O-type foot-and-mouth disease nanoparticle antigen NP-OVPn, preparation method and application

Abstract

The invention provides an O-type foot-and-mouth disease nanoparticle antigen NP-OVPn as well as a preparation method and application thereof. The NP-OVPn provided by the invention is obtained by the following steps: after intracellular self-assembled O-type foot-and-mouth disease nanoparticle antigen NP-OVPn-T is subjected to TEV enzyme cleavage, removing T fusion-promoting tag protein through nickel affinity chromatography purification, and enabling antigen epitopes to be fully exposed on the surface of nanoparticle carrier protein NP. According to the FMDV O-type NP-OVPn nanoparticle antigen, three structural proteins of FMDV O-type VP1, VP2 and VP3 are displayed at the same time in the form of nanoparticles, the FMDV O-type NP-OVPn nanoparticle antigen can well and uniformly react with antibodies generated after immunization or infection of three animals including pigs, cattle and sheep, and the problem that the source of the FMDV O-type antigen is limited in FMDV clinical diagnosis is solved. The invention also provides a vaccine developed on the basis of the NP-OVPn nanoparticle antigen, and an FMDV O-type LB-ELISA antibody detection kit. The kit can be simultaneously used for quantitative detection of FMDV O-type specific antibodies from three different animals, namely pigs, cows and sheep, and has the advantages of high sensitivity, good specificity and high accuracy.

Description

Technical Field

[0001] The present invention relates to an O-type foot-and-mouth disease nanoparticle antigen, and specifically to an O-type foot-and-mouth disease nanoparticle antigen NP-OVPn, a preparation method and an application thereof. Technical Background

[0002] Foot-and-mouth disease virus (FMDV) is a highly pathogenic infectious virus that can infect cloven-hoofed animals such as pigs, cattle, and sheep. Diseased animals will show symptoms such as fever, lameness, and vesicular eruptions on the skin and mucous membranes, causing great harm to the breeding industry.

[0003] FMDV belongs to the Picornaviridae family and is divided into 7 serotypes: O, A, C, SAT1, SAT2, SAT3 (i.e., South African types 1, 2, and 3), and Asia1 (Asian type 1). There is almost no cross-immune protection between different serotypes. The FMDV genome is a single-stranded positive-sense RNA encoding four structural proteins: VP4, VP2, VP3, and VP1. It is reported that one molecule of VP2, VP3, and VP1 proteins can assemble into a 5S monomer, 5 5S monomers can assemble to form a 12S pentamer, and 12 pentamers can assemble into a complete 75S foot-and-mouth disease VLPs (Virus like particals). Research shows that the pentamer is the most basic unit for inducing protective antibodies in the body, while the complete 75S virus capsid can induce a higher level of neutralizing antibodies in the host, playing a better immune protection role. However, the efficiency of in vitro assembly of VP2, VP3, and VP1 proteins into 75S VLPs is generally low, and the industrialization is difficult, which greatly limits the research and development of FMDV-related subunit vaccines and their effective diagnosis in clinical practice.

[0004] In recent years, nanoparticles have become an emerging antigen preparation technology, and nanoparticle antigens possess many advantages that single or monovalent antigens do not have. Firstly, they can effectively increase the antigen valence; secondly, by attaching to a larger scaffold carrier, the antigen effectively improves its uptake and retention in lymphoid follicles after being presented by APCs; thirdly, through repeated arrangement, the antigen can achieve effective binding and activation of multiple B cell receptors. Currently, there are mainly three ways to display antigens on nanoparticles, namely encapsulation chemical coupling, gene fusion, and tag coupling. Among them, SpyTag / SpyCatcher has been widely used in the research and development of nanoparticle antigens due to its mild reaction conditions and stable structure. Currently, there have been studies on tandem display and expression of epitopes of different structural proteins of FMDV using nanoparticles, such as patent application CN 116286682A. However, new antigen epitopes are easily formed after epitope splicing and fusion, and the corresponding relationship between the fusion epitope and the antibody induced by the native structural protein is relatively poor, resulting in many deficiencies such as easy false positives and low detection accuracy in the application of diagnostic reagents.

[0005] Liquid blocking ELISA (LB-ELISA) is one of the most commonly used methods for accurately evaluating the FMDV antibody level clinically at present. However, due to the relatively limited source of its raw materials, and it is difficult for conventional raw materials to meet the need for simultaneously quantitatively detecting antibody levels from three different animal sources, pigs, cattle, and sheep, the quality of many commercially available kits for quantitatively detecting the clinical antibody level of foot-and-mouth disease is uneven. Summary of the Invention

[0006] In view of this, in order to overcome the deficiencies of the prior art, the present invention provides a nanoparticle antigen NP-OVPn of foot-and-mouth disease serotype O. The NP-OVPn nanoparticle antigen provided by the present invention contains all the antigenic epitopes of the structural proteins VP1, VP2, and VP3 of FMDV serotype O, and can react well and uniformly with the antibodies produced after immunization or infection of three animals, pigs, cattle, and sheep.

[0007] The NP-OVPn provided by the present invention is obtained by cleaving the intracellular self-assembled nanoparticle antigen NP-OVPn-T of foot-and-mouth disease serotype O with TEV protease, and then purifying and removing the T fusion tag protein by nickel affinity chromatography, so that the antigenic epitopes are fully exposed on the surface of the nanoparticle carrier protein NP. The antigenic epitopes of the NP-OVPn nanoparticle antigen are activated, and the antigen reactivity is greatly improved compared with the nanoparticle NP-OVPn-T.

[0008] The NP-OVPn-T is self-assembled by dehydration condensation reaction after the recombinant proteins SpyC-NP and foot-and-mouth disease type O recombinant antigen SpyT-OVPn-T are expressed in host cells; wherein NP is a nanoparticle carrier protein, OVPn is at least one of foot-and-mouth disease type O structural proteins VP1, VP2, and VP3, n represents an integer from 1 to 3, and T is a fusogenic tag protein; NP and OVPn-T are connected by an amide bond.

