A type foot-and-mouth disease virus polyepitope recombinant nanoprotein and application thereof

Abstract

The present application relates to the field of foot-and-mouth disease virus type A detection technology, and more particularly to a foot-and-mouth disease virus type A multi-epitope recombinant nanoprotein and its application. The present application provides foot-and-mouth disease virus type A related proteins and antibodies, and establishes a detection kit based on competitive enzyme-linked immunosorbent assay and magnetic particle chemiluminescence immunoassay technology, which has the advantages of high sensitivity, wide detection range, short detection time, simple operation, low cost, etc. It is also matched with automatic instruments and standard curves, and can realize precise quantification and full automation detection, and has important value and good popularization prospect in foot-and-mouth disease immune antibody level detection, epidemic situation monitoring, epidemiological investigation and clinical large sample screening, etc.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection of foot-and-mouth disease virus type A, and in particular to a multi-epitope recombinant nanoprotein of foot-and-mouth disease virus type A and its application. BACKGROUND

[0002] Foot-and-mouth diease (FMD) is an acute, febrile, highly contagious disease caused by foot-and-mouth diease virus (FMDV). Pigs, cattle, sheep and more than 70 kinds of wild even-toed animals are susceptible. The clinical symptoms are high fever, depression, loss of appetite, oral mucosa, skin and hoof of the hoof, and different degrees of blisters and erosion. It can be transmitted through air and pollutants, spread fast, spread widely, and the mortality rate of young even-toed animals is very high.

[0003] FMDV belongs to the family of small RNA viruses (Picornaviridae) and the genus of foot-and-mouth disease virus (Aphthovirus), and is a single-stranded positive-sense RNA virus, which is composed of 5' untranslated regions (UTR) and a large open reading frame (ORF) and 3' UTR 3 parts. The ORF encodes a polyprotein, which can form four structural proteins (VP4, VP2, VP3 and VP1) and ten non-structural proteins (L pro 2A, 2B, 2C, 3A, 3B1, 3B2, 3B3, 3C pro and 3D pol ) after multiple cleavages. The structural proteins form the virus capsid, and the non-structural proteins are related to virus replication. Among them, the antigen variation of VP1 is the largest, while the difference between VP2 and VP3 is relatively small. Most of the VP1 protein is exposed on the surface of the virus particle, containing the major antigenic epitopes of foot-and-mouth disease virus 141-160 and 200-213 amino acid residues, wherein the G-H loop composed of amino acid residues 141-160 of VP1 protrudes from the surface of the virus capsid and forms a highly conserved Arg-Gly-Asp (RGD) sequence on the 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 test and serological test, foot-and-mouth disease virus can be divided into 7 serotypes, i.e. A type, A type, C type, SAT1 type, SAT2 type, SAT3 type and Asia 1 type, each of which is divided into different subtypes, and there is no cross protection between types. Among them, A, O and Asia 1 type are the main epidemic serotypes of foot-and-mouth disease in China, and in recent years, A type is more. A type has the most antigen spectrum and genetic diversity. According to the genetic divergence relationship of VP1 gene sequence, FMDV-A strains can be divided into 3 topological types and 26 genotypes. The prevalence of a large number of antigen spectrum and genetic diversity strains brings great challenges to the prevention and control of foot-and-mouth disease.

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

[0006] Due to the diversity of A type FMDV antigen spectrum, antibody detection methods are often used to evaluate the immune effect of FMD vaccine. Mainly including virus neutralization test, liquid phase blocking ELISA and solid phase competitive ELISA. Virus neutralization test needs to prepare monolayer cells, which is complex in operation, 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 on the market have poor sensitivity, narrow detection spectrum, complicated operation steps, high requirement for detection personnel, long reaction time, cannot realize full-automatic detection, and are difficult to realize rapid detection of large quantities in a short time, therefore, the application provides a kind of A type foot-and-mouth disease virus multi-epitope recombinant nanoprotein and its application. SUMMARY

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

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

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

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

[0011] In a fourth aspect, the application provides a kit for detecting foot-and-mouth disease virus type A antibody based on enzyme-linked immunosorbent assay, which comprises a labeled antibody and a foot-and-mouth disease virus type A antigen.

