 30 minutes. The supernatant was collected to obtain the initial extract.

[0056] The initial extract was purified to obtain a purified extract. The initial extract contained various proteins from the bacteria, and the purpose of purification was to remove these contaminating proteins. Purification methods could include various approaches, such as utilizing protein molecular weight or pre-labeling the target protein. This embodiment uses a tag sequence approach. To avoid affecting the folding of proteins such as VPO, a tag sequence was included before the Sumo fragment of the first recombinant expression vector. Based on this tag sequence, the initial extract was purified to obtain the purified extract.

[0057] Tag sequences can be His tags, Flag tags, HA tags, etc. This example uses the His tag as an example to describe the purification process. Based on the specificity of the His tag, metal chelation chromatography, such as a Ni²⁺ affinity column, is used to purify the initial extract. First, imidazole is added to the initial extract to achieve a preset low concentration of imidazole, such as 75 mM. Then, the sample is loaded onto a chromatography column (Bio-Link Ni Chromstar FF chromatography medium, 50 / 200 column, column volume 185 ml) pre-equilibrated with Ni²⁺ affinity chromatography resin (20 mM PB, 1 M NaCl, 75 mM imidazole, pH 7.5). The low concentration of imidazole can compete with impurities that do not bind tightly to Ni²⁺, while the target protein remains stably bound to Ni²⁺ based on the His tag. After eluting impurities, the chromatography column is eluted again with elution buffer to obtain the initially purified protein solution A. In this embodiment, the elution buffer used was 0%-100% buffer B (20mM PB, 1M NaCl, 500mM imidazole, pH 7.5), with linear elution of 10 CV to purify the extract. SDS-PAGE was used to assess the purification effect. Figure 2 .

[0058] To reduce the impact of salt in the protein solution, the liquid purified by the tag sequence can be used as the primary purification solution for secondary purification to remove salt. Secondary purification can be performed using methods such as dialysis or ultrafiltration. In this example, G25 desalting is used for secondary purification. The primary purification solution is passed through a gel medium (such as Sephadex G-25 Fine chromatography medium, 50 / 300 column, column volume 432 ml). Small molecules (such as salt ions) can pass through, while large protein molecules are blocked outside the gel medium, thus achieving desalting and obtaining the purified extract. In this example, the buffer used for G25 desalting is 40 mM Tris, 500 mM NaCl, 5% glycerol, 5 mM CaCl2, pH 8.0.

[0059] 2.3 Enzyme digestion and assembly The purified extract was digested with Sumo enzyme, and the Sumo enzyme was removed to obtain self-assembled type O foot-and-mouth disease virus-like particles. The purified extract was then digested to obtain a digestion buffer. To ensure controllable digestion, the concentration of the digestion buffer was adjusted to 1 mg / mL, and then digestion was performed according to the recommended digestion ratio of SUMO enzyme:protein = 1:100-1:500 (m:m). In this embodiment, a recombinant protein:Sumo enzyme ratio of 1:150 was used to prepare the digestion buffer, and digestion was performed. The digestion temperature was 15℃, and the digestion system was placed on a shaker overnight (approximately 8 hours). The digestion buffer consisted of 5-50 mM Tris-HCl, 250-1000 mM NaCl, 0-20 mM CaCl2, and 0-20% glycerol, with a pH of 7.0-9.0.

[0060] Following enzyme digestion, similar to the protein purification methods described above, Sumo enzyme removal can be achieved by adjusting protein quality and tags. Since this example constructs a His-tagged Sumo vector, Sumo protein removal can be performed via the His tag. The enzyme digestion solution was passed through a Nie metal ion chelation column to remove the His-Sumo tag, thereby collecting a protein solution containing VP0, VP1, and VP3. Using this protein solution as a flow-through buffer, VLPs were assembled overnight at 15°C in assembly buffer to obtain self-assembled type O foot-and-mouth disease virus-like particles. The SDS-PAGE electrophoresis image of the enzyme digestion assembly is shown below. Figure 3 .

[0061] 2.4 Electron Microscopy Observation Finally, the self-assembled type O foot-and-mouth disease virus-like particles were examined by electron microscopy to determine whether the assembly was successful. Two drops of the assembled VLPs were placed on a 300-mesh copper grid using a burette, allowed to absorb at room temperature for 2-3 minutes, and then the remaining liquid on the copper grid was blotted dry with filter paper. The grid was then stained with 1% phosphotungstic acid, and the assembled VLPs were observed under a transmission electron microscope. Figure 4As shown in the electron micrographs, this embodiment can be confirmed to achieve efficient protein assembly. Furthermore, because the protein in this embodiment is soluble and has a low inclusion body content, high-yield virus-like particle preparation can be achieved.