[0009] Most preferably, when n is 3, the antigen displayed by SpyC-NP has richer antigenic epitopes and higher antigen valence, which is beneficial to improving the positive detection rate of clinical serum samples.

[0010] The T is selected from the nucleotide sequence encoding the SUMO tag as shown in SEQ ID NO.12 or the nucleotide sequence encoding the GST tag as shown in SEQ ID NO.13.

[0011] Preferably, the nucleotide sequence encoding T is as shown in SEQ ID NO.12, and its encoded product SUMO protein is more conducive to the subsequent activation of antigenic epitopes.

[0012] The SpyT-OVPn-T is formed by fusing the coding sequences of SpyT, OVPn, and T in the "5'→3'" direction. In this fusion mode, it is not only beneficial to the full exposure of SpyT in the spatial structure to promote its effective binding with SpyC, but also beneficial to the effective cleavage of the T protein, thereby enabling the epitopes of OVPn to be fully exposed and activated.

[0013] The SpyC is selected from the nucleotide sequence encoding SpyCatcher003 as shown in SEQ ID NO.1, the nucleotide sequence encoding SpyCatcher002 as shown in SEQ ID NO.2, or the nucleotide sequence encoding SpyCatcher001 as shown in SEQ ID NO.3.

[0014] Most preferably, the nucleotide sequence encoding SpyC is as shown in SEQ ID NO.1, and its encoded product SpyCatcher003 has the highest efficiency of dehydration condensation reaction with SpyT.

[0015] Furthermore, the nanoparticle carrier protein NP is selected from ferritin Fn, bacteriophage AP205 coat protein, riboflavin synthase LS, dihydrolipoamide acetyltransferase E2p, or dodecahedral nanoparticle mi3, wherein the nucleotide sequence encoding ferritin Fn is as shown in SEQ ID NO.4, the nucleotide sequence encoding bacteriophage AP205 coat protein is as shown in SEQ ID NO.5, the nucleotide sequence encoding riboflavin synthase LS is as shown in SEQ ID NO.6, the nucleotide sequence encoding dihydrolipoamide acetyltransferase E2p is as shown in SEQ ID NO.7, or the nucleotide sequence encoding dodecahedral nanoparticle mi3 is as shown in SEQ ID NO.8.

[0016] Most preferably, the nucleotide sequence encoding NP is as shown in SEQ ID NO.8, and its encoded product mi3 can display a higher antigen valence of the expressed antigen, with less steric hindrance and a more stable nanoparticle structure formed.

[0017] Furthermore, the preparation method of the foot-and-mouth disease O nanoparticle antigen NP-OVPn-T includes the steps:

[0018] 1) Synthesis of the recombinant protein SpyC-NP: It is formed by fusing protein SpyC and protein NP. The nucleotide sequence encoding SpyC is as shown in SEQ ID NO.1.

[0019] 2) Synthesis of the recombinant antigen SpyT-OVPn-T: The nucleotide sequence encoding SpyT-OVP1-T is as shown in SEQ ID NO.14, the nucleotide sequence encoding SpyT-OVP2-T is as shown in SEQ ID NO.15, and the nucleotide sequence encoding SpyT-OVP3-T is as shown in SEQ ID NO.16.

[0020] 3) Cloning the recombinant protein SpyC-NP and the recombinant antigen SpyT-OVPn-T into the same expression vector or different expression vectors respectively.

[0021] 4) Transferring the expression vector into the same competent cell by the chemical transformation method.

[0022] 5) Under different temperature and inducer conditions, the competent cells transfected with the expression vector are induced to express the SpyC-NP and the SpyT-OVPn-T intracellularly successively; a dehydration reaction occurs between the SpyC-NP and the SpyT-OVPn-T and they self-assemble into NP-OVPn-T.

[0023] The SpyC at the N-terminus of the SpyC-NP protein contains an amino group "-NH2", which can react with the carboxyl group "-COOH" contained in SpyT at the N-terminus of the SpyT-OVPn-T protein to undergo the dehydration condensation reaction to form an amide bond "-NH—C=O-".

[0024] The SpyC-NP is formed by fusing SpyC and NP in the "5'→3'" direction, and its nucleotide coding sequence is preferably as shown in SEQ ID NO.9 (the fusion of SEQ ID NO.1 and SEQ ID NO.8). When SpyC is fused to the 5'-end of NP, it is more conducive to the self-assembly of SpyC-NP to form nanoparticles.

[0025] The SpyT is selected from the nucleotide sequence encoding SpyTag003 as shown in SEQ ID NO.10 and the nucleotide sequence encoding SpyTag001 as shown in SEQ ID NO.11.

[0026] Most preferably, when the nucleotide sequence encoding SpyT is as shown in SEQ ID NO.10, the efficiency of the dehydration condensation reaction between the encoded product SpyTag003 and SpyC is the highest.

[0027] Furthermore, the dehydration condensation reaction is spontaneously carried out between the recombinant protein SpyC-NP and at least one of the three recombinant antigens SpyT-OVP1-T, SpyT-OVP2-T, and SpyT-OVP3-T in the same host cell to achieve the display expression of at least one antigen protein among OVP1-T, OVP2-T, and OVP3-T on the surface of the nanoparticle carrier protein NP.

[0028] Furthermore, the expression vector in step 3) of the preparation method is selected from the pET28a vector or the pG-Tf2 vector.

[0029] Furthermore, the host cell in step 4) of the preparation method is selected from one of Escherichia coli BL21(DE3), BL21(DE3)Rosetta, BL21(DE3)Star, or one of Vero cells and Human T cells.

[0030] Furthermore, in step 5) of the preparation method, first regulate the expression of the SpyC-NP protein in the host cell, the final concentration range of the inducer tetracycline is 5 - 50 ng / mL, and the regulation temperature is 37℃±5℃; then regulate the expression of the SpyT-OVPn-T protein in the host cell, the final concentration range of the inducer isopropyl-β-D-thiogalactopyranoside IPTG is 50 - 200 mmol / L, and the regulation temperature is 20℃±5℃.

[0031] The present invention also provides a vaccine based on the above-mentioned O-type foot-and-mouth disease nanoparticle antigen NP-OVPn.

