Intracellular self-assembly type A foot-and-mouth disease nanoparticle antigen NP-AVPn, preparation method and application

Abstract

The invention provides a type A foot-and-mouth disease nanoparticle antigen NP-AVPn, a preparation method and an application. The NP-AVPn provided by the invention is obtained by carrying out TEV enzyme cracking on intracellular self-assembled A type foot-and-mouth disease nano-particle antigen NP-AVPn-T, and then carrying out nickel affinity chromatography purification to remove T fusion-promoting tag protein, so that antigen epitopes are fully exposed on the surface of nano-particle carrier protein NP. According to the FMDVA type NP-AVPn nanoparticle antigen disclosed by the invention, three structural proteins of FMDVA type VP1, VP2 and VP3 are simultaneously displayed in a nanoparticle form, so that the FMDVA type NP-AVPn nanoparticle antigen can be simultaneously subjected to good and uniform reaction with antibodies generated after immunization or infection of three animals of pigs, cattle and sheep, and the problem that the FMDVA type antigen is limited in source in FMDV clinical diagnosis is solved. The invention also provides a vaccine developed based on the NP-AVPn nanoparticle antigen and an FMDVA type LB-ELISA antibody detection kit, and the kit can be simultaneously used for quantitative detection of FMDVA 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 a nano-particle antigen of foot-and-mouth disease type A, and specifically relates to an intracellular self-assembled nano-particle antigen NP-AVPn of foot-and-mouth disease type A, a preparation method and an application thereof. Background Art

[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, which poses a great threat to the livestock 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 and play 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 have many advantages that single or monovalent antigens do not possess. First, they can effectively increase the antigen valence; second, by attaching to a larger scaffold carrier, the antigen is effectively improved in its uptake and retention in lymphoid follicles after being presented by APCs; third, through repeated arrangement, the antigen can achieve effective binding and activation of multiple B cell receptors. At present, 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. At present, 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 needs of simultaneously quantitatively detecting antibody levels from three different animal sources, pigs, cattle, and sheep, the quality of many commercially available kits in the quantitative detection of foot-and-mouth disease clinical antibody levels 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-AVPn of foot-and-mouth disease type A. The NP-AVPn nanoparticle antigen provided by the present invention contains all the antigenic epitopes of the VP1, VP2, and VP3 structural proteins of FMDV type A, and can simultaneously have a good and uniform reaction with the antibodies produced after immunization or infection of three animals, pigs, cattle, and sheep.

[0007] The NP-AVPn provided by the present invention is obtained by cleaving the intracellular self-assembled foot-and-mouth disease type A nanoparticle antigen NP-AVPn-T with TEV enzyme and then purifying and removing the T-promoting 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-AVPn nanoparticle antigen are activated, and there is a significant improvement in antigen reactivity compared to the nanoparticle NP-AVPn-T.

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

[0012] The SpyT-AVPn-T is formed by fusing the coding sequences of SpyT, AVPn, 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 AVPn 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, phage 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 phage 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 number of the expressed antigen, with less steric hindrance, and the formed nanoparticle structure is more stable.

[0017] Furthermore, the preparation method of the foot-and-mouth disease type A nanoparticle antigen NP-AVPn-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-AVPn-T: The nucleotide sequence encoding SpyT-AVP1-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-AVP3-T is as shown in SEQ ID NO.16.

[0020] 3) Cloning the recombinant protein SpyC-NP and the recombinant antigen SpyT-AVPn-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-AVPn-T intracellularly successively; a dehydration reaction occurs between the SpyC-NP and the SpyT-AVPn-T and they self-assemble into NP-AVPn-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-AVPn-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-AVP1-T, SpyT-AVP2-T, and SpyT-AVP3-T in the same host cell, so as to achieve the display and expression of at least one antigen protein among AVP1-T, AVP2-T, and AVP3-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°C ± 5°C; then regulate the expression of the SpyT-AVPn-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.

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

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

[0033] Among them, the foot-and-mouth disease type A NP-AVPn 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-AVPn nanoparticle antigen; the experimental animals are New Zealand white rabbits, Japanese white rabbits or guinea pigs; preferably New Zealand white rabbits.

