Foot and mouth disease virus type A multi-epitope recombinant nano protein and application thereof

Abstract

The invention relates to the technical field of A type foot-and-mouth disease virus detection, in particular to A type foot-and-mouth disease virus multi-epitope recombinant nano protein and application thereof. The invention provides the foot-and-mouth disease virus type A related protein and the antibody, establishes a detection kit based on a competitive enzyme-linked immunosorbent assay technology and a magnetic particle chemiluminescence immunoassay technology, has the advantages of high sensitivity, wide detection range, short detection time, simplicity and convenience in operation, low cost and the like, and is also matched with an automatic instrument and a standard curve; the kit can realize accurate quantification and full-automatic detection, and has important value and good popularization prospect in the aspects of type A foot-and-mouth disease immune antibody level detection, epidemic situation monitoring, epidemiological investigation, clinical mass sample screening and the like.

Description

Technical Field

[0001] The invention relates to the technical field of type A foot-and-mouth disease virus detection, and in particular to a type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein and an application thereof. Background Art

[0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious disease caused by the foot-and-mouth disease virus (FMDV). Domestic animals such as pigs, cattle, and sheep, as well as more than 70 species of wild even-toed ungulates are susceptible. Clinical symptoms include high fever, depression, loss of appetite, blisters of varying degrees and erosions on the oral mucosa, mammary skin, and hoof forks and heels. It can be transmitted through the air and pollutants, with a fast transmission speed and a wide transmission area. The mortality rate of young even-toed ungulates is very high.

[0003] FMDV belongs to the genus Aphthovirus of the family Picornaviridae. It is a single-stranded positive-strand RNA virus consisting of a 5' untranslated region (UTR), a large open reading frame (ORF) and 3'-UTR. ORF encodes a polyprotein that can be cleaved multiple times to form four structural proteins (VP4, VP2, VP3 and VP1) and 10 non-structural proteins (L pro ,2A,2B,2C,3A,3B1,3B2,3B3,3C pro and 3D pol ), structural proteins make up the viral capsid, and non-structural proteins are related to viral replication. Among them, VP1 has the largest antigenic variation, while the difference between VP2 and VP3 is relatively small. Most of the VP1 protein is exposed on the surface of the virus particle, including the main antigenic epitopes of foot-and-mouth disease virus at positions 141 to 160 and 200 to 213 amino acid residues. The GH loop composed of amino acid residues 141 to 160 of VP1 protrudes from the surface of the viral capsid, and forms a highly conserved Arg-Gly-Asp (RGD) sequence on its top. It can induce the body to produce neutralizing antibodies, so the antigenic epitope of VP1 protein is often used as a diagnostic antigen.

[0004] According to animal cross-immunity tests and serological tests, foot-and-mouth disease virus can be divided into 7 serotypes, namely type A, type A, type C, type SAT1, type SAT2, type SAT3 and type Asia 1. Each serotype is divided into different subtypes, and there is no cross-protection between types. Among them, types A, O and Asia 1 are the main foot-and-mouth disease serotypes prevalent in my country, especially type A in recent years. Type A has the most antigenic spectrum and genetic diversity. According to the genetic evolution relationship of the VP1 gene sequence, FMDV-A strains can be divided into 3 topological types and 26 genotypes. The prevalence of numerous antigenic spectrum and genetically diverse strains has brought great challenges to the prevention and control of foot-and-mouth disease.

[0005] Ferritin is a regular octahedral nanoparticle (8nm inner diameter, 12nm outer diameter) consisting of an outer shell composed of protein and an iron core composed of hydrated iron oxide. It has the characteristics of simple and stable structure, tolerance to various denaturants, and high temperature resistance, which is conducive to carrying and presenting antigens. It is widely present in animals, plants, microorganisms (except yeast cells), etc. The spherical nanocage-like protein shell is self-assembled by 24 identical ferritin subunits. Antigens can be connected to the surface of ferritin nanoparticles or encapsulated in ferritin, and the antigen protein can be packaged in the capsid, which can not only be delivered to the target cells in a targeted manner, assist the target cells in the uptake and processing of the corresponding antigens, but also enhance the antigen stability and immunogenicity of the target protein, and can increase the loading and efficiency of target proteins such as antibodies or drugs.

[0006] Due to the diversity of the antigen spectrum of type A FMDV, antibody detection methods are often used to evaluate the immune effect of FMD vaccines. Mainly including virus neutralization test, liquid phase blocking ELISA and solid phase competitive ELISA. Virus neutralization test requires the preparation of monolayer cells, which is complicated, time-consuming and low in sensitivity; liquid phase blocking ELISA and solid phase competitive ELISA are internationally recognized FMDV antibody detection methods, but the liquid phase blocking ELISA and solid phase competitive ELISA detection kits currently on the market have poor sensitivity, narrow detection spectrum, cumbersome operation steps, high requirements for detection personnel, long reaction time, and cannot achieve full-automatic detection. It is difficult to achieve rapid detection of large quantities in a short time. Therefore, this application proposes a multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus and its application. Summary of the invention

[0007] The purpose of the present invention is to address the problems of low sensitivity and poor stability of LPB-ELISA and SPC-ELISA detection kits currently on the market in the background technology, and to propose a multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus and its application.