[0032] The present invention also provides a liquid-phase blocking ELISA antibody detection kit based on the above-mentioned O-type foot-and-mouth disease nanoparticle antigen NP-OVPn. The ELISA antibody detection kit includes: foot-and-mouth disease O-type NP-OVPn nanoparticle antigen, an enzyme-labeled plate coated with a polyclonal antibody specific to the NP-OVPn nanoparticle antigen, a sample diluent, a washing solution, a negative control, a positive control, a monoclonal antibody specific to the NP-OVPn nanoparticle antigen crosslinked with horseradish peroxidase conjugate, a chromogenic solution, and a termination solution.

[0033] Among them, the foot-and-mouth disease O-type NP-OVPn nanoparticle antigen is a recombinant antigen used in a liquid phase system, that is, a liquid-phase antigen.

[0034] The polyclonal antibody is prepared by immunizing experimental animals with the NP-OVPn nanoparticle antigen; the experimental animals are New Zealand white rabbits with big ears, Japanese white rabbits with big ears, or guinea pigs; preferably New Zealand white rabbits with big ears.

[0035] The monoclonal antibody is prepared by immunizing BALB / c mice with the NP-OVPn nanoparticle antigen and then through processes such as cell fusion, screening of hybridoma cells, and purification of ascites.

[0036] The foot-and-mouth disease O-type LB-ELISA antibody detection kit based on the nanoparticle antigen NP-OVPn can be used for the quantitative detection of FMDV O-type specific antibodies from three different animal sources, namely pigs, cattle, and sheep.

[0037] The beneficial effects of the present invention are as follows:

[0038] 1. The FMDV O-type NP-OVPn nanoparticle antigen described in the present invention simultaneously displays three structural proteins, VP1, VP2, and VP3 of FMDV O-type, in the form of nanoparticles, and can react well and uniformly with the antibodies produced after immunization or infection of three animals, pigs, cattle, and sheep, overcoming the problem of limited sources of FMDV O-type antigens in the clinical diagnosis of FMDV.

[0039] 2. The FMDV O-type LB-ELISA antibody detection kit developed based on the FMDV O-type NP-OVPn nanoparticle antigen in the present invention can be used for the quantitative detection of FMDV O-type specific antibodies from three different animal sources, pigs, cattle, and sheep, and has the advantages of high sensitivity, good specificity, and high accuracy.

[0040] 3. The NP-OVPn nanoparticles of the present invention simultaneously contain all the antigenic epitopes of three structural proteins. In terms of epitope abundance, epitope arrangement, and epitope accuracy, they not only retain all the advantages of natural virus antigens but also avoid relevant biosafety issues and research and development limiting factors. Among them, the epitope abundance is reflected in more abundant antigenic epitopes compared with tandem epitopes, representing a higher and more authentic positive detection rate in product applications and their performance; the epitope arrangement is reflected in a more natural spatial conformation compared with tandem epitopes, that is, a higher affinity between the antigenic epitope and the corresponding antibody; the epitope accuracy is reflected in that no new non-self antigenic epitopes are formed compared with tandem epitopes, representing a higher detection accuracy in product applications and their performance.

[0041] 4. The antigenic epitopes contained in the NP-OVPn nanoparticles of the present application are highly consistent in terms of reactivity and specificity with bovine, ovine, and porcine antibodies after optimization, that is, the same antigen can be used for the detection of FMDV antibody levels in bovine, ovine, and porcine sources simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present invention will be further described below with reference to the drawings:

[0043] Figure 1 It is a photo of the SDS-PAGE map for detecting the molecular weight of SpyC-NP and NP-OVPn-T nanoparticle proteins. M is the protein molecular weight standard, lane 1 is the non-induced supernatant, lane 2 is the SpyC-NP induced expression supernatant, and lane 3 is the NP-OVPn-T induced expression supernatant.

[0044] Figure 2 It is a photo of the SDS-PAGE map for detecting the molecular weight of NP-OVPn nanoparticle proteins. M is the protein molecular weight standard, lane 1 is the purified NP-OVPn-T, lane 2 is the NP-OVPn-T after digestion, and lane 3 is the NP-OVPn after removing the tag.

[0045] Figure 3 It is a photo of the transmission electron microscope observation result of NP-OVPn nanoparticles.

[0046] Figure 4This is a color comparison photo of NP-OVPn nanoparticles antigen on the NC membrane. M is the protein molecular weight standard. Lane 1 is porcine FMDV serotype O positive serum. Lane 2 is bovine FMDV serotype O positive serum. Lane 3 is ovine FMDV serotype O positive serum. Lane 4 is porcine FMDV serotype O negative serum. Lane 5 is bovine FMDV serotype O negative serum. Lane 6 is ovine FMDV serotype O negative serum. Lane 7 is classical swine fever virus positive serum. Lane 8 is porcine reproductive and respiratory syndrome virus positive serum. Lane 9 is pseudorabies virus positive serum. Lane 10 is porcine circovirus type 2 positive serum. Detailed implementation mode

[0047] Term explanation:

[0048] The term "Foot and mouth disease virus (FMDV)" is a picornavirus with high pathogenicity, which can infect cloven-hoofed animals such as pigs, cattle, and sheep. After infection, the diseased animals will show symptoms such as fever, lameness, and vesicular eruptions on the skin and mucous membranes.

[0049] The term "nanoparticle" refers to a protein complex with a certain spatial structure and in a granular shape assembled by a single subunit or multiple subunits. Under the electron microscope, its diameter is between 10nm and 500nm. It should be well-known to professionals in this field that nanoparticles can be used as a carrier to display other antigens through chemical coupling, gene fusion, and tag coupling. Generally, the nanoparticles after display have higher immunogenicity and immunoreactivity than the initial antigen.

[0050] The term "lysis" refers to the cleavage of the target protein by a specific enzyme under certain conditions, so that it is completely separated into two independent proteins.

[0051] The term "self-assembly" refers to some antigens with a high-level structure, whose structural proteins or constituent subunits can spontaneously assemble together to form a protein with a higher-level spatial structure under specific conditions.