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

[0036] The foot-and-mouth disease type A LB-ELISA antibody detection kit based on the nanoparticle antigen NP-AVPn can be used for the quantitative detection of FMDV type A-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 foot-and-mouth disease type A NP-AVPn nanoparticle antigen described in the present invention simultaneously displays three structural proteins, namely VP1, VP2, and VP3 of foot-and-mouth disease type A in the form of nanoparticles, and can react well and uniformly with the antibodies produced after immunization or infection of three animals, namely pigs, cattle, and sheep, overcoming the problem of limited sources of foot-and-mouth disease type A antigens in the clinical diagnosis of FMDV.

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

[0040] 3. The NP-AVPn 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-AVPn 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] Figure 1 The SDS-PAGE map photo for detecting the molecular weight of SpyC-NP and NP-AVPn-T nanoparticle proteins. M is the protein molecular weight standard. Lane 1 is the non-induced supernatant, lane 2 is the induced expression supernatant of SpyC-NP, and lane 3 is the induced expression supernatant of NP-AVPn-T.

[0043] Figure 2 The SDS-PAGE map photo for detecting the molecular weight of NP-AVPn nanoparticle proteins. M is the protein molecular weight standard. Lane 1 is the purified NP-AVPn-T, lane 2 is the NP-AVPn-T after digestion, and lane 3 is the NP-AVPn after removing the tag.

[0044] Figure 3 The transmission electron microscopy observation result photo of NP-AVPn nanoparticles.

[0045] Figure 4 The color development comparison photo of NP-AVPn nanoparticle antigen on the NC membrane. M is the protein molecular weight standard. Lane 1 is the porcine FMDV type A positive serum, lane 2 is the bovine FMDV type A positive serum, lane 3 is the ovine FMDV type A positive serum, lane 4 is the porcine FMDV type A negative serum, lane 5 is the bovine FMDV type A negative serum, lane 6 is the ovine FMDV type A negative serum, lane 7 is the classical swine fever virus positive serum, lane 8 is the porcine reproductive and respiratory syndrome virus positive serum, lane 9 is the pseudorabies virus positive serum, and lane 10 is the porcine circovirus type 2 positive serum. DETAILED DESCRIPTION OF THE INVENTION

[0046] Glossary of Terms:

[0047] The term "Foot-and-Mouth Disease Virus (FMDV)" is a picornavirus with high pathogenicity that 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.

[0048] The term "nanoparticle" refers to a protein complex with a certain spatial structure and a granular shape assembled from a single subunit or multiple subunits. Under an electron microscope, its diameter ranges from 10 nm to 500 nm. It should be well-known to professionals in the field that nanoparticles can be used as a carrier to display other antigens through methods such as chemical coupling, gene fusion, and tag coupling. Generally, the nanoparticles after display have higher immunogenicity and immunoreactivity than the initial antigen.

[0049] The term "lysis" refers to the cleavage of a target protein by a specific enzyme under certain conditions to completely separate it into two independent proteins.

[0050] The term "self-assembly" refers to some antigens with a high-level structure, whose structural proteins or constituent subunits can spontaneously assemble together under specific conditions to form a protein with a higher-level spatial structure.

[0051] 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 the field that the peptide segment should be in a specific spatial conformation to have good specific binding with antibodies.

[0052] Operations such as "coating", "blocking", "washing", "drying", "pat-drying", and "incubating" involved in the present invention are all routine experimental operations in the field. Relevant technical personnel in the field should understand the specific referents, implementation processes, and methods of these operations.

[0053] Unless otherwise specified, the chemical reagents used in this application are all of analytical grade and obtained through commercial channels.

[0054] Unless otherwise specified, the biological materials used in this application can be obtained through commercial channels.

[0055] Unless otherwise specified, the experimental methods used in this application are all conventional methods.

[0056] The clinical serum samples involved in the present invention are all prepared by separating the serum from the collected whole blood by centrifugation at 4000 r / min for 5 min at 4°C and adding Proclin 300 (48914-U, SIGMA) with a final concentration of 2% (W / W).

[0057] 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.

[0058] 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.

[0059] 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).