[0008] In a first aspect, the present application provides a type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein, wherein the encoding gene of the type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein is an amino acid sequence as shown in SEQ ID NO.14.

[0009] In a second aspect, the present application provides a recombinant plasmid, wherein the recombinant plasmid comprises the coding gene as described in the first aspect.

[0010] In a third aspect, the present application provides a type A foot-and-mouth disease virus polyclonal antibody, wherein the polyclonal antibody is prepared using the multi-epitope recombinant nanoprotein described in the first aspect as an immunogen.

[0011] In a fourth aspect, the present application provides a kit for detecting type A foot-and-mouth disease virus antibodies based on an enzyme-linked immunosorbent assay, comprising a labeled antibody and a type A foot-and-mouth disease virus antigen; The labeled antibody is the HRP-labeled polyclonal antibody of the type A foot-and-mouth disease virus described in the third aspect; The type A foot-and-mouth disease virus antigen is the type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein described in the first aspect.

[0012] In a fifth aspect, the present application provides a kit for detecting type A foot-and-mouth disease virus antibodies based on a magnetic particle chemiluminescent immunoassay, which comprises a labeled polyclonal antibody and a labeled antigen; The labeled polyclonal antibody is the type A foot-and-mouth disease virus polyclonal antibody of the third aspect labeled with an acridinium ester; The labeled antigen is the multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus labeled with biotin as described in the first aspect.

[0013] Compared with the prior art, the beneficial technical effects of this application are: The present invention firstly provides a type A foot-and-mouth disease virus VP1 recombinant nanoparticle protein and a type A foot-and-mouth disease virus polyclonal antibody, which provide a technical platform for type A foot-and-mouth disease virus immune antibody level detection, epidemic situation monitoring and epidemiological investigation.

[0014] Secondly, the present invention adopts competitive enzyme-linked immunosorbent assay technology to establish a kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay.

[0015] It has the following advantages: (1) This kit has the advantages of strong specificity and good stability. Compared with the liquid phase blocking ELISA antibody detection kit for foot-and-mouth disease virus type A produced by Lanzhou Zoological Research Biotechnology Co., Ltd., the compliance rate is 95.33%. It can be used for the detection of type A foot-and-mouth disease serum antibodies.

[0016] (2) The recombinant nanoparticle protein of type A foot-and-mouth disease virus VP1 is used as the antigen, which has high sensitivity and can cover all genotypes of type A foot-and-mouth disease virus in the detection range to avoid missed detection.

[0017] (3) The detection time is short, the operation is simple, and the cost is low. It is suitable for rapid detection of large quantities of samples and has a high promotion value in the monitoring and diagnosis of foot-and-mouth disease.

[0018] Furthermore, the present invention adopts magnetic particle chemiluminescent immunoassay technology combined with biotin labeling technology and acridinium ester labeling technology to establish a kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic particle chemiluminescent immunoassay. FMDV-A antibody detection can be performed quickly, accurately and in large quantities, providing new technical support for the rapid diagnosis of FMDV-A.

[0019] It has the following advantages: (1) The acridinium ester reaction system is used, which has instant luminescence, short reaction time and good stability.

[0020] (2) The specific binding process between antigen and antibody adopts the competitive method, which is a one-step reaction with short time and simple operation.

[0021] (3) The multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus is used as the coating source and its corresponding polyclonal antibody is used as the labeling antibody, which has strong specificity.

[0022] (4) The chemiluminescent immunoassay technique was used, which had good sensitivity and was compared with the liquid-phase blocking ELISA antibody detection kit for foot-and-mouth disease virus type A produced by Lanzhou Zoological Research Biotechnology Co., Ltd., with a compliance rate of 97.16%.

[0023] (5) Equipped with a fully automatic chemiluminescence immunoassay analyzer, it can achieve high sensitivity, high specificity, and accurate quantitative detection. Fully automated detection greatly reduces labor and time costs and is suitable for screening a large number of samples.