[0052] The term "antigenic epitope" refers to a peptide segment that can stimulate the body to produce specific antibodies. It should be understood by professionals in this field that the peptide segment should be in a specific spatial conformation to have good specific binding with the antibody.

[0053] The operations such as "coating", "blocking", "washing", "drying", "patting dry", and "incubating" involved in the present invention are all conventional test operations in this field. Relevant technical personnel in this field should understand the specific referents, implementation processes, and methods of these operations.

[0054] Unless otherwise specified, the chemical reagents used in this application are all of analytical grade and obtained from commercial channels.

[0055] Unless otherwise specified, the biomaterials used in this application are all commercially available.

[0056] Unless otherwise specified, the experimental methods used in this application are all conventional methods.

[0057] The clinical serum samples involved in the present invention are all prepared by centrifuging the collected whole blood at 4°C and 4000 r / min for 5 min to separate the serum and adding Proclin 300 (48914-U, SIGMA) with a final concentration of 2% (W / W).

[0058] Unless otherwise specified, the preparation process of the phosphate (PBS) buffer used in this application is as follows: 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and the volume is made up to 1 L with water.

[0059] Unless otherwise specified, the preparation process of the PBST buffer used in this application: Add 0.05% (v / v) of Tween-20 to the PBS buffer.

[0060] Unless otherwise specified, the preparation process of the skim milk blocking solution used in this application: The skim milk powder is diluted with the PBST buffer, and the concentration of the skim milk blocking solution is 5% (w / v).

[0061] The present invention discloses an FMDV O type NP-OVPn nanoparticle antigen, a preparation method, as well as a vaccine based on this antigen and an FMDV O type LB-ELISA antibody detection kit. The following specific embodiments are used to further describe the present invention, but these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that without departing from the spirit and scope of the present invention, the details and forms of the technical solutions of the present invention can be modified or replaced, but these modifications and replacements all fall within the protection scope of the present invention.

[0062] Example 1 Coding gene sequence of NP-OVPn nanoparticle antigen

[0063] In this example, the SpyC-NP gene is formed by fusing SpyC and NP in the "5'→3'" direction, and its nucleotide coding sequence is shown in SEQ ID NO.9. The SpyT-OVPn-T gene is formed by fusing the coding sequences of SpyT, OVPn, and T in the "5'→3'" direction. The nucleotide sequence of SpyT-OVP1-T is shown in SEQ ID NO.14, the nucleotide sequence of SpyT-OVP2-T is shown in SEQ ID NO.15, and the nucleotide sequence of SpyT-OVP3-T is shown in SEQ ID NO.16.

[0064] Unless otherwise specified, this application commissioned General Biology (Anhui) Co., Ltd. to synthesize SpyT-OVP1-T, SpyT-OVP2-T and SpyT-OVP3-T genes, and simultaneously ligated the three coding genes to the pET28a vector, named pET28a:SpyT-OVPn-T.

[0065] Unless otherwise specified, this application commissioned General Biology (Anhui) Co., Ltd. to synthesize the SpyC-NP gene, and cloned the coding gene into the pG-Tf2 vector, replacing the coding gene of the original Tf protein on the vector. The constructed recombinant vector was named pG:SpyC-NP.

[0066] Table 1 Nucleotide sequence information of the coding gene:

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Example 2 Induced Expression of SpyC-NP and SpyT-OVPn-T Recombinant Antigens

[0074] First, the pET28a:SpyT-OVPn-T recombinant vector and the pG:SpyC-NP recombinant vector described in Example 1 were simultaneously transferred into the same BL21(DE3) competent cells by chemical transformation method. The obtained recombinant strain was named BL21(DE3) / pET28a:SpyT-OVPn-T / pG:SpyC-NP.

[0075] Secondly, the recombinant strain BL21(DE3) / pET28a:SpyT-OVPn-T / pG:SpyC-NP was inoculated into LB liquid medium (added with kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 20 μg / mL). After culturing at 37°C and 220 r / min for about 10 h, it was inoculated into an LB shake flask (a 500 mL shake flask with 200 mL of LB liquid medium) at an inoculation ratio of 1%. When the OD 600 reached about 0.4, tetracycline with a final concentration of 20 ng / mL (W / V) was added to the medium, and the culture was continued at 37°C and 200 r / min for 2 h to induce the expression of SpyC-NP protein (theoretical molecular weight is 38 KDa).

[0076] After the induction expression of SpyC-NP protein ended, the culture temperature was reduced to 20 °C, the rotation speed was kept at 200 r / min unchanged, and isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 150 mmol / L (M / V) was added to the culture medium to further induce the expression of SpyT-OVPn-T protein, and the induction duration was 8 h. Among them, the theoretical molecular weight of SpyT-OVP1-T protein is 38 KDa, the theoretical molecular weight of SpyT-OVP2-T protein is 39 KDa, and the theoretical molecular weight of SpyT-OVP3-T protein is 39 KDa.

[0077] When SpyT-OVP1-T, SpyT-OVP2-T, and SpyT-OVP3-T proteins were induced to express, they could undergo a dehydration condensation reaction with the SpyC-NP protein already expressed in the cells to form NP-OVPn-T nanoparticles, and its theoretical molecular weight is the sum of the molecular weights of SpyT-OVP1-T, SpyT-OVP2-T, SpyT-OVP3-T, and SpyC-NP proteins.

[0078] The bacterial cells were collected by centrifugation at 8000 r / min for 10 min, resuspended with PBS (8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, made up to 1 L), broken by an ultrasonic crusher (800 w, ultrasonic for 2 s, stopped for 5 s, ultrasonic duration 20 min), then centrifuged at 13000 r / min for 30 min to separate the supernatant and precipitate. The precipitate was resuspended with the above PBS buffer in equal proportion for SDS-PAGE detection. The results showed that a target protein band with a size of about 150 KDa could be detected in the supernatant, and the protein expression level was about 0.4 mg / mL ( Figure 1 as shown).