[0012] The labeled antibody is an HRP-labeled foot-and-mouth disease virus type A polyclonal antibody as described in the third aspect.

[0013] The foot-and-mouth disease virus type A antigen is the foot-and-mouth disease virus type A multi-epitope recombinant nanoprotein as described in the first aspect.

[0014] In a fifth aspect, the application provides a kit for detecting foot-and-mouth disease virus type A antibody based on magnetic microparticle chemiluminescence immunoassay, which comprises a labeled polyclonal antibody and a labeled antigen.

[0015] The labeled polyclonal antibody is an acridinium ester-labeled foot-and-mouth disease virus type A polyclonal antibody as described in the third aspect.

[0016] The labeled antigen is a biotin-labeled foot-and-mouth disease virus type A multi-epitope recombinant nanoprotein as described in the first aspect.

[0017] Compared with the prior art, the application has the following beneficial technical effects:

[0018] The application first provides a foot-and-mouth disease virus type A VP1 recombinant nanoparticle protein and a foot-and-mouth disease virus type A polyclonal antibody, which provides a technical platform for foot-and-mouth disease virus type A immune antibody level detection, epidemic situation monitoring and epidemiological investigation.

[0019] Secondly, the application establishes a kit for detecting foot-and-mouth disease virus type A antibody based on enzyme-linked immunosorbent assay by using competitive enzyme-linked immunosorbent technology.

[0020] The application has the following advantages:

[0021] (1) The kit has the advantages of high specificity and good stability, and compared with the foot-and-mouth disease virus type A liquid blocking ELISA antibody detection kit produced by Lanzhou Animal Research Biotechnology Co., Ltd., the coincidence rate is 95.33%. It can be used for detection of foot-and-mouth disease virus type A serum antibody.

[0022] (2) The recombinant nanometer protein of foot-and-mouth disease virus type A VP1 is used as antigen, which has high sensitivity and can cover all genotypes of foot-and-mouth disease virus type A, avoiding missed detection.

[0023] (3) The detection time is short, the operation is simple, the cost is low, and it is suitable for rapid detection of large number of samples, and has high popularization value in the monitoring and diagnosis of foot-and-mouth disease.

[0024] Further, the application adopts magnetic particle chemiluminescence immunoassay technology combined with biotin labeling technology and acridinium ester labeling technology to establish a kit for detecting foot-and-mouth disease virus type A antibody based on magnetic particle chemiluminescence immunoassay. The FMDV-A antibody detection can be carried out quickly, accurately and in large quantities, which provides new technical support for rapid diagnosis of FMDV-A.

[0025] Has the following advantages:

[0026] (1) The acridinium ester reaction system is used for instantaneous luminescence, and the reaction time is short and the stability is good.

[0027] (2) The specific binding process of antigen and antibody adopts competition method, one-step reaction, short time and simple operation.

[0028] (3) The multi-epitope recombinant nanometer protein of foot-and-mouth disease virus type A is used as coating agent, and the corresponding polyclonal antibody is used as labeled antibody, which has high specificity.

[0029] (4) The chemiluminescence immunoassay technology has good sensitivity, and compared with the foot-and-mouth disease virus type A liquid blocking ELISA antibody detection kit produced by Lanzhou Animal Research Biotechnology Co., Ltd., the coincidence rate is 97.16%.

[0030] (5) The matched automatic chemiluminescence immunoassay analyzer can realize precise quantitative detection with high sensitivity and high specificity. The automatic detection greatly reduces the labor and time cost, and is suitable for screening of large number of samples.