[0024] In summary, the present invention provides type A foot-and-mouth disease virus-related proteins and antibodies, and establishes a detection kit based on competitive enzyme-linked immunosorbent assay technology and magnetic microparticle chemiluminescent immunoassay technology. The former has the advantages of strong specificity, good stability, high sensitivity, wide detection range, short detection time, simple operation, and low cost; the latter adopts an acridinium ester reaction system, combined with magnetic microparticle separation technology and chemiluminescent immunoassay technology, and has the advantages of short reaction time, good stability, strong specificity, and high sensitivity. At the same time, it is equipped with automated instruments and standard curves to achieve accurate quantification and fully automated detection. It has important value and good promotion prospects in the detection of type A foot-and-mouth disease virus immune antibody levels, epidemic monitoring, epidemiological surveys, and large-scale clinical sample screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of PCR identification of multi-epitope recombinant plasmid of type A foot-and-mouth disease virus; Figure 2 This is a diagram showing the expression results of multi-epitope nano-recombinant protein of foot-and-mouth disease virus type A. DETAILED DESCRIPTION

[0026] The present application discloses a type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

[0027] In the first aspect of the present application, the present application provides a type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein, the encoding gene of the type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein has a nucleotide sequence as shown in SEQ ID NO.13, corresponding to the amino acid sequence as shown in SEQ ID NO.14.

[0028] In the second aspect of the present application, the present application provides a recombinant plasmid, which comprises the coding gene of the first aspect.

[0029] In the third aspect of the present application, the present application provides a type A foot-and-mouth disease virus polyclonal antibody, which is prepared using the multi-epitope recombinant nanoprotein of the first aspect as an immunogen.

[0030] In a fourth aspect of the present application, the present application provides a kit for detecting type A foot-and-mouth disease virus antibodies based on an enzyme-linked immunosorbent assay, comprising a labeled antibody and a type A foot-and-mouth disease virus antigen; The labeled antibody is a third aspect A-type foot-and-mouth disease virus polyclonal antibody labeled with HRP; The type A foot-and-mouth disease virus antigen is the type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein in the first aspect.

[0031] In a fifth aspect of the present application, the present application provides a kit for detecting type A foot-and-mouth disease virus antibodies based on a magnetic particle chemiluminescence immunoassay, which comprises a labeled polyclonal antibody and a labeled antigen; The labeled polyclonal antibody is a third aspect of a foot-and-mouth disease virus type A polyclonal antibody labeled with an acridinium ester; In the first aspect, the labeled antigen is a biotin-labeled multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus.

[0032] Example 1: Preparation of coding genes, the specific scheme is as follows: S101. The full-length sequences of VP1 genes of 40 strains of type A foot-and-mouth disease virus retrieved from Genbank were analyzed, and the dominant strains A / AF72 (Genbank accession number: MT447399.1), A / WH / CHA / 2009 (Genbank accession number: JF792355.1) and A / GDMM / CHA / 2013 (Genbank accession number: KF450794.1) prevalent in China were selected. Antigenic epitope prediction analysis was performed using the biological online software Immunomedicine Group and SVMTriP, and the sequences selected based on the literature and analysis results were as follows: A / AF72 strain The dominant linear antigen epitope sequence 1, 22-34aa, specifically the nucleotide sequence shown in SEQ ID NO.1; The dominant linear antigen epitope sequence 2, 137-149aa is specifically the nucleotide sequence shown in SEQ ID NO.2; A / WH / CHA / 2009 strain (Sea-97-G1) The dominant linear antigen epitope sequence 3, 90-106aa, specifically the nucleotide sequence shown in SEQ ID NO.3; The dominant linear antigen epitope sequence 4, 130-148aa, specifically the nucleotide sequence shown in SEQ ID NO.4; A / GDMM / CHA / 2013 strain (Sea-97-G2) VP1 full-length sequence 5, 1-212aa, specifically the nucleotide sequence shown in SEQ ID NO.5; Ferritin nucleotide sequence 6 (Genbank accession number: AY072939.1), specifically the nucleotide sequence shown in SEQ ID NO.6; According to the preferred codons of Escherichia coli, the optimized nucleotide sequences were obtained, specifically: The nucleotide sequence shown in SEQ ID NO.7; The nucleotide sequence shown in SEQ ID NO.8; The nucleotide sequence shown in SEQ ID NO.9; The nucleotide sequence shown in SEQ ID NO.10; The nucleotide sequence shown in SEQ ID NO.11; S102, the nucleotide sequence of ferritin after truncated optimization is the nucleotide sequence shown in SEQ ID NO.12; S103, the above sequences are connected with a flexible connecting peptide in the order of 7-8-9-10-11-12, the flexible connecting peptide nucleotide sequence is specifically the nucleotide sequence shown in SEQ ID NO.15, and BamHI and HindIII restriction sites are inserted at both ends. The optimized nucleotide sequence is obtained, specifically the nucleotide sequence shown in SEQ ID NO.13. Among them, the BamHI restriction site is the nucleotide sequence shown in SEQ ID NO.16, and the HindIII restriction site is the nucleotide sequence shown in SEQ ID NO.17.

[0033] S104. The above recombinant nucleotide sequence with restriction sites is synthesized into a recombinant plasmid pUC57-FMDV-Af by Jiaotong Yongyong Biological (Anhui) Co., Ltd.