[0079] Example 3 Preparation and reactivity identification of NP-OVPn-T and NP-OVPn nanoparticles

[0080] The supernatant containing the NP-OVPn-T protein described in Example 2 was filtered through a 0.22 μm filter, and the target protein was purified using a protein purifier (Taidu Biotechnology Co., Ltd.). Because the NP-OVPn-T protein contains a His tag at the C-terminus, Ni NTABeads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) were used as filler. The column volume was approximately 10 mL, and the sample loading flow rate was 2 mL / min. Contaminants were eluted using a buffer containing 50 mmol / L imidazole, and the target protein was eluted using a buffer containing 500 mmol / L imidazole. After elution, the target protein was identified using SDS-PAGE, which showed that the purified NP-OVPn-T recombinant antigen was approximately 90% pure.

[0081] The purified NP-OVPn-T protein was digested with 1% (V / V) TEV enzyme at 20°C for 6 hours. The system after digestion contained two components, NP-OVPn nanoparticles and T protein. Ni NTABeads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) filler (column volume of about 5 mL) was used for purification, and the flow-through fraction, i.e., NP-OVPn protein, was collected for SDS-PAGE analysis. The results showed that the molecular weight of NP-OVPn protein was about 100KDa ( Figure 2 The prepared NP-OVPn nanoparticles were detected using a transmission electron microscope, and it was found that nanoparticles of about 150 nm were observed under the electron microscope, and the OVPn protein was uniformly distributed on the surface of the NP nanoparticles ( Figure 3 shown).

[0082] The reactivity and specificity of the purified NP-OVPn nanoparticle antigen were preliminarily verified using the Western blot method. First, the NP-OVPn antigen was subjected to SDS-PAGE and transferred to a NC membrane by wet transfer after electrophoresis. After blocking with 5% skim milk (prepared with PBST, where PBST is PBS supplemented with 0.05% Tween 20) at room temperature for 1 hour, FMDV O-type positive serum (pig serum, bovine serum, and sheep serum) obtained from clinical isolation, FMDV O-type negative serum (pig serum, bovine serum, and sheep serum), swine fever virus-positive serum, porcine blue ear disease virus-positive serum, porcine pseudorabies virus-positive serum, and porcine circovirus type 2 positive serum were used as primary antibodies. The serum was diluted 1:100 with 5% skim milk. After incubation with the primary antibody for 1 hour at room temperature, the membrane was washed three times with PBST. HRP-conjugated goat anti-pig, rabbit anti-bovine, or rabbit anti-goat antibodies were used as secondary antibodies. The secondary antibodies were diluted 1:50,000 in 5% skim milk and incubated at room temperature for 1 h. After washing three times with PBST, the cells were developed using DAB colorimetric solution (Kangwei Century Biotechnology Co., Ltd.).

[0083] As a result, the NP-OVPn nanoparticles antigen could react with FMDV type O positive sera (porcine serum, bovine serum, and ovine serum) to produce a uniform specific color reaction, and did not react with FMDV type O negative sera (porcine serum, bovine serum, and ovine serum), classical swine fever virus positive serum, porcine reproductive and respiratory syndrome virus positive serum, pseudorabies virus positive serum, and porcine circovirus type 2 positive serum, demonstrating that the NP-OVPn nanoparticles antigen had good specificity ( Figure 4 as shown).

[0084] Example 4 Preparation of Antibodies Specific to FMDV O-Type NP-OVPn Nanoparticle Antigen

[0085] In this example, the preparation process of the FMDV type O NP-OVPn nanoparticles antigen-specific polyclonal antibody mainly included the following steps:

[0086] Step S401: Prepare the immunogen. After diluting the NP-OVPn nanoparticles antigen described in Example 3 to 1 mg / mL, mix it with Freund's adjuvant (Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the immunizations after the first immunization) at a ratio of 1:1 (V / V), and use the push-pin method to emulsify and prepare the immunogen.

[0087] Step S402: Immunize the experimental animals by multi-point subcutaneous injection in the back using the emulsified immunogen described in Step S401. The protein immunization dose for experimental rabbits was 0.5 mg / rabbit, and the emulsified immunization volume was 1 mL / rabbit; the protein immunization dose for guinea pigs was 0.2 mg / guinea pig, and the emulsified immunization volume was 0.4 mL / guinea pig.

[0088] Step S403: Boost immunize the experimental animals described in Step S402 14 - 28 days after the first immunization. The immunization method and dose were the same as those for the first immunization. Collect the serum antibody titer 7 - 10 days after the boost immunization. After meeting the requirements, serum collection can be carried out.

[0089] Step S404: After collecting the serum in Step S403, use Protein A packing material to purify and prepare the FMDV type O NP-OVPn nanoparticles antigen-specific polyclonal antibody.

[0090] In this example, the preparation process of the FMDV type O NP-OVPn nanoparticles antigen-specific monoclonal antibody mainly included the following steps:

[0091] Step S405: The same as Step S401.

[0092] Step S406: Immunize mice by multi-point subcutaneous injection in the back with the emulsified immunogen described in Step S405, at a dose of 0.2 mL per mouse. Boost immunization is carried out 14 - 28 days after the first immunization, with the same method and dose as the first immunization. 14 - 60 days after the boost immunization, take 0.2 mL of antigen and perform boost immunization by intraperitoneal injection.

[0093] Step S407: PEG-fuse cells. Take the spleen of the immunized mice in Step S406 and mix it with the resuscitated SP2 / 0 cells at a ratio of spleen cells:SP2 / 0 cells = 5:1, and centrifuge at 1200 r / min for 3 min. Add 1 mL of PEG fusogen within 1 minute and stir the cells. After standing at room temperature for 90 s, add PBS to terminate.

[0094] Step S408: Spread the fused cells in Step S407 onto a 96-well cell culture dish and statically culture it in an incubator at 37°C and 5% CO2.

[0095] Step S409: Perform subcloning by the limiting dilution method. For the first subcloning of positive cell wells in Step S408, seed 2 cells per well and screen using HAT cell medium. For the second and third subclonings, seed 1 cell per well and screen using HT cell medium.

[0096] Step S410: After the three subcloning screenings described in Step S409 and with all the supernatant detection results of monoclonal wells being positive, change the medium to complete medium and continue to expand the culture and cryopreserve.