[0060] The present invention discloses an FMDV A-type NP-AVPn nanoparticle antigen, a preparation method, as well as a vaccine based on this antigen and an FMDV A-type LB-ELISA antibody detection kit. The following specific examples are used to further describe the present invention, but these examples are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0061] Gene sequence encoding NP-AVPn nanoparticle antigen in Example 1

[0062] 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 as shown in SEQ ID NO.9. The SpyT-AVPn-T gene is formed by fusing the coding sequences of SpyT, AVPn, and T in the "5'→3'" direction. The nucleotide sequence of SpyT-AVP1-T is as shown in SEQ ID NO.14, the nucleotide sequence of SpyT-AVP2-T is as shown in SEQ ID NO.15, and the nucleotide sequence of SpyT-AVP3-T is as shown in SEQ ID NO.16.

[0063] Unless otherwise specified, this application entrusts General Biology (Anhui) Co., Ltd. to synthesize the SpyT-AVP1-T, SpyT-AVP2-T, and SpyT-AVP3-T genes, and simultaneously ligate the 3 coding genes to the pET28a vector, named pET28a:SpyT-AVPn-T.

[0064] Unless otherwise specified, this application entrusts General Biology (Anhui) Co., Ltd. to synthesize the SpyC-NP gene, and clone the coding gene into the pG-Tf2 vector to replace the original coding gene of the Tf protein on the vector. The completed recombinant vector is named pG:SpyC-NP.

[0065] Nucleotide sequence information of the coding gene in Table 1:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] Induced expression of SpyC-NP and SpyT-AVPn-T recombinant antigens in Example 2

[0073] First, the pET28a:SpyT-AVPn-T recombinant vector and the pG:SpyC-NP recombinant vector described in Example 1 were simultaneously transformed into the same BL21(DE3) competent cells by the chemical transformation method, and the resulting recombinant strain was named BL21(DE3) / pET28a:SpyT-AVPn-T / pG:SpyC-NP.

[0074] Second, the recombinant strain BL21(DE3) / pET28a:SpyT-AVPn-T / pG:SpyC-NP was inoculated into LB liquid medium (supplemented with kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 20 μg / mL), and incubated at 37 °C with constant shaking at 220 r / min for about 10 h. Then, 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%. It was cultured at 37 °C and 220 r / min until the OD 600 reached about 0.4, and then 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 the SpyC-NP protein (theoretical molecular weight is 38 KDa).

[0075] After the induction of the expression of the SpyC-NP protein was completed, 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 medium to further induce the expression of the SpyT-AVPn-T protein, and the induction duration was 8 h. Among them, the theoretical molecular weight of the SpyT-AVP1-T protein is 37.3 KDa, the theoretical molecular weight of the SpyT-AVP2-T protein is 37.8 KDa, and the theoretical molecular weight of the SpyT-AVP3-T protein is 38.2 KDa.

[0076] After the induced expression of SpyT-AVP1-T, SpyT-AVP2-T, and SpyT-AVP3-T proteins, they can undergo a dehydration condensation reaction with the pre-expressed SpyC-NP protein in the cell to form NP-AVPn-T nanoparticles. The theoretical molecular weight of the nanoparticles is the sum of the molecular weights of SpyT-AVP1-T, SpyT-AVP2-T, SpyT-AVP3-T, and SpyC-NP proteins.

[0077] Collect the bacterial cells by centrifugation at 8000 r / min for 10 min, resuspend the cells 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), disrupt the cells using an ultrasonic disruptor (800 w, ultrasonic for 2 s, stop for 5 s, total ultrasonic time 20 min), then centrifuge at 13000 r / min for 30 min to separate the supernatant and precipitate. Resuspend the precipitate with an equal volume of the above PBS buffer and perform SDS-PAGE detection. The results show that a target protein band with a size of about 150 KDa can be detected in the supernatant, and the protein expression level is about 0.4 mg / mL ( Figure 1 as shown).

[0078] Preparation and reactivity identification of NP-AVPn-T and NP-AVPn nanoparticles in Example 3

[0079] Filter the supernatant containing NP-AVPn-T protein described in Example 2 using a 0.22 μm filter, and then purify the target protein from the treated sample using a protein purifier (Taidu Biotechnology Co., Ltd.). Since the C-terminus of the NP-AVPn-T protein contains a His tag, Ni NTABeads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) packing material is used, with a column volume of about 10 mL. The sample loading flow rate is 2 mL / min. Use a buffer containing 50 mmol / L imidazole to elute the impurity proteins, and use a buffer containing 500 mmol / L imidazole to elute the target protein. After eluting the target protein, identify it using SDS-PAGE. The results show that the purity of the purified NP-AVPn-T recombinant antigen is about 90%.