[0034] S105. The amino acid sequence of the multi-epitope recombinant nanoparticle protein encoded by sequence SEQ ID NO.13 is the amino acid sequence shown in SEQ ID NO.14.

[0035] Table 1 Sequence information of nucleotide sequences

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] Table 2 Sequence information of amino acid sequences

[0043] Example 2: Construction of a recombinant plasmid, comprising the following steps: S201. Cultivate the A-type foot-and-mouth disease virus recombinant protein gene-positive bacteria (Amp+) and Escherichia coli containing pET-His-MBP (Kan+) and pET-28b(+) (Kan+) respectively according to the culture method of Escherichia coli, and extract the A-type foot-and-mouth disease virus recombinant protein gene-positive plasmid, pET-His-MBP and pET-28b(+) plasmid according to the instructions of the plasmid extraction kit (purchased from Quanshijin Biotechnology Co., Ltd.).

[0044] S202. According to the instructions of restriction endonucleases (purchased from Quanshijin Biotechnology Co., Ltd.), pET-His-MBP and pET-28b(+) plasmids were double digested with XbaI / BamHI. The products were subjected to agarose gel electrophoresis (120V, 30min). The His-MBP nucleic acid fragment and pET-28b(+) vector were recovered according to the instructions of Tiangen Agarose Gel DNA Recovery Kit.

[0045] S203. According to the instruction of T4 DNA ligase (purchased from Quanshijin Biotechnology Co., Ltd.), the His-MBP nucleic acid fragment after enzyme digestion and the pET-28b(+) vector were ligated. The ligated product was named pET-28b-His-MBP.

[0046] S204. The ligated product was transferred into DH5α competent cells (full gold) for culture. The cultured DH5α was coated on LB plates (Kan+) and cultured at 37°C overnight.

[0047] S205. According to the instructions of restriction endonucleases (purchased from Quanshijin Biotechnology Co., Ltd.), the positive plasmid of the recombinant protein gene of type A foot-and-mouth disease virus and the pET-28b-His-MBP plasmid were double-digested with BamHI / HindIII. The products were subjected to agarose gel electrophoresis (120v, 30min) and the recombinant protein nucleic acid of type A foot-and-mouth disease virus and the pET-28b-His-MBP plasmid were recovered according to the Tiangen agarose gel DNA recovery kit.

[0048] S206. According to the instruction of T4 DNA ligase (purchased from Quanshijin Biotechnology Co., Ltd.), the digested type A foot-and-mouth disease virus recombinant protein nucleic acid and pET-28b-His-MBP were ligated.

[0049] S207. The ligation product pET-28b-FMDV-A-His-MBP was transformed into DH5α competent cells (Full Gold) for culture. The cultured DH5α was coated on LB plates (Kan+) and cultured at 37°C overnight.

[0050] S208. Select a monoclonal bacterial strain as a template, use the universal primers T7 / T7ter of the pET-28b vector as primers, perform PCR amplification, and perform agarose gel electrophoresis (120v 30min) on the product. A 317bp band of the empty vector control (lane 2) and a 2858bp band of the pET-28b-FMDV-A-His-MBP target band (lane 3) are visible. Figure 1 As shown, the product fragment size is consistent with the expected.

[0051] Example 3: Expression, identification and purification of recombinant nanoparticle protein, specifically comprising the following steps: Recombinant plasmid transformation: The verified recombinant plasmid pET-28b-FMDV-A-His-MBP was transformed into competent E. coli cells BL21 (DE3), coated on LB plates (Kan+), and cultured at 37°C overnight.

[0052] S301. Pick a single colony containing the recombinant plasmid and add it to liquid LB medium (Kan+), and culture it at 37°C overnight.

[0053] S302, take 1 ml of bacterial solution and add it to 100 ml of fresh liquid LB medium (Kan+) and culture it in a shaker at 220 rpm and 37°C until OD 600 It is between 0.6-0.8.

[0054] S303, IPTG-induced expression and identification of recombinant nanoparticle protein: ①Add IPTG at a final concentration of 0.5 mM and express overnight at 15°C and 195 rpm.

[0055] ②After induction, take 1 ml of bacterial solution and centrifuge at 4 degrees, 8000 rpm for 5 minutes. Resuspend the bacteria with an appropriate amount of PBS (pH 8.0), then add 2× SDS-PAGE loading buffer, mix well, incubate at 95℃ for 10 minutes, and perform SDS-PAGE electrophoresis analysis.

[0056] ③ The remaining bacterial solution was centrifuged at 8000 rpm for 5 min at 4°C. The precipitate was collected and resuspended in 10 ml PBS (pH 8.0). Lysozyme was added at a final concentration of 1 mg / mL. After ice bathing for 30 min, the bacteria were broken by low-temperature ultrasound (ultrasonic time 4 s, interval 9 s, 120 times).

[0057] ④ After crushing, centrifuge at 4℃, 12000 rpm for 30 min. Collect the supernatant solution and precipitate. Take 100μl of the supernatant for SDS-PAGE analysis.