[0097] Step S411: Inject the hybridoma cells after the expansion culture described in Step S410 into the abdominal cavity of mice to prepare ascites. After collecting the ascites, purify it using Protein A packing material to prepare a monoclonal antibody specific to the NP-OVPn nanoparticle antigen of foot-and-mouth disease.

[0098] Label the purified monoclonal antibody described in Step S411 with HRP using the sodium periodate labeling method.

[0099] Example 5 FMDV O-Type LB-ELISA Antibody Detection Kit

[0100] In this example, the FMDV O type LB-ELISA antibody detection kit mainly includes the following components: foot-and-mouth disease O type NP-OVPn nanoparticle antigen, an enzyme-labeled plate coated with a polyclonal antibody specific to the NP-OVPn nanoparticle antigen, sample diluent, washing solution, negative control, positive control, a conjugate of a monoclonal antibody specific to the NP-OVPn nanoparticle antigen crosslinked with horseradish peroxidase, chromogenic solution (Solution A and Solution B), and termination solution.

[0101] Foot-and-mouth disease O type NP-OVPn nanoparticle antigen: The preparation process is as described in Examples 1 - 3.

[0102] Enzyme-linked immunosorbent assay (ELISA) plate coated with NP-OVPn nanoparticle antigen-specific polyclonal antibody

[0103] Coat the microplate with the NP-OVPn nanoparticle antigen-specific polyclonal antibody prepared in Example 4 at a coating concentration of 1-5 μg / mL and a coating amount of 0.1-0.5 μg / well using carbonate buffer at pH 9.6, and incubate overnight at 2-8°C; discard the coating solution, wash 2-4 times with phosphate buffer, and pat dry or aspirate dry; add blocking solution (phosphate buffer supplemented with 1% casein and 0.1% BSA) and incubate overnight at 2-8°C; discard the blocking solution, wash 3 times with phosphate buffer, and pat dry or aspirate dry; dry at 37°C for 2-3 h and then place in an aluminum foil bag containing desiccant, and store at 4°C for later use.

[0104] NP-OVPn nanoparticle antigen-specific monoclonal antibody cross-linked horseradish peroxidase conjugate

[0105] Dilute the horseradish peroxidase-labeled NP-OVPn nanoparticle antigen-specific monoclonal antibody described in Example 4 using phosphate buffer containing 1% (W / V) casein and 1% (W / V) sucrose at a dilution ratio of 1:(70000-80000) (V / V) to obtain the NP-OVPn nanoparticle antigen-specific monoclonal antibody cross-linked horseradish peroxidase conjugate.

[0106] Sample diluent: Phosphate buffer

[0107] Washing solution: Phosphate buffer containing 0.05% V / V Tween-20

[0108] Chromogenic solution: Comprising Solution A and Solution B. Among them, Solution A is prepared by adding 20 mg of TMB to 10 mL of absolute ethanol, making up the volume to 100 mL with ddH2O, mixing well and then aliquoting aseptically; Solution B is prepared by dissolving 2.1 g of citric acid, 2.82 g of anhydrous Na2HPO4, and 0.64 mL of 0.75% urea hydrogen peroxide in ddH2O, making up the volume to 100 mL, mixing well and then aliquoting aseptically.

[0109] Stop solution: 2 mol / L H2SO4

[0110] Positive control: NP-OVPn nanoparticle antigen

[0111] Negative control: Serum from pigs or sheep or cattle that is negative for FMDV detected by PCR method, or replaced by sample diluent.

[0112] In this example, the usage method of the foot-and-mouth disease type O liquid-phase blocking ELISA antibody detection kit based on NP-OVPn nanoparticle antigen mainly includes the following steps:

[0113] Step S501: Before use, all components of the kit should be restored to 20 - 26 °C. The reagents should be gently rotated or shaken to mix evenly.

[0114] Step S502: React the antigen and antibody (positive and negative controls and samples to be tested). According to different needs, select one of the layouts in Table 2 or Table 3 and operate on the serum dilution plate. Table 2 is the layout diagram for antibody titer determination (quantitative) to detect 10 samples; Table 3 is the layout diagram for antibody titer determination (quantitative) to detect 20 samples.

[0115] Table 2 Layout of 96 - well plate for detecting 10 samples

[0116]

[0117] Table 3 Layout of 96 - well plate for detecting 20 samples

[0118]

[0119] Step S503: Dilute the NP - OVPn nanoparticle antigen. Use the prepared washing solution to dilute the NP - OVPn nanoparticle antigen 450 - fold outside the wells (i.e., 1 part of NP - OVPn nanoparticle antigen plus 449 parts of washing solution). Taking Table 2 in Step S502 as an example for operation, add 75 μL of washing solution to each well of A1 - A10, add 50 μL of washing solution to each well of A11, A12, and B12, and add 50 μL of washing solution to each well of B1 - B11, C1 - C11, D1 - D11, E1 - E11, F1 - F11, G1 - G11, and H1 - H11. Add 25 μL of the sample to be tested to each well of A1 - A10, add 50 μL of the positive control to well A11, add 50 μL of the negative control to well A12, and use a multi - channel pipette to blow and mix evenly (pipette each well 6 - 7 times). The samples to be tested A1 - A10 are serially diluted 2 - fold from A to H at a volume of 50 μL / well; the positive control A11 is serially diluted 2 - fold from A to H at a volume of 50 μL / well; the negative control A12 is serially diluted 2 - fold from A to B at a volume of 50 μL / well. The samples to be tested are diluted from 1:4 to 1:512 in columns 1 - 10; the positive control is diluted from 1:2 to 1:256. Since the positive control provided by the kit is already diluted 1:8, it becomes diluted from 1:16 to 1:2048; the negative control is diluted from 1:2 to 1:4.

[0120] Step S504: Add the proportionally diluted NP-OVPn nanoparticle antigen described in step S503, 50 μL to each well in columns 1-11, 50 μL to each of wells A12 and B12, and 100 μL to each of wells E12, F12, G12, and H12. After adding the samples, double all dilutions, with the samples to be tested ranging from 1:8 to 1:1024, the positive control ranging from 1:32 to 1:4096, and the negative control ranging from 1:4 to 1:8. Apply a sealing film and mix well by shaking.