[0031] In summary, the application provides a type A foot-and-mouth disease virus related protein and antibody, and establishes a detection kit based on competitive enzyme-linked immunosorbent assay and magnetic particle chemiluminescence immunoassay technology. The former has the advantages of high specificity, good stability, high sensitivity, wide detection range, short detection time, simple operation, low cost, etc. The latter uses an acridinium ester reaction system, combines magnetic particle separation technology and chemiluminescence immunoassay technology, has the advantages of short reaction time, good stability, high specificity, high sensitivity, and can realize precise quantification, automatic detection, etc. with the support of automatic instruments and standard curves. It has important value and good popularization prospect in the detection of type A foot-and-mouth disease virus immune antibody level, epidemic situation monitoring, epidemiological investigation and large-scale clinical sample screening. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 PCR identification result graph of the type A foot-and-mouth disease virus multi-epitope recombinant plasmid;

[0033] Figure 2 Type A foot-and-mouth disease virus multi-epitope nano recombinant protein expression result graph. DETAILED DESCRIPTION

[0034] The application discloses a type A foot-and-mouth disease virus multi-epitope recombinant nano protein and application thereof, and those skilled in the art can refer to the content herein and appropriately improve process parameters to realize. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0035] In the first aspect of the application, the application provides a type A foot-and-mouth disease virus multi-epitope recombinant nano protein, and the coding gene of the type A foot-and-mouth disease virus multi-epitope recombinant nano protein is a nucleotide sequence as shown in SEQ ID NO. 13, and corresponds to an amino acid sequence as shown in SEQ ID NO. 14.

[0036] In the second aspect of the application, the application provides a recombinant plasmid, and the recombinant plasmid comprises the coding gene according to the first aspect.

[0037] In the third aspect of the application, the application provides a type A foot-and-mouth disease virus polyclonal antibody, and the polyclonal antibody is prepared by taking the multi-epitope recombinant nano protein according to the first aspect as an immunogen.

[0038] In the fourth aspect of the application, the application provides a kit for detecting type A foot-and-mouth disease virus antibodies based on enzyme-linked immunosorbent assay, and the kit comprises a labeled antibody and a type A foot-and-mouth disease virus antigen.

[0039] The labeled antibody is a third aspect of an HRP-labeled type A foot-and-mouth disease virus polyclonal antibody.

[0040] The type A foot-and-mouth disease virus antigen is a first aspect of a type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein.

[0041] 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 magnetic particle chemiluminescence immunoassay, comprising a labeled polyclonal antibody and a labeled antigen.

[0042] The labeled polyclonal antibody is a third aspect of an acridinium ester-labeled foot-and-mouth disease virus type A polyclonal antibody.

[0043] The labeled antigen is a first aspect of a biotin-labeled type A foot-and-mouth disease virus multi-epitope recombinant nanoprotein.

[0044] Example 1: Preparation of a coding gene, the specific scheme is as follows:

[0045] S101, analyze the full-length sequences of 40 type A foot-and-mouth disease virus VP1 genes retrieved from Genbank, select the dominant strains A / AF72 (Genbank accession number: MT447399.1), A / WH / CHA / 2009 (Genbank accession number: JF792355.1), A / GDMM / CHA / 2013 (Genbank accession number: KF450794.1) prevalent in China, and use the online software Immunomedicine Group and SVMTriP to perform antigen epitope prediction analysis, and select the sequences according to the literature data and analysis results as follows:

[0046] A / AF72 strain

[0047] The dominant linear antigen epitope sequence 1 is 22-34aa, and the nucleotide sequence is specifically shown in SEQ ID NO. 1.

[0048] The dominant linear antigen epitope sequence 2 is 137-149aa, and the nucleotide sequence is specifically shown in SEQ ID NO. 2.

[0049] A / WH / CHA / 2009 strain (Sea-97-G1)

[0050] The dominant linear antigen epitope sequence 3 is 90-106aa, and the nucleotide sequence is specifically shown in SEQ ID NO. 3.

[0051] The dominant linear antigen epitope sequence 4 is 130-148aa, and the nucleotide sequence is specifically shown in SEQ ID NO. 4.