[0058] ⑤ In the supernatant sample, the expressed multi-epitope recombinant nanoprotein has an obvious 94KD protein band, which is the same as the expected size. It is named as multi-epitope recombinant nanoprotein FMDV-A-His-MBP. Figure 2 As shown, lane 1 is an empty vector control, lane 2 is protein Ruler, and lane 3 is the target protein.

[0059] S304. Purification of recombinant nanoparticle protein ① Add sample: All the remaining supernatant protein solution was added to the affinity purification Ni-NTA chromatography column treated in advance with 5 volumes of Binding Buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), and incubated for 4 h at 4°C and 200 rpm.

[0060] ②Remove unbound proteins: After the incubation, the supernatant-Ni-NTA mixture was washed three times with 5 volumes of Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) to fully remove non-specific proteins and unbound target proteins.

[0061] ③Elute the target protein: Finally, the target protein was eluted with 5 volumes of Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0), incubated at room temperature for 10-15 min, and the eluted protein solution was collected by centrifugation and repeated this operation 4 times.

[0062] ④Desalting treatment to remove imidazole and ultrafiltration concentration: a. Balance the desalting column in advance, take 2.5 mL of the target protein sample and load it onto the column. Elute the target protein with 3.5 mL of PBS filtered through a 0.22μm filter membrane, and collect the eluted protein sample until all the protein solution is replaced and collected.

[0063] b. Transfer the desalted protein solution to a 3 KD ultrafiltration tube and centrifuge at 3000 rpm at 4°C for 5 min. Centrifuge several times until about 2 mL of liquid remains.

[0064] c. The target protein concentration was determined using the BCA protein quantitative detection kit: 1.526 mg / mL.

[0065] Example 4: Preparation and titer determination of FMDV-A-VP1 polyclonal antibodies S401, immunize 2-month-old female New Zealand rabbits with purified FMDV-A-His-MBP protein as immunization antigen. For the first immunization, 1 mg of multi-epitope recombinant nanoprotein was emulsified with an equal amount of Freund's complete adjuvant and injected subcutaneously at multiple points on the back; booster immunization was performed every two weeks, with 1 mg of multi-epitope recombinant nanoprotein emulsified with an equal amount of Freund's incomplete adjuvant and injected subcutaneously at multiple points on the back; one week after the third booster immunization, blood was collected from the ear vein of the rabbits, centrifuged at 12000rpm for 5 minutes. After a large amount of blood was collected from the heart, it was placed at room temperature overnight, centrifuged at 12000rpm for 20 minutes, and the serum was packaged and stored at -80℃.

[0066] S402, polyclonal antibodies were obtained by ammonium sulfate precipitation of the immunized serum. OD was detected by indirect ELISA 450nm Value. The judgment standard is: OD 450nm ≥0.2, judged as positive; OD 450nm If the value is less than 0.2, it is judged as negative. The maximum dilution multiple of the positive serum sample is taken as the titer of the serum sample.

[0067] Example 5: A reagent for detecting antibodies to type A foot-and-mouth disease virus based on enzyme-linked immunosorbent assay, the main components of which are: coated ELISA plate, horseradish peroxidase-labeled polyclonal antibody (HRP-labeled antibody), positive quality control product, negative quality control product, diluent, washing solution, chromogenic substrate solution A, chromogenic substrate solution B, and stop solution.

[0068] The method for preparing the coated ELISA plate specifically comprises the following steps: S501, Antigen coating: Use CBS buffer (pH=9.6) to dilute the A-type foot-and-mouth disease multi-epitope recombinant nanoparticle protein of the present application into a coating solution with a final concentration of 2μg / mL, and add the coating solution to the ELISA plate, 100μL / well. Coat at 4°C for 12-18h, discard the solution in the ELISA plate wells; add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300μL / well, wash 5 times, 3min / time; S502, blocking: add blocking solution to the ELISA plate obtained in step S501, 200 μL / well, block at 37° C. for 2 h, discard the solution in the wells of the ELISA plate, and obtain a coated ELISA plate.

[0069] The blocking solution is bovine serum albumin (BSA) diluted with PBS buffer (pH=7.4), and the concentration of BSA is 2% (mass volume ratio w / v).

[0070] S503. Colorimetric substrate solution A: prepared from 200 mg of 3,3',5,5'-tetramethylbenzidine (TMB) and 100 ml of anhydrous ethanol.

[0071] S504, color substrate solution B: add ultrapure water to 900 ml in 14.6 g Na2HPO4 and 9.33 g citric acid, adjust the pH to 5.0-5.4, and add more ultrapure water to make up to 1000 ml.