[0080] Digest the purified NP-AVPn-T protein with 1% (V / V) TEV enzyme at 20 °C for 6 h. The digested system contains two components, NP-AVPn nanoparticles and T protein. Purify the system using Ni NTABeads 6FF (Changzhou Tiandi Renhe Biotechnology Co., Ltd.) packing material (column volume about 5 mL), and collect the flow-through fraction, which is the NP-AVPn protein, for SDS-PAGE detection. The results show that the molecular weight of the NP-AVPn protein is about 100 KDa ( Figure 2As shown). Transmission electron microscopy was used to detect the prepared NP-AVPn nanoparticles, and it was found that nanoparticles of about 150 nm could be observed under the electron microscope, and the AVPn protein was uniformly distributed on the surface of the NP nanoparticles ( Figure 3 As shown).

[0081] The Western blot method was used to preliminarily verify the reactivity and specificity of the purified NP-AVPn nanoparticle antigen. First, the NP-AVPn antigen was subjected to SDS-PAGE. After electrophoresis, it was transferred to a NC membrane by wet transfer method. After blocking with 5% skim milk (prepared with PBST, where PBST is PBS added with 0.05% Tween 20) at room temperature for 1 h, clinical isolate-derived FMDV A positive sera (porcine serum, bovine serum, and ovine serum), FMDV A 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 virus positive serum were used as primary antibodies, and the sera were all diluted at a ratio of 1:100 with 5% skim milk. After incubating the primary antibody at room temperature for 1 h, it was washed 3 times with PBST. HRP (horseradish peroxidase)-labeled goat anti-pig or rabbit anti-bovine or rabbit anti-sheep antibody was used as the secondary antibody, and the secondary antibody was diluted at a ratio of 1:50000 with 5% skim milk and incubated at room temperature for 1 h. After washing 3 times with PBST, color development treatment was carried out using DAB chromogenic solution (ComWin Biotech Co., Ltd.).

[0082] The results showed that the NP-AVPn nanoparticle antigen could undergo a uniform specific color reaction with FMDV A positive sera (porcine serum, bovine serum, and ovine serum), and had no reaction with FMDV A 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 virus positive serum, proving that the NP-AVPn nanoparticle antigen had good specificity ( Figure 4 As shown).

[0083] Preparation of FMDV A type NP-AVPn nanoparticle antigen-specific antibodies in Example 4

[0084] In this embodiment, the preparation process of the FMDV A NP-AVPn nanoparticle antigen-specific polyclonal antibody mainly includes the following steps:

[0085] Step S401: Prepare the immunogen. After diluting the NP-AVPn nanoparticle antigen described in Example 3 to 1 mg / mL, it was mixed with Freund's adjuvant (Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the immunization after the first immunization) at a ratio of 1:1 (V / V), and the immunogen was prepared by emulsification using the push needle method.

[0086] Step S402: Immunize experimental animals by multi-point subcutaneous injection in the back with the emulsified immunogen described in Step S401. The protein immunization dose for experimental rabbits is 0.5 mg per rabbit, and the emulsified immunization volume is 1 mL per rabbit; the protein immunization dose for guinea pigs is 0.2 mg per guinea pig, and the emulsified immunization volume is 0.4 mL per guinea pig.

[0087] Step S403: Perform booster immunization 14 - 28 days after the first immunization of the experimental animals described in Step S402. The immunization method and dose are the same as those for the first immunization. Collect blood serum antibody titer 7 - 10 days after the booster immunization. Serum collection can be carried out after meeting the requirements.

[0088] Step S404: After the serum is collected in Step S403, use Protein A packing material to purify and prepare NP-AVPn nanoparticle antigen-specific polyclonal antibody.

[0089] In this embodiment, the preparation process of the FMDV A type NP-AVPn nanoparticle antigen-specific monoclonal antibody mainly includes the following steps:

[0090] Step S405: The same as Step S401.

[0091] Step S406: Immunize mice by multi-point subcutaneous injection in the back with the emulsified immunogen described in Step S405, with a dose of 0.2 mL per mouse. Perform booster immunization 14 - 28 days after the first immunization. The method and dose are the same as those for the first immunization. 14 - 60 days after the booster immunization, take 0.2 mL of antigen and perform boost immunization by intraperitoneal injection.