[0052] A / GDMM / CHA / 2013 strain (Sea-97-G2)

[0053] VP1 full-length sequence 5, 1-212aa, the nucleotide sequence is specifically shown as SEQ ID NO. 5;

[0054] Ferritin nucleotide sequence 6 (Genbank accession number: AY072939.1), the nucleotide sequence is specifically shown as SEQ ID NO. 6;

[0055] According to the preferred codon of E. coli, the optimized nucleotide sequence is obtained, which is specifically shown as follows:

[0056] The nucleotide sequence is specifically shown as SEQ ID NO. 7;

[0057] The nucleotide sequence is specifically shown as SEQ ID NO. 8;

[0058] The nucleotide sequence is specifically shown as SEQ ID NO. 9;

[0059] The nucleotide sequence is specifically shown as SEQ ID NO. 10;

[0060] The nucleotide sequence is specifically shown as SEQ ID NO. 11;

[0061] S102, the optimized nucleotide sequence of ferritin truncation is specifically shown as SEQ ID NO. 12;

[0062] S103, the above sequences are connected in the order of 7-8-9-10-11-12 by using flexible connection peptide, the nucleotide sequence of the flexible connection peptide is specifically shown as SEQ ID NO. 15, and BamHI and HindIII enzyme cutting sites are inserted at both ends. The optimized nucleotide sequence is specifically shown as SEQ ID NO. 13. The nucleotide sequence of the BamHI enzyme cutting site is specifically shown as SEQ ID NO. 16, and the nucleotide sequence of the HindIII enzyme cutting site is specifically shown as SEQ ID NO. 17.

[0063] S104, the recombinant nucleotide sequence with enzyme cutting site is synthesized into recombinant plasmid pUC57-FMDV-A-f by Bio (Anhui) Co., Ltd.

[0064] S105, the amino acid sequence of the multi-epitope recombinant nanoparticle protein encoded by the sequence SEQ ID NO. 13 is specifically shown as SEQ ID NO. 14.

[0065] Table 1 sequence information of nucleotide sequence

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Table 2 Sequence information of amino acid sequence

[0074]

[0075] Example 2: Construction of recombinant plasmid, including the following steps:

[0076] S201, according to the culture mode of E. coli, culture the A type foot-and-mouth disease virus recombinant protein gene positive strain (Amp+), E. coli containing pET-His-MBP (Kan+) and pET-28b(+) (Kan+) respectively, according to the operation manual of plasmid extraction kit (purchased from Zominer Biotech Co., Ltd.), extract the A type foot-and-mouth disease virus recombinant protein gene positive plasmid, pET-His-MBP and pET-28b(+) plasmid.

[0077] S202, according to the instruction manual of restriction endonuclease (purchased from Zominer Biotech Co., Ltd.), XbaI / BamHI double enzyme digestion was performed on pET-His-MBP and pET-28b(+) plasmid respectively. Agarose gel electrophoresis (120v, 30min) was performed on the product. According to the instruction manual of Tian Gen agarose gel DNA recovery kit, His-MBP nucleic acid fragment and pET-28b(+) vector were recovered.

[0078] S203, according to the instruction manual of T4 DNA ligase (purchased from Zominer Biotech Co., Ltd.), the digested His-MBP nucleic acid fragment and pET-28b(+) vector were ligated, and the product after ligation was named as pET-28b-His-MBP.

[0079] S204, the product after ligation was transferred into DH5α competent cells (Zominer) for culture, and the cultured DH5α was coated on LB plate (Kan+) and cultured at 37℃ overnight.

[0080] S205. Following the instructions of the restriction endonucleases (purchased from TransGen Biotech Ltd.), the recombinant protein gene positive plasmid of foot-and-mouth disease virus type A and the pET-28b-His-MBP plasmid were double-digested with BamHI / HindIII. The products were then subjected to agarose gel electrophoresis (120V, 30min), and the recombinant protein nucleic acid of foot-and-mouth disease virus type A and the pET-28b-His-MBP plasmid were recovered according to the Tiangen Agarose Gel DNA Recovery Kit.

[0081] S206. Following the instructions of T4 DNA ligase (purchased from TransGen Biotech Ltd.), ligate the enzyme-digested recombinant protein nucleic acid of type A foot-and-mouth disease virus with pET-28b-His-MBP.

[0082] S207. The ligation product pET-28b-FMDV-A-His-MBP was transformed into DH5α competent cells (Full Gold) and cultured. The cultured DH5α cells were plated on LB plates (Kan+) and cultured overnight at 37°C.