[0072] In this embodiment, the working principle of the kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay is as follows: a competitive method is used to determine whether a sample contains type A foot-and-mouth disease virus antibodies. Type A foot-and-mouth disease virus antigen (i.e., type A foot-and-mouth disease virus recombinant nanoparticle protein of the present application) is coated in the microwells of the ELISA plate to prepare a stationary phase; the sample is added to the microwells of the coated ELISA plate, and then HRP-labeled type A foot-and-mouth disease virus polyclonal antibody is added, and the antibody in the sample and the HRP-labeled type A foot-and-mouth disease virus polyclonal antibody competitively bind to the type A foot-and-mouth disease virus recombinant nanoparticle protein on the coated ELISA plate to form an antigen-antibody complex; after washing, a two-component 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution is added; TMB is converted into blue under the catalysis of HRP, and finally converted into yellow under the action of acid. The depth of color is negatively correlated with the content of type A foot-and-mouth disease virus antibodies in the sample; the absorbance (OD value) is measured at a wavelength of 450nm using an enzyme marker, and the OD value is used to calculate whether the sample contains type A foot-and-mouth disease virus antibodies.

[0073] The working process of the kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay specifically includes the following steps: S601, sample addition: add the pre-mixed negative control, positive control and sample to be tested to the coated ELISA plate, 5 μL / well; S602, adding enzyme-labeled antibody: add HRP-labeled polyclonal antibody solution to the sample well and control well, 95 μL / well, and mix well; seal the ELISA plate with a sealing film and incubate at 37°C for 30 min; The HRP-labeled polyclonal antibody solution is prepared by diluting the HRP-labeled polyclonal antibody with PBST buffer (0.05% Tween-20, pH=7.4) at a dilution ratio of 1:10000 (mass to volume ratio w / v). S603, washing: discard the solution in the wells of the ELISA plate, add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL / well, wash 4 times, 3 min / time; S604, color development and termination: first add 50 μL / well color development substrate solution A to the ELISA plate, then add 50 μL / well color development substrate solution B, mix well, and color development at 37°C in the dark for 15 min; then add 50 μL / well of the stop solution, and gently shake the ELISA plate until the color is uniform; The stop solution is prepared by diluting 98% H2SO4 with deionized water to obtain a concentration of 2M H2SO4.

[0074] S605. Reading: Place the ELISA plate into an ELISA reader, and perform dual wavelength measurement and read the absorbance OD value under the conditions of a test wavelength of 450nm and a reference wavelength of 630nm.

[0075] Result judgment: The OD value of the negative control is recorded as ODN, the OD value of the positive control is recorded as ODP, and the OD value of the sample to be tested is recorded as ODS; Test establishment conditions: ODN value>0.5 and ODP value<0.2, the test result is valid, otherwise retest; Result determination: if the ODS value of the sample to be tested / ODN value of the negative control is ≤0.25, the result is judged to be positive; if the ODS value of the sample to be tested / ODN value of the negative control is >0.35, the result is judged to be negative; if 0.25 <ODS value of the sample to be tested / ODN value of the negative control is ≤0.35, the result is judged to be suspicious and the OD value of the sample to be tested needs to be retested. If the retested OD value is ≤0.25, the result is judged to be positive; if the retested OD value is >0.25, it is judged to be negative.

[0076] Example 6: Kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic microparticle chemiluminescence immunoassay In this embodiment, the working principle of the kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic particle chemiluminescence immunoassay is as follows: the competitive immunoassay principle is combined with magnetic particle separation technology, the sample to be tested is mixed and incubated with biotin-labeled type A foot-and-mouth disease multi-epitope recombinant protein (antigen), acridinium ester-labeled polyclonal antibody and streptavidin magnetic beads to form an immune complex, after the unbound impurities are removed by washing with solid phase carrier magnetic particles, a luminescent substrate is added to promote luminescence, and the relative luminescence intensity (RLU) is measured. Within a certain range, RLU is inversely proportional to the titer of type A foot-and-mouth disease virus antibodies, and RLU=corresponding type A foot-and-mouth disease virus antibody titer value is output through the built-in standard curve of the instrument.

[0077] In this embodiment, the main components of the kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic particle chemiluminescence immunoassay are: magnetic bead working solution, antigen working solution, acridinium ester marker working solution, pre-excitation solution, excitation solution, calibrator, positive quality control product, and negative quality control product.

[0078] Magnetic bead working solution: prepared from 225 μl of streptavidin affinity magnetic beads and 4275 μl of PBS-BSA solution (1% BSA, pH=7.4).

[0079] Antigen working solution: prepared from 5 μg of biotin-labeled multi-epitope recombinant protein (antigen) and 9995 μl of PBS-BSA solution (1% BSA, pH=7.4).

[0080] Acridinium ester labeling working solution: prepared from 2ug of acridinium ester labeled polyclonal antibody and 9998μl of PBS-BSA solution (1% BSA, pH=7.4).

[0081] Pre-stimulation solution: 0.1 mol / L nitric acid; 0.13 mol / L urea peroxide.