[0092] 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, stir the cells, and add PBS to terminate after standing at room temperature for 90 s.

[0093] Step S408: Spread the fused cells in Step S407 onto a 96-well cell culture dish and statically culture them in an incubator at 37°C and 5% CO2.

[0094] Step S409: Perform subcloning by the limiting dilution method. For the first subcloning of the positive cell wells in Step S408, seed 2 cells per well and use HAT cell medium for screening. For the second and third subclonings, seed 1 cell per well and use HT cell medium for screening.

[0095] Step S410: After the three subcloning screenings described in Step S409 and the supernatant detection results of the monoclonal wells are all positive, change the medium to complete medium, continue to expand the culture and freeze.

[0096] Step S411: Inject the hybridoma cells after the expansion culture described in step S410 into the peritoneal cavity of a mouse to prepare ascites. After collecting the ascites, use Protein A packing material for purification to prepare the NP-AVPn nanoparticle antigen-specific monoclonal antibody.

[0097] Label the purified monoclonal antibody described in step S411 with HRP using the sodium periodate labeling method.

[0098] FMDV A type LB-ELISA antibody detection kit in Example 5

[0099] In this embodiment, the FMDV A type LB-ELISA antibody detection kit mainly includes the following components: foot-and-mouth disease type A NP-AVPn nanoparticle antigen, an enzyme-labeled plate coated with a polyclonal antibody specific to the NP-AVPn nanoparticle antigen, a sample diluent, a washing solution, a negative control, a positive control, an NP-AVPn nanoparticle antigen-specific monoclonal antibody cross-linked horseradish peroxidase conjugate, a chromogenic solution (solution A and solution B), and a termination solution.

[0100] Foot-and-mouth disease type A NP-AVPn nanoparticle antigen: The preparation process is as described in Examples 1-3.

[0101] Enzyme-labeled plate coated with a polyclonal antibody specific to the NP-AVPn nanoparticle antigen:

[0102] Use a carbonate buffer solution with a pH of 9.6 to coat the NP-AVPn nanoparticle antigen-specific polyclonal antibody prepared in Example 4 on the microplate at a coating concentration of 1-5 μg / mL and a coating amount of 0.1-0.5 μg / well, and coat overnight at 2-8°C; discard the coating solution, wash 2-4 times with a phosphate buffer solution, and pat dry or aspirate dry; add a blocking solution (phosphate buffer solution added with 1% casein and 0.1% BSA) and block overnight at 2-8°C; discard the blocking solution, wash 3 times with a phosphate buffer solution, and pat dry or aspirate dry; dry at 37°C for 2-3 h and then put it into an aluminum foil bag containing a desiccant and store at 4°C for standby.

[0103] NP-AVPn nanoparticle antigen-specific monoclonal antibody cross-linked horseradish peroxidase conjugate:

[0104] Use a phosphate buffer solution containing 1% (W / V) casein and 1% (W / V) sucrose to dilute the NP-AVPn nanoparticle antigen-specific monoclonal antibody labeled with horseradish peroxidase described in Example 4 at a ratio of 1:(70000-80000) (V / V) to form an NP-AVPn nanoparticle antigen-specific monoclonal antibody cross-linked horseradish peroxidase conjugate.

[0105] Sample diluent: Phosphate buffer solution.

[0106] Washing solution: Phosphate buffer containing 0.05% V / V Tween-20.

[0107] 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 aseptically aliquoting; 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% hydrogen peroxide urea in ddH2O, making up the volume to 100 mL, mixing well and then aseptically aliquoting.

[0108] Stop solution: 2 mol / L H2SO4.

[0109] Positive control: NP-AVPn nanoparticle antigen.

[0110] Negative control: Serum from pigs or sheep or cattle that is negative for FMDV detected by PCR method, or replaced by sample diluent.

[0111] In this example, the usage method of the foot-and-mouth disease type A liquid-phase blocking ELISA antibody detection kit based on NP-AVPn nanoparticle antigen mainly includes the following steps:

[0112] 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 well.

[0113] Step S502: Antigen-antibody (positive and negative controls and test samples) reaction. According to different needs, select one of the layouts in Table 2 or Table 3 for operation on the serum dilution plate. Table 2 is the layout diagram for detecting 10 samples in the antibody titer determination (quantitative); Table 3 is the layout diagram for detecting 20 samples in the antibody titer determination (quantitative).