[0062] Example 3: Immunogenicity experiment of foot-and-mouth disease VLP vaccine in rabbits To verify the immunogenicity of the O-type foot-and-mouth disease virus-like particles constructed in this embodiment, rabbits were used as immunized animals. The constructed O-type foot-and-mouth disease virus-like particles were injected into the rabbits, and their antibody levels were observed.

[0063] 3.1 Preparation of vaccine composition A foot-and-mouth disease (FMD) VLP vaccine composition was prepared in advance. The vaccine composition contained an immunizing dose of type O FMD virus-like particle antigen and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included biphasic adjuvants, such as 206 adjuvant; and aqueous adjuvants of polymeric nanoparticles, such as Freemix aqueous adjuvant.

[0064] This embodiment uses two different pharmaceutically acceptable carriers: 206 adjuvant and aqueous adjuvant. For 206 adjuvant, emulsification was performed at an antigen:adjuvant volume ratio of (1-3):(1-7); for aqueous adjuvant, emulsification was performed at an aqueous phase:oil phase volume ratio of (7-9):(3:1). Specifically, in 206 adjuvant emulsification, type O foot-and-mouth disease virus-like particles were slowly added to 206 adjuvant at an antigen:adjuvant volume ratio of 1:1, and emulsified at 220 rpm for 15 minutes. The emulsified vaccine was stored at 4°C. In aqueous adjuvant emulsification, type O foot-and-mouth disease virus-like particles were slowly added to aqueous adjuvant at an antigen:adjuvant ratio of 4:1, and emulsified at room temperature at 220 rpm for 15 minutes. The emulsified vaccine was stored at 4°C. The aqueous adjuvant used was Freemix (Luoyang Saiwei Biotechnology Co., Ltd.).

[0065] 3.2 Immunity Eleven rabbits were randomly divided into three groups: a 206 group, a water adjuvant group, and a control group. Immunization was administered via intramuscular injection of 50 mg / 1 ml / head in the hind leg. Serum was collected from the rabbits after two immunizations.

[0066] 3.3 Neutralizing antibody level detection The collected serum was analyzed for neutralizing antibodies using the MYA98 / BY / 2010 strain, and the results are shown in Table 1. In the control group, all rabbits were negative for neutralizing antibodies during both the first and second immunization periods, while in the experimental groups (both using 206 adjuvant and water adjuvant), the level of neutralizing antibodies rose slowly during the first immunization period. After the second immunization, the level of neutralizing antibodies in the experimental groups increased significantly from day 7. Therefore, the type O foot-and-mouth disease virus-like particles prepared in Example 2 can form high levels of specific antibodies. Within the experimental groups, compared with water adjuvant, the average neutralizing antibody level of 206 adjuvant was approximately 1:264 21 days after the second immunization, while the average level of water adjuvant was only 1:58. This further demonstrates that the vaccine composition consisting of type O foot-and-mouth disease virus-like particles obtained in Example 2 and a biphasic adjuvant, such as 206 adjuvant, has a better immunogenic effect than water adjuvant.

Claims

1. A method for preparing type O foot-and-mouth disease virus-like particle antigen, characterized in that: The specific steps of the preparation method are as follows: S1. Construct the structural protein of type O foot-and-mouth disease virus. The recombinant expression vector includes the Sumo-VP1 sequence, Sumo-VP3 sequence, Sumo-VP0 sequence, and molecular chaperone sequence. S2. The S1 recombinant expression vector was transformed into genetically engineered bacteria and expressed Sumo-VP1 protein, Sumo-VP3 protein, Sumo-VP0 protein and molecular chaperone protein; S3. The S2 genetically engineered bacteria were disrupted to obtain the initial extract; S4. Purify the initial extract from S3 to obtain a purified extract; S5. The purified extract from S4 was digested with Sumo enzyme and the Sumo enzyme was removed to obtain self-assembled type O foot-and-mouth disease virus-like particle antigen.