[0082] Provocative solution: 0.4 mol / L sodium hydroxide, 0.02 mol / L tritonx-100, and normal saline.

[0083] The working process of the kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic particle chemiluminescence immunoassay specifically includes the following steps: S701, sample / negative control / positive control (10 μL) and antigen working solution (50 μL) + magnetic bead working solution (20 μL) + acridinium ester marker working solution (50 μL), react at 37°C for 15 min, wash with 0.1 mol / L PBS buffer solution, add pre-excitation solution (100 μL) and excitation solution (100 μL) and react at 37°C for 2 min, and detect the luminescence value.

[0084] S702, Establishment of standard curve: The positive serum of type A foot-and-mouth disease virus with known antibody titer (1:8192) detected by the antibody neutralization test method in GB / T 18935 was diluted 2-fold with SPF pig serum as diluent, and the positive serum with antibody titer of 1:4096, 1:2048, 1:1024, 1:512, 1:256, 1:128, 1:64, 1:32, 1:16, 1:8 was obtained respectively. The positive serum with antibody titer of 1:512, 1:256, 1:128, 1:64, 1:32, 1:16 was selected as calibrator 1, calibrator 2, calibrator 3, calibrator 4, calibrator 5, calibrator 6 respectively. When testing, add them in order, the instrument automatically recognizes the test, and generates a standard curve.

[0085] S703, result determination: Test establishment conditions: The titer of the positive control antibody is between 1:512 and 1:2048, and the titer of the negative control antibody is less than 1:8 Result judgment: When the antibody titer is ≥1:128, it is judged as positive for type A foot-and-mouth disease antibody; When the antibody titer is less than 1:64, it is judged as negative for type A foot-and-mouth disease antibody; When the antibody titer is 1:64≤<1:128, it is considered suspicious and retesting is recommended. When the antibody titer is ≥1:128, it is considered positive. When the antibody titer is <1:128, it is considered negative. S704, kit performance test: (1) Sensitivity testing: The kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay of the present application, the kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic microparticle chemiluminescence immunoassay of the present application, and the liquid phase blocking ELISA detection kit for type A foot-and-mouth disease virus antibodies were used to detect the sensitivity control products respectively. Among them, the sensitivity control products were prepared by 2-fold gradient dilution of type A foot-and-mouth disease virus antibody positive serum with known neutralizing antibody titer, and the neutralizing antibody titers of the sensitivity control products were 1:8192, 1:4096, 1:2048, 1:1024, 1:512, 1:256, 1:128, 1:64, 1:32, 1:16, and 1:8, respectively. The test results are shown in Table 3.

[0086] Table 3, the sensitivity test results of the kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay, the kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic microparticle chemiluminescence immunoassay, and the liquid phase blocking ELISA test kit for foot-and-mouth disease virus type A antibodies of the present application:

[0087] (2) Specificity (cross-reaction) test: The labeled antigen working solution was used to simultaneously detect positive sera of foot-and-mouth disease virus type A, Asia type I, type A, swine fever virus, porcine reproductive and respiratory syndrome virus, and canine parvovirus. The results showed that this method had no cross-reaction with other susceptible animal viruses.

[0088] (3) Repeatability test: Calibrators 1, 3, and 5 were tested for repeatability according to the A-type foot-and-mouth disease virus magnetic microparticle chemiluminescent antibody detection method. Each calibrator was tested 20 times. The results showed that the stability of the A-type foot-and-mouth disease virus magnetic microparticle chemiluminescent antibody detection method was in line with expectations, and the CV values ​​were all less than 3%.

[0089] (4) Analysis of compliance rate: A total of 238 serum clinical samples of cattle, sheep and pigs were tested using the established enzyme-linked immunosorbent assay and magnetic microparticle chemiluminescent immunoassay kits for detecting type A foot-and-mouth disease virus antibodies and the standard method for detecting foot-and-mouth disease virus antibodies recommended by the World Organization for Animal Health (WOAH) and the international trade designated method. The results showed that the compliance rates of the kit for detecting type A foot-and-mouth disease virus antibodies established by the present application based on enzyme-linked immunosorbent assay and magnetic microparticle chemiluminescent immunoassay and the type A foot-and-mouth disease virus liquid phase blocking ELISA antibody detection kit produced by Lanzhou Animal Research Biotechnology Co., Ltd. reached 95.33% (Table 4) and 97.16% (Table 5), respectively.