[0083] S208. Select a single-clone bacterial strain as a template, and use the universal primers T7 / T7ter for the pET-28b vector as primers for PCR amplification. Perform agarose gel electrophoresis on the products (120V 30min). A 317bp band (lane 2) is visible for the empty vector control, and approximately 2858bp (lane 3) is visible for the pET-28b-FMDV-A-His-MBP target band. Figure 1 As shown, the product fragment size is as expected.

[0084] Example 3: Expression, identification, and purification of recombinant nanoparticle proteins, specifically including the following steps:

[0085] Recombinant plasmid transformation:

[0086] The validated recombinant plasmid pET-28b-FMDV-A-His-MBP was transformed into E. coli BL21(DE3) competent cells. The cells were plated on LB agar plates (Kan+) and incubated overnight at 37°C.

[0087] S301. Pick a single colony containing the recombinant plasmid and add it to liquid LB medium (Kan+), and incubate overnight at 37°C.

[0088] S302. Add 1 ml of bacterial culture to 100 ml of fresh liquid LB medium (Kan+) and incubate in a shaker at 220 rpm and 37°C until OD reaches 100%. 600 It is between 0.6 and 0.8.

[0089] IPTG-induced expression and identification of S303 and recombinant nanoparticle proteins:

[0090] ①Add IPTG with final concentration of 0.5 mM, express at 15℃, 195 rpm overnight.

[0091] ②After induction, centrifuge 1 ml of bacterial solution at 8000 rpm for 5 min at 4℃. Resuspend the bacterial body with appropriate PBS (pH 8.0), then add 2x SDS-PAGE loading buffer and incubate at 95℃ for 10 min for SDS-PAGE analysis.

[0092] ③Centrifuge the remaining bacterial solution at 8000 rpm for 5 min at 4℃. Collect the precipitate, resuspend the bacterial body with 10 ml of PBS (pH 8.0), and add lysozyme with a final concentration of 1 mg / mL. After ice bath for 30 min, ultrasonically break the bacterial body (ultrasonic time 4 s, intermittent 9 s, 120 times).

[0093] ④After breaking, centrifuge at 12000 rpm for 30 min at 4℃. Collect the supernatant and precipitate. Take 100 μl of supernatant for SDS-PAGE analysis.

[0094] ⑤In the supernatant sample, the expressed multi-epitope recombinant nanoprotein has a clear 94 KD protein band, which is the same as the expected size. It is named multi-epitope recombinant nanoprotein FMDV-A-His-MBP, as shown in Figure 2 Lane 1 is empty vector control, Lane 2 is protein ruler, and Lane 3 is the target protein.

[0095] S304、Purification of recombinant nanoparticle protein

[0096] ①Add sample:

[0097] Add the remaining supernatant protein solution to the affinity purification Ni-NTA column treated with 5 times the volume of Binding Buffer (50 mM NaH2P04, 300 mM NaCl, 10 mM imidazole, pH 8.0) and incubate at 4℃, 200 rpm for 4 h.

[0098] ②Remove unbound proteins:

[0099] After incubation, wash the supernatant-Ni-NTA mixture with 5 times the volume of Wash buffer (50 mM NaH2P04, 300 mM NaCl, 20 mM imidazole, pH 8.0) for 3 times to remove non-specific proteins and unbound target proteins.

[0100] ③Elute the target protein:

[0101] Finally, the target protein was eluted with 5 volumes of Elution buffer (50 mM NaH2P04, 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. This operation was repeated 4 times.

[0102] ④ Desalination treatment to remove imidazole and ultrafiltration concentration:

[0103] a. The desalination column was equilibrated in advance, and 2.5 mL of the target protein sample was loaded onto the column. The target protein was eluted and replaced with 3.5 mL of 0.22 μm filtered PBS. The eluted protein sample was collected until all the protein solution was replaced.

[0104] b. The desalted protein solution was transferred to a 3 KD ultrafiltration tube, centrifuged at 4°C, 3000 rpm for 5 min, and repeated several times until the remaining liquid was about 2 mL.

[0105] c. The BCA protein quantitative detection kit was used to determine the concentration of the target protein: 1.526 mg / mL.