[0121] Step S505: Incubate the serum dilution plate with the added samples in step S504 at 37 °C for 2 h.

[0122] Step S506: Transfer the samples in the serum dilution plate described in step S505 to the corresponding wells on the coated plate in sequence, 50 μL per well. Apply a sealing film and incubate at 37 °C for 30 min.

[0123] Step S507: Discard the liquid in each well after incubation in step S506 into the waste liquid cylinder, wash the plate wells with 300 μL of washing solution for a total of 5 times. After each wash, discard the liquid in the well. After discarding the last wash solution, pat dry the remaining wash solution in the well on absorbent paper.

[0124] Step S508: Add the NP-OVPn nanoparticle antigen-specific monoclonal antibody conjugated with horseradish peroxidase to the dried plate wells in step S507, 50 μL per well. Apply a sealing film and incubate at 37 °C for 35 min.

[0125] Step S509: Repeat the operation of step S407 on the enzyme-labeled plate obtained in step S508, add the mixed chromogenic solution (chromogenic solution A and chromogenic solution B mixed at 1:1) at 50 μL per well, and incubate at 37 °C in the dark for 15 min.

[0126] Step S510: Add 50 μL of stop solution to each well of the enzyme-labeled plate obtained in step S509 to terminate the reaction. Measure and record the OD values (450 nm) of the samples and controls, and the readings are valid within 15 min.

[0127] According to the test results, when the antibody titer is between 1:64 and 1:128, it is judged as suspicious. The suspicious samples to be tested need to be retested. If the retested antibody titer ≥ 1:128, it is judged as positive; if < 1:128, it is judged as negative.

[0128] According to the actual corresponding relationship between the FMDV O-type LB-ELISA antibody detection kit provided in this application and the immune protection effect during clinical application, the FMDV O-type LB-ELISA antibody detection kit can provide the following guidance for the clinical immune prevention and control of FMDV O-type: when the antibody titer in bovine and ovine serum samples is ≥1:128, it can provide more than 99% protection; when the antibody titer is ≤1:16, there is no protection; when the antibody titer is between 1:22 and 1:90, the protection rate is 50%. When the antibody titer in porcine serum samples is ≥1:64, it can provide more than 99% protection; when the antibody titer <1:4, there is no protection; when the antibody titer is between 1:4 and 1:45, the protection rate is 50%.

[0129] Example 6 Specificity Study of FMDV O-Type LB-ELISA Antibody Detection Kit Based on NP-OVPn Nanoparticle Antigen Specificity Study

[0130] Collect FMDV porcine antibody-positive serum, FMDV bovine antibody-positive serum, FMDV ovine antibody-positive serum, Porcine circovirus type 2 (PCV-2) antibody-positive serum, Porcine Pseudorabies Virus (PRV) antibody-positive serum, African Swine fever virus (ASFV) antibody-positive serum, Porcine epidemic diarrheavirus (PEDV) positive serum, and Porcine reproductive and respiratory syndrome virus antibody-positive serum (PRRSV) from clinical sources. All the above collected clinical positive serum samples were verified by corresponding commercial ELISA antibody detection kits, and the verification results were all positive.

[0131] Use the FMDV O-type LB-ELISA antibody detection kit based on NP-OVPn nanoparticle antigen in Example 5 to detect the above different virus antibody-positive sera, and the detection results are shown in Table 4.

[0132] Table 4 Specificity detection results of different virus antibody-positive sera

[0133]

[0134]

[0135] It can be seen from the detection results in Table 4 that the FMDV O-type LB-ELISA antibody detection kit based on NP-OVPn nanoparticle antigen provided in this application has good specificity.

[0136] Example 7 Sensitivity Study of FMDV O-Type LB-ELISA Antibody Detection Kit Based on NP-OVPn Nanoparticle Antigen Sensitivity Study

[0137] Randomly select 1 portion of FMDV O-type positive serum (bovine origin), and perform serial dilution with PBS buffer at a dilution factor of 1:(2 - 1024) (V / V). Use the FMDV O-type LB-ELISA antibody detection kit based on NP-OVPn nanoparticles antigen in Example 5 to detect the positive sera at different dilution factors. The detection results are shown in Table 5. Use a commercially available kit to detect the positive sera at different dilution factors, and the detection results are shown in Table 6.

[0138] Table 5 Detection results of the FMDV O-type LB-ELISA antibody detection kit

[0139]

[0140] Table 6 Detection results of the commercially available kit

[0141]

[0142] According to the detection results, when the serum sample is diluted at 1:256 (V / V) for detection, the serum titer detection result of the FMDV O-type LB-ELISA antibody detection kit provided in this application is 1:128, while the serum titer detection result of the commercially available kit is 1:32. That is, the sensitivity of the FMDV O-type LB-ELISA antibody detection kit is about 4 times that of the commercially available kit.

[0143] The results of the sensitivity detection of FMDV O-type porcine origin positive serum and FMDV O-type ovine origin positive serum are relatively consistent with the detection results of the FMDV O-type bovine origin positive serum provided in this example. Therefore, in this example, only the FMDV O-type bovine origin positive serum is used as an example to elaborate on the advantages of the FMDV O-type LB-ELISA antibody detection kit provided in this application in terms of sensitivity.

[0144] Example 8 Clinical Application of FMDV O-Type LB-ELISA Antibody Detection Kit Based on NP-OVPn Nanoparticle Antigen Clinical Application

[0145] From a pig farm positive for FMDV O-type vaccine immunization and a negative pig farm for FMDV, 20 pigs were randomly selected from each for blood collection and serum separation. From a sheep farm positive for FMDV O-type vaccine immunization and a negative sheep farm for FMDV, 20 sheep were randomly selected from each for blood collection and serum separation. From a cattle farm positive for FMDV O-type vaccine immunization and a negative cattle farm for FMDV, 20 cattle were randomly selected from each for blood collection and serum separation. After randomly mixing the above 60 clinical positive serum samples from different species of animals and 60 clinical negative serum samples from different species of animals, the FMDV O-type LB-ELISA antibody detection kit provided by this application was used for antibody titer detection to investigate the compliance of serum titer with the clinical immunization background.