[0090] Table 4: Comparison results of 238 serum clinical samples using the A-type foot-and-mouth disease virus antibody detection kit established by the present application based on enzyme-linked immunosorbent assay and the A-type foot-and-mouth disease virus liquid phase blocking ELISA antibody detection kit of Lanzhou Zouyan Biotechnology Co., Ltd.:

[0091] Table 5: Comparison results of 238 serum clinical samples using the magnetic particle chemiluminescent detection kit for type A foot-and-mouth disease virus antibodies established by the present application based on the magnetic particle chemiluminescent immunoassay and the liquid phase blocking ELISA antibody detection kit for type A foot-and-mouth disease virus of Lanzhou Zoological Research Biotechnology Co., Ltd.:

[0092] It can be seen from Table 4 that the conformity rate between the A-type foot-and-mouth disease virus antibody detection kit established by the present application based on the enzyme-linked immunosorbent assay and the foot-and-mouth disease virus A liquid phase blocking antibody detection kit of Lanzhou Animal Research Biotechnology Co., Ltd. is 95.33%; it can be seen from Table 5 that the A-type foot-and-mouth disease virus antibody magnetic particle chemiluminescence detection kit established by the present application based on the magnetic particle chemiluminescence immunoassay and the foot-and-mouth disease virus A liquid phase blocking antibody detection kit of Lanzhou Animal Research Biotechnology Co., Ltd. is 97.16%. Both can significantly improve the detection sensitivity of A-type foot-and-mouth disease virus antibodies.

[0093] The present invention first provides a recombinant nanoparticle protein of type A foot-and-mouth disease virus VP1 and a polyclonal antibody of type A foot-and-mouth disease virus, providing a technical platform for the detection of type A foot-and-mouth disease immune antibody levels, epidemic monitoring and epidemiological investigation.

[0094] A kit for detecting antibodies against type A foot-and-mouth disease virus was established based on enzyme-linked immunosorbent assay (ELISA) using competitive ELISA technique.

[0095] It has the following advantages: (1) This kit has the advantages of strong specificity and good stability. Compared with the LPB-ELISA test kit produced by Lanzhou Zoological Research Biotechnology Co., Ltd., the compliance rate is 95.33%. It can be used for the detection of serum antibodies to type A foot-and-mouth disease virus.

[0096] (2) The recombinant nanoparticle protein of type A foot-and-mouth disease virus VP1 is used as the antigen, which has high sensitivity and can cover all genotypes of type A foot-and-mouth disease virus in the detection range to avoid missed detection.

[0097] (3) The detection time is short, the operation is simple, and the cost is low. It is suitable for large-scale rapid detection and has great promotion value in the monitoring and diagnosis of foot-and-mouth disease.

[0098] A kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic particle chemiluminescence immunoassay was established by combining magnetic particle chemiluminescence immunoassay technology with biotin labeling technology and acridinium ester labeling technology. It can quickly, accurately and massively detect FMDV-A serum antibodies, providing new technical support for the rapid diagnosis of FMDV-A.

[0099] It has the following advantages: (1) The acridinium ester reaction system is used, which has instant luminescence, short reaction time and good stability.

[0100] (2) The specific binding process between antigen and antibody adopts the competitive method, which is a one-step reaction with short time and simple operation.

[0101] (3) Multiple strains of type A foot-and-mouth disease virus VP1 multi-epitope recombinant nanoproteins are used as coating agents and labeled antibodies, which have strong specificity.

[0102] (4) The chemiluminescent immunoassay technique was used, which had good sensitivity and was compared with the liquid-phase blocking ELISA antibody detection kit for foot-and-mouth disease virus type A produced by Lanzhou Zoological Research Biotechnology Co., Ltd., with a compliance rate of 97.16%.

[0103] At the same time, it is equipped with an automated chemiluminescence immunoassay analyzer and a standard curve, which can achieve accurate quantitative and fully automated detection with high sensitivity and specificity, and can be used for the screening of type A foot-and-mouth disease virus antibodies in a large number of clinical samples.

[0104] The above specific embodiments are only several optional embodiments of the present invention. Based on the technical solutions of the present invention and the relevant inspirations of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. A multi-epitope recombinant nanoprotein of foot-and-mouth disease virus type A, characterized in that: The encoding gene of the multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus is the amino acid sequence shown in SEQ ID NO.

14.

2. A recombinant plasmid, characterized in that: The recombinant plasmid comprises the coding gene according to claim 1.

3. A polyclonal antibody against type A foot-and-mouth disease virus, characterized in that: The polyclonal antibody is prepared using the multi-epitope recombinant nanoprotein according to claim 1 as an immunogen.

4. A kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay, characterized in that: It contains a marker antibody and a type A foot-and-mouth disease virus antigen; The labeled antibody is the HRP-labeled polyclonal antibody of type A foot-and-mouth disease virus according to claim 3; The type A foot-and-mouth disease virus antigen is the type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein according to claim 1.

5. A kit for detecting type A foot-and-mouth disease virus antibodies based on magnetic microparticle chemiluminescence immunoassay, characterized in that: comprising a labeled polyclonal antibody and a labeled antigen; The labeled polyclonal antibody is the type A foot-and-mouth disease virus polyclonal antibody of claim 3 labeled with an acridinium ester; The labeled antigen is the multi-epitope recombinant nanoprotein of type A foot-and-mouth disease virus according to claim 1 labeled with biotin.

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