[0106] Example 4: Preparation and titer determination of FMDV-A-VP1 polyclonal antibody

[0107] S401, the purified FMDV-A-His-MBP protein was used as an immunizing antigen to immunize 2-month-old female New Zealand rabbits. The first immunization was performed by subcutaneous injection of 1 mg of multi-epitope recombinant nanoprotein emulsified with an equal amount of Freund's complete adjuvant in multiple points on the back; booster immunization was performed every two weeks by subcutaneous injection of 1 mg of multi-epitope recombinant nanoprotein emulsified with an equal amount of Freund's incomplete adjuvant in multiple points on the back; one week after the third booster immunization, the rabbits were bled from the marginal vein, and centrifuged at 12000 rpm for 5 min. After collecting a large amount of blood from the heart, it was placed at room temperature overnight, centrifuged at 12000 rpm for 20 min, and the serum was stored at -80°C.

[0108] S402, the polyclonal antibody was obtained by ammonium sulfate precipitation of the immunized serum. The OD value was detected by indirect ELISA. 450nm The judgment criteria are: OD 450nm ≥ 0.2, positive; OD 450nm < 0.2, negative. The maximum dilution factor of the positive serum sample is used as the titer of the serum sample.

[0109] Example 5: reagent for detecting A type foot-and-mouth disease virus antibody based on enzyme-linked immunosorbent assay, main components: coated enzyme-labeled plate, horseradish peroxidase labeled polyclonal antibody (HRP labeled antibody), positive quality control, negative quality control, diluent, washing solution, color developing substrate solution A, color developing substrate solution B, and stop solution.

[0110] The preparation method of the coated enzyme-labeled plate specifically includes the following steps:

[0111] S501, antigen coating: the A type foot-and-mouth disease multi-epitope recombinant nanoparticle protein of the application is diluted into a coating solution with a final concentration of 2 μg / mL using CBS buffer (pH=9.6), and the coating solution is added to the enzyme-labeled plate at 100 μL / well. 4°C coating for 12-18 h, discard the solution in the enzyme-labeled plate wells; add PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL / well, wash 5 times, 3 min / time;

[0112] S502, blocking: add blocking solution to the enzyme-labeled plate obtained in step S501, 200 μL / well, 37°C blocking for 2 h, discard the solution in the enzyme-labeled plate wells, and obtain the coated enzyme-labeled plate.

[0113] The blocking solution is bovine serum albumin (BSA) diluted with PBS buffer (pH=7.4), and the concentration of BSA is 2% (w / v).

[0114] S503, color developing substrate solution A: prepared from 3,3',5,5'-tetramethylbenzidine (TMB) 200 mg and anhydrous ethanol 100 ml.

[0115] S504, color developing substrate solution B: to 14.6 g of Na2HPO4 and 9.33 g of citric acid, add ultrapure water to 900 ml, adjust the pH to 5.0-5.4, and add ultrapure water to 1000 ml.

[0116] In this embodiment, the working principle of the kit for detecting foot-and-mouth disease virus type A antibody based on enzyme-linked immunosorbent assay is as follows: whether the sample contains foot-and-mouth disease virus type A antibody is determined by using a competitive method. The foot-and-mouth disease virus type A antigen (i.e., the foot-and-mouth disease recombinant nanoparticle protein of the present application) is coated in the microholes of the enzyme-labeled plate to form a fixed phase; the sample is added to the microholes of the coated enzyme-labeled plate, and then the HRP-labeled foot-and-mouth disease virus type A polyclonal antibody is added; the antibody in the sample and the HRP-labeled foot-and-mouth disease virus type A polyclonal antibody competitively bind to the foot-and-mouth disease virus type A recombinant nanoparticle protein coated on the enzyme-labeled plate to form an antigen-antibody complex; after washing, the two-component 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution is added; TMB is converted into blue under the catalysis of HRP and is finally converted into yellow under the action of acid. The color depth is negatively correlated with the content of foot-and-mouth disease virus type A antibody in the sample; the absorbance (OD value) is determined at 450 nm wavelength by using an enzyme-labeled instrument, and whether the sample contains foot-and-mouth disease virus type A antibody is calculated by the OD value.