[0146] Table 7 Statistical results of antibody titer detection of 120 clinical serum samples

[0147]

[0148] According to the antibody titer statistical results described in Table 7 in this example, the FMDV O-type antibody levels in different farms are completely consistent with the actual immunization background, indicating that the use of the FMDV O-type LB-ELISA antibody detection kit provided by this application has certain guiding significance for the detection and evaluation of FMDV O-type clinical immunization effects or the infection screening of FMDV-negative farms.

[0149] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. An intracellular self-assembled foot-and-mouth disease O-type nanoparticle antigen NP-OVPn-T, characterized in that, The NP-OVPn-T is self-assembled by dehydration condensation reaction after the recombinant proteins SpyC-NP and foot-and-mouth disease type O recombinant antigen SpyT-OVPn-T are expressed in host cells; wherein NP is a nanoparticle carrier protein, OVPn is at least one of foot-and-mouth disease type O structural proteins VP1, VP2, and VP3, n represents an integer from 1 to 3, and T is a fusogenic tag protein; NP and OVPn-T are connected by an amide bond.

2. A nano-particle antigen NP-OVPn of foot-and-mouth disease of O type, characterized in that, The NP-OVPn is obtained by cleaving the intracellular self-assembled foot-and-mouth disease type O nanoparticle antigen NP-OVPn-T described in claim 1 with TEV enzyme, and removing the T fusogenic tag protein by nickel affinity chromatography, so that the antigenic epitope is fully exposed on the surface of the nanoparticle carrier protein NP.

3. The O-type foot-and-mouth disease nanoparticle antigen according to claim 1 or 2, characterized in that, The nanoparticle carrier protein NP is selected from ferritin Fn, bacteriophage AP205 coat protein, riboflavin synthase LS, dihydrolipoamide acetyltransferase E2p, or icosahedral nanoparticle mi3, wherein the nucleotide sequence encoding ferritin Fn is as shown in SEQ ID NO.4, the nucleotide sequence encoding bacteriophage AP205 coat protein is as shown in SEQ ID NO.5, the nucleotide sequence encoding riboflavin synthase LS is as shown in SEQ ID NO.6, the nucleotide sequence encoding dihydrolipoamide acetyltransferase E2p is as shown in SEQ ID NO.7, or the nucleotide sequence encoding icosahedral nanoparticle mi3 is as shown in SEQ ID NO.

8.

4. The preparation method of the O-type foot-and-mouth disease nanoparticle antigen NP-OVPn-T according to claim 1, wherein, The method includes the steps: 1) Synthesis of the recombinant protein SpyC-NP: It is formed by fusing protein SpyC and protein NP, and the coding nucleotide sequence of SpyC is as shown in SEQ ID NO.

1. 2) Synthesis of the recombinant antigen SpyT-OVPn-T: The coding nucleotide sequence of SpyT-OVP1-T is as shown in SEQ ID NO.14, the coding nucleotide sequence of SpyT-OVP2-T is as shown in SEQ ID NO.15, and the coding nucleotide sequence of SpyT-OVP3-T is as shown in SEQ ID NO.

16. 3) Cloning the recombinant protein SpyC-NP and the recombinant antigen SpyT-OVPn-T into the same expression vector or different expression vectors respectively. 4) Transferring the expression vector into the same competent cell by chemical transformation method. 5) Under different temperature and inducer conditions, the competent cells transfected with the expression vector are induced to express the SpyC-NP and the SpyT-OVPn-T intracellularly successively; a dehydration reaction occurs between the SpyC-NP and the SpyT-OVPn-T and they self-assemble into NP-OVPn-T.

5. The preparation method of the foot-and-mouth disease type O nanoparticle antigen NP-OVPn-T according to claim 4, characterized in that The dehydration condensation reaction is spontaneously carried out by the recombinant protein SpyC-NP and at least one of the three recombinant antigens SpyT-OVP1-T, SpyT-OVP2-T, and SpyT-OVP3-T in the same host cell to achieve the display and expression of at least one antigen protein among OVP1-T, OVP2-T, and OVP3-T on the surface of the nanoparticle carrier protein NP.

6. The preparation method of the foot-and-mouth disease O type nanoparticle antigen NP-OVPn-T according to claim 4, wherein the expression vector in step 3) is selected from the pET28a vector or the pG-Tf2 vector.

7. The preparation method of the foot-and-mouth disease O type nanoparticle antigen NP-OVPn according to claim 4, wherein the host cell in step 4) is selected from one of Escherichia coli BL21(DE3), BL21(DE3)Rosetta, BL21(DE3)Star, or one of Vero cells and Human T cells.

8. The preparation method of the foot-and-mouth disease O type nanoparticle antigen NP-OVPn according to claim 4, wherein in step 5), first regulate the expression of the SpyC-NP protein in the host cell, the final concentration range of the inducer tetracycline is 5-50 ng / mL, and the regulation temperature is 37°C ± 5°C; then regulate the expression of the SpyT-OVPn-T protein in the host cell, the final concentration range of the inducer isopropyl-β-D-thiogalactopyranoside IPTG is 50-200 mmol / L, and the regulation temperature is 20°C ± 5°C.

9. A vaccine based on the foot-and-mouth disease O type nanoparticle antigen NP-OVPn according to claim 2.

10. A liquid-phase blocking ELISA antibody detection kit based on the O-type foot-and-mouth disease nanoparticle antigen NP-OVPn described in claim 2, wherein the ELISA antibody detection kit comprises: Foot-and-mouth disease O type NP-OVPn nanoparticle antigen, enzyme-linked immunosorbent assay plate coated with polyclonal antibodies specific to the NP-OVPn nanoparticle antigen, sample diluent, washing solution, negative control, positive control, NP-OVPn nanoparticle antigen-specific monoclonal antibody cross-linked horseradish peroxidase conjugate, chromogenic solution, and termination solution.

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