[0114] Table 2 Layout of 96-well plate for detecting 10 samples

[0115]

[0116]

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

[0118]

[0119] Step S503: Dilute the NP-AVPn nanoparticle antigen. Dilute the NP-AVPn nanoparticle antigen 450-fold outside the wells using the prepared washing solution (i.e., 1 part of NP-AVPn nanoparticle antigen plus 449 parts of washing solution). Taking Table 2 in Step S502 as an example, 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 positive control to well A11, add 50 μL of negative control to well A12, and mix well with a multichannel pipette (pipette 6 - 7 times for each well). The samples to be tested in A1 - A10 are serially diluted 2-fold from A to H at a volume of 50 μL / well; the positive control in A11 is serially diluted 2-fold from A to H at a volume of 50 μL / well; the negative control in 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 NP-AVPn nanoparticle antigen diluted in proportion as described in Step S503. Add 50 μL to each well of columns 1 - 11, add 50 μL to each of wells A12 and B12, and add 100 μL to each of wells E12, F12, G12, and H12. After adding the samples, all dilution factors are doubled. The samples to be tested are from 1:8 - 1:1024, the positive control is from 1:32 - 1:4096, and the negative control is from 1:4 - 1:8. Stick on the sealing film and mix well by oscillation.

[0121] Step S505: Place the serum dilution plate with the added samples in Step S504 in an incubator 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 / well. Stick on the 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, and wash a total of 5 times. After each washing, discard the liquid in the well. After discarding the last washing solution, pat dry the remaining washing solution in the well on the absorbent paper.

[0124] Step S508: Add NP-AVPn nanoparticle antigen-specific monoclonal antibody conjugated horseradish peroxidase conjugate into the plate holes dried in step S507, 50 μL per well, apply a sealing plate membrane, and incubate at 37 °C for 35 min.

[0125] Step S509: Repeat the operation according to step S407 for the enzyme-labeled plate obtained in step S508, add the mixed chromogenic solution (chromogenic solution A and chromogenic solution B are 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 the termination 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 A type LB-ELISA antibody detection kit provided in this application and the immune protection effect during clinical application, the FMDV A type LB-ELISA antibody detection kit can provide the following guidance for FMDV A type clinical immunoprevention and control: when the antibody titer in bovine and ovine serum samples ≥ 1:128, it can provide more than 99% protection; when the antibody titer ≤ 1:16, there is no protection; when the antibody titer is between 1:22 and 1:90, it is 50% protection. When the antibody titer in porcine serum samples ≥ 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, it is 50% protection.

[0129] Specificity of FMDV A type LB-ELISA antibody detection kit based on NP-AVPn nanoparticle antigen in Example 6 Research

[0130] Collect clinically sourced positive sera of FMDV antibodies from pigs, cattle, sheep, positive sera of antibodies against Porcine circovirus type 2 (PCV-2), positive sera of antibodies against Porcine Pseudorabies Virus (PRV), positive sera of antibodies against African Swine fever virus (ASFV), positive sera of Porcine epidemic diarrheavirus (PEDV), and positive sera of antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV). 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 type A LB-ELISA antibody detection kit based on NP-AVPn nanoparticle antigen in Example 5 to detect the above positive sera of different virus antibodies, and the detection results are shown in Table 4.

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

[0133]

[0134]

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

[0136] Sensitivity of FMDV A type LB-ELISA antibody detection kit based on NP-AVPn nanoparticle antigen in Example 7 Research

[0137] Arbitrarily select 1 positive serum of FMDV type A (from cattle), and perform serial dilution with PBS buffer at a dilution factor of 1:(2 - 1024) (V / V). Use the FMDV type A LB-ELISA antibody detection kit based on NP-AVPn nanoparticle antigen in Example 5 to detect the positive sera of the above different dilution factors, and the detection results are shown in Table 5. Use a commercially available kit to detect the positive sera of the above different dilution factors, and the detection results are shown in Table 6.

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

[0139]

[0140] Table 6 Detection Results of Commercial Kits on Sale

[0141]

[0142]

[0143] According to the detection results, after the serum samples were diluted at 1:256 (V / V) for detection, the serum titer detection result of the FMDV A type LB-ELISA antibody detection kit provided by this application was 1:128, while the serum titer detection result of the commercial kit on sale was 1:48. That is, the sensitivity of the FMDV A type LB-ELISA antibody detection kit was about 4 times that of the commercial kit on sale.