[0117] The working process of the kit for detecting foot-and-mouth disease virus type A antibody based on enzyme-linked immunosorbent assay specifically includes the following steps:

[0118] S601, adding sample: adding the pre-mixed negative quality control, positive quality control and sample to be tested into the coated enzyme-labeled plate, 5 μL / hole;

[0119] S602, adding enzyme-labeled antibody: adding the HRP-labeled polyclonal antibody solution to the sample hole and the control hole, 95 μL / hole, and mixing; the enzyme-labeled plate is sealed with a sealing film, and incubated at 37°C for 30 min;

[0120] 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 1:10,000 (mass / volume ratio w / v);

[0121] S603, washing: discarding the solution in the holes of the enzyme-labeled plate, adding PBST buffer (0.05% Tween-20, pH=7.4) for washing, 300 μL / hole, washing 4 times, 3 min / time;

[0122] S604, color development and termination: adding 50 μL / hole of color developing substrate liquid A to the enzyme-labeled plate, and then adding 50 μL / hole of color developing substrate liquid B, mixing, color developing at 37°C for 15 min in the dark; then adding the termination liquid, 50 μL / hole, and gently shaking the enzyme-labeled plate until the color development is uniform;

[0123] The termination liquid is prepared by diluting 98% H2SO4 with deionized water to a concentration of 2M H2SO4.

[0124] S605, reading: the enzyme-labeled plate was put into the microplate reader, and the absorbance OD value was read by double-wavelength determination under the condition that the test wavelength was 450 nm and the reference wavelength was 630 nm.

[0125] Result determination:

[0126] The OD value of the negative quality control was recorded as ODN, the OD value of the positive quality control was recorded as ODP, and the OD value of the sample to be tested was recorded as ODS.

[0127] The detection condition is that the ODN value is greater than 0.5 and the ODP value is less than 0.2, the detection result is valid, otherwise the detection is re-performed.

[0128] Result determination: the ODS value of the sample to be tested / the ODN value of the negative quality control is less than or equal to 0.25, the result is positive; the ODS value of the sample to be tested / the ODN value of the negative quality control is greater than 0.35, the result is negative; 0.25

[0129] Example 6: kit for detecting A-type foot-and-mouth disease virus antibody based on magnetic particle chemiluminescence immunoassay

[0130] In this embodiment, the working principle of the kit for detecting A-type foot-and-mouth disease virus antibody based on magnetic particle chemiluminescence immunoassay is as follows: using the principle of competitive immunoassay and combining with magnetic particle separation technology, the sample to be tested, biotin-labeled A-type foot-and-mouth disease multi-epitope recombinant protein (antigen), acridinium ester-labeled polyclonal antibody and streptavidin magnetic beads are mixed and incubated to form an immune complex. After removing the unbound impurities by solid-phase carrier magnetic particles, the 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 A-type foot-and-mouth disease virus antibody, and the standard curve built in the instrument is used to output RLU=corresponding A-type foot-and-mouth disease virus antibody titer.

[0131] In this embodiment, the main components of the kit for detecting A-type foot-and-mouth disease virus antibody based on magnetic particle chemiluminescence immunoassay are magnetic bead working solution, antigen working solution, acridinium ester label working solution, pre-activation solution, activation solution, calibration, positive quality control, negative quality control.

[0132] Magnetic bead working solution: prepared from 225 μl of streptavidin magnetic beads and 4275 μl of PBS-BSA solution (1% BSA, pH=7.4).

[0133] Antigen working solution: biotin-labeled multi-epitope recombinant protein (antigen) 5 μg and PBS-BSA solution (1% BSA, pH = 7.4) 9995 μl.

[0134] Acridinester-labeled working solution: 2ug of acridinester-labeled polyclonal antibody and 9998 μl of PBS-BSA solution (1% BSA, pH = 7.4).

[0135] Pre-priming solution: 0.1 mol / L nitric acid; 0.13 mol / L urea peroxide.

[0136] Primed solution: 0.4 mol / L sodium hydroxide, 0.02 mol / L tritonx-100, physiological saline.