2. The method for preparing type O foot-and-mouth disease virus-like particle antigen according to claim 1, characterized in that: The amino acid sequence corresponding to the Sumo-VP0 sequence is as shown in SEQ ID NO.2 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.2; and / or, The SEQ ID NO.2: GAGQSSPATG SQNQSGNTGS IINNYYMQQY QNSMDTQLGD NAISGGSNEG STDTTSTHTT NTQNNDWFSK LASSAFSGLF GALLADKKTE ETTLLEDRIL TTRNGHTTST TQSSVGVTYG YATAEDFVSG PNTSGLETRV VQAERFFKTH LFDWVTSDPF GRCHQLELPT DHKGVYGSLT DSYAYMRNGW DVEVTAVGNQ FNGGCLLVAM VPELCSIDKR GLYQLTLFPH QFINPRTNMT AHITVPFVGV NRYDQYKVHK PWTLVVMVVA PLIVNTEGAP QIKVYANIAP TNVHVAGEFP SKE; The amino acid sequence corresponding to the Sumo-VP1 sequence is as shown in SEQ ID NO.3 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.3; and / or, The SEQ ID NO.3: GIFPVACSDG YGGLVTTTDPK TADPAYGKVF NPPRNMLPGR FTNFLDVAEA CPTFLHFEGD VPYVTTKTDS DRVLAQFDLS LAAKHMSNTF LAGLAQYYTQ YSGTINLHFM FTGPTDAKAR YMIAYAPPGM EPPKTPEAAA HCIHAEWDTG LNSKFTFSIP YLSAADYAYT ASDAAETTNV QGWVCLFQIT HGKADGDALV VLASAGKDFE LRLPVDARTQ; The amino acid sequence corresponding to the Sumo-VP3 sequence is as shown in SEQ ID NO.4 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO.4; The SEQ ID NO.4: TTSTGESADP VTATVENYGV ETQVQRRQHT DVSFILDRFV KVTPKDQINV LDLMQTPAHT LVGALLRTAT YYFADLEVAV KHEGNLTWVP NGAPETALDN TTNPTAYHKA PLTRLALPYT APHRVLATVY NGNCKYGESP VTNARGDLQV LAQKAARALP TSFNYGAIKA TRVTELLYRM KRAETYCPRP LLAIHPSAAR HKQKIVAPVK Q.

3. The method for preparing type O foot-and-mouth disease virus-like particle antigen according to claim 1, characterized in that: The recombinant expression vector includes a first shell recombinant vector, a second shell recombinant vector, and a molecular chaperone recombinant expression vector, wherein the first shell recombinant vector contains a Sumo-VP1-Sumo-VP3 sequence, and the second shell recombinant vector contains a Sumo-VP0 sequence; and / or, The recombinant expression vector includes a first recombinant vector and a second recombinant vector, wherein the first recombinant vector contains the Sumo-VP1-Sumo-VP3 sequence, and the second shell recombinant vector contains the Sumo-VP0 sequence and a molecular chaperone sequence.

4. The method for preparing type O foot-and-mouth disease virus-like particle antigen according to claim 1, characterized in that: The recombinant expression vector also includes a molecular chaperone recombinant expression vector, wherein the molecular chaperone protein is DsbA protein, pG-KJE8 protein, pGro7 protein, pKJE7 protein, or pTf16 protein.

5. The method for preparing type O foot-and-mouth disease virus-like particle antigen according to claim 4, characterized in that: The DsbA protein sequence is as shown in SEQ ID NO. 5 or has one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO. 5; The SEQ ID NO.5: MAQYEDGKQY TTLEKPVAGA PQVLEFFSFF CPHCYQFEEV LHISDNVKKK LPEGVKMTKY HVNFMGGDLG KDLTQAWAVA MALGVEDKVT VPLFEGVQKT QTIRSASDIR DVFINAGIKG EEYDAAWNSF VVKSLVAQQE KAAADVQLRG VPAMFVNGKY QLNPQGMDTS NMDVFVQQYA DTVKYLSEKK.

6. The method for preparing type O foot-and-mouth disease virus-like particle antigen according to claim 1, characterized in that: The first recombinant expression vector includes a tag sequence preceding the Sumo fragment; the purification of the initial extract to obtain a purified extract includes: Based on the tag sequence, the initial extract is purified to obtain a purified extract.

7. The application of the type O foot-and-mouth disease virus-like particle antigen according to any one of claims 1 to 6, characterized in that: The type O foot-and-mouth disease virus-like particle antigen is used to prepare vaccine compositions.

8. A vaccine composition, characterized in that: The vaccine composition comprises an immunizing dose of the type O foot-and-mouth disease virus-like particle antigen of claim 6 and a pharmaceutically acceptable carrier.

9. A vaccine composition according to claim 8, characterized in that: The pharmaceutically acceptable carrier is a biphasic adjuvant.

10. The use of the vaccine composition according to any one of claims 8 to 9 in the preparation of a medicament for the prevention and / or treatment of foot-and-mouth disease.

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