[0144] The results of the FMDV A type porcine positive serum and the FMDV A type ovine positive serum in the sensitivity detection were relatively consistent with the detection results of the FMDV A type bovine positive serum provided in this example. Therefore, in this example, only the FMDV A type bovine positive serum was taken as an example to elaborate on the advantages of the FMDV A type LB-ELISA antibody detection kit provided by this application in terms of sensitivity.

[0145] Clinical application of FMDV A type LB-ELISA antibody detection kit based on NP-AVPn nanoparticle antigen in Example 8 Application

[0146] 18 animals were randomly selected from a certain FMDV A type vaccine-immunized positive pig farm and a certain FMDV negative pig farm for blood collection and serum separation. 18 animals were randomly selected from a certain FMDV A type vaccine-immunized positive sheep farm and a certain FMDV negative sheep farm for blood collection and serum separation. 18 animals were randomly selected from a certain FMDV A type vaccine-immunized positive cattle farm and a certain FMDV negative cattle farm for blood collection and serum separation. After randomly mixing the above 54 clinical positive serum samples from different animal species and 54 clinical negative serum samples from different animal species, the antibody titer detection was carried out using the FMDV A type LB-ELISA antibody detection kit provided by this application to examine the compliance of the serum titer with the clinical immune background.

[0147] Table 7 Statistics of Antibody Titer Detection Results of 108 Clinical Serum Samples

[0148]

[0149]

[0150] According to the antibody titer statistical results described in Table 7 of this embodiment, the FMDV A antibody levels in different farms are completely consistent with the actual immunization background, indicating that the use of the FMDV A LB-ELISA antibody detection kit provided by this application has certain guiding significance for the detection and evaluation of the clinical immunization effect of FMDV A or the infection screening of FMDV-negative farms.

[0151] 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-assembling foot-and-mouth disease type A nanoparticle antigen NP-AVPn-T, characterized in that, The NP-AVPn-T is self-assembled by dehydration condensation reaction after the recombinant proteins SpyC-NP and foot-and-mouth disease type A recombinant antigen SpyT-AVPn-T are expressed in host cells; wherein NP is a nanoparticle carrier protein, AVPn is at least one of the foot-and-mouth disease type A structural proteins VP1, VP2, and VP3, n represents an integer from 1 to 3, and T is a fusogenic tag protein; an amide bond is formed between NP and AVPn-T.

2. A foot-and-mouth disease type A nanoparticle antigen NP-AVPn, characterized in that, The NP-AVPn is obtained by cleaving the intracellular self-assembled foot-and-mouth disease type A nanoparticle antigen NP-AVPn-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 foot-and-mouth disease A 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 dodecahedron 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 dodecahedron nanoparticle mi3 is as shown in SEQ ID NO.

8.

4. The preparation method of the foot-and-mouth disease A nanoparticle antigen NP-AVPn-T according to claim 1, characterized in that, 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-AVPn-T: The coding nucleotide sequence of SpyT-AVP1-T is as shown in SEQ ID NO.14, the coding nucleotide sequence of SpyT-AVP2-T is as shown in SEQ ID NO.15, and the coding nucleotide sequence of SpyT-AVP3-T is as shown in SEQ ID NO.

16. 3) Cloning the recombinant protein SpyC-NP and the recombinant antigen SpyT-AVPn-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-AVPn-T in the cells successively; a dehydration reaction occurs between the SpyC-NP and the SpyT-AVPn-T and they self-assemble into NP-AVPn-T.

5. The preparation method of the foot-and-mouth disease type A nanoparticle antigen NP-AVPn-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-AVP1-T, SpyT-AVP2-T, and SpyT-AVP3-T in the same host cell to achieve the display expression of at least one antigen protein among AVP1-T, AVP2-T, and AVP3-T on the surface of the nanoparticle carrier protein NP.

6. The preparation method of the foot-and-mouth disease A nanoparticle antigen NP-AVPn-T according to claim 4, characterized in that, 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 type A nanoparticle antigen NP-AVPn according to claim 4, characterized in that 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 type A nanoparticle antigen NP-AVPn according to claim 4, characterized in that 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-AVPn-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 type A nanoparticle antigen NP-AVPn according to claim 2.

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

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