[0137] The working process of the kit for detecting A-type foot-and-mouth disease virus antibody based on magnetic particle chemiluminescence immunoassay method specifically includes the following steps:

[0138] S701, sample / negative quality control / positive quality control (10 μL) and antigen working solution (50 μl) + magnetic bead working solution (20 μl) + acridinester-labeled working solution (50 μl), 37°C reaction for 15 min, 0.1 mol / L PBS buffer solution washing, adding pre-priming solution (100 μl) and priming solution (100 μl) at 37°C for 2 min, detecting the luminescence value.

[0139] S702, standard curve establishment: the A-type foot-and-mouth disease virus positive serum with known antibody titer (1:8192) detected by the antibody neutralization experiment method in GB / T 18935 is diluted by 2 times gradient with SPF pig serum as diluent, and positive serum with antibody titers of 1:4096, 1:2048, 1:1024, 1:512, 1:256, 1:128, 1:64, 1:32, 1:16 and 1:8 is obtained respectively. The positive serum with antibody titers of 1:512, 1:256, 1:128, 1:64, 1:32 and 1:16 is selected as calibration 1, calibration 2, calibration 3, calibration 4, calibration 5 and calibration 6 respectively. During detection, they are added in order, and the instrument automatically recognizes and detects to generate a standard curve.

[0140] S703, result determination:

[0141] Detection condition:

[0142] The antibody titer of the positive quality control is between 1:512 and 1:2048, and the antibody titer of the negative quality control is <1:8

[0143] Result determination:

[0144] The antibody titer ≥ 1:128 is determined as positive for type A foot-and-mouth disease antibody;

[0145] The antibody titer < 1:64 is determined as negative for type A foot-and-mouth disease antibody;

[0146] 1:64≤antibody titer < 1:128 is determined as suspicious, and retesting is recommended. When the retested antibody titer ≥ 1:128, it is determined as positive, and when the retested antibody titer < 1:128, it is determined as negative;

[0147] S704, kit performance detection:

[0148] (1) Sensitivity detection:

[0149] The sensitivity quality control is detected by the kit for detecting type A foot-and-mouth disease virus antibody based on enzyme-linked immunosorbent method, the kit for detecting type A foot-and-mouth disease virus antibody based on magnetic particle chemiluminescence immunoassay, and the liquid-phase blocking ELISA detection kit for foot-and-mouth disease type A antibody. The sensitivity quality control is prepared by 2-fold gradient dilution of type A foot-and-mouth disease virus antibody positive serum with known neutralizing antibody titer. The neutralizing antibody titers of the sensitivity quality control are 1:8192, 1:4096, 1:2048, 1:1024, 1:512, 1:256, 1:128, 1:64, 1:32, 1:16, and 1:8. The detection results are shown in Table 3.

[0150] Table 3, sensitivity detection results of the kit for detecting type A foot-and-mouth disease virus antibody based on enzyme-linked immunosorbent method, the kit for detecting type A foot-and-mouth disease virus antibody based on magnetic particle chemiluminescence immunoassay, and the liquid-phase blocking ELISA detection kit for foot-and-mouth disease type A antibody:

[0151]

[0152] (2) Specificity (cross reaction) test: The labeled antigen working solution is used to detect type A foot-and-mouth disease virus, Asia I type, A type, porcine fever virus, porcine reproductive and respiratory syndrome virus, and canine parvovirus positive serum. The results show that the method has no cross reaction with other susceptible animal viruses.

[0153] (3) Reproducibility test: The calibration 1, calibration 3, and calibration 5 are repeatedly detected according to the type A foot-and-mouth disease virus magnetic particle chemiluminescence antibody detection method. Each calibration is detected for 20 times. The results show that the stability of the type A foot-and-mouth disease virus magnetic particle chemiluminescence antibody detection method meets the expectation, and the CV values are all less than 3%.

[0154] (4) Coincidence rate analysis: The A-type foot-and-mouth disease virus antibody reagent kit established by the enzyme-linked immunosorbent method and the magnetic particle chemiluminescence immunoassay method was used to detect 238 serum clinical samples of cattle, sheep and pigs together with the A-type foot-and-mouth disease virus liquid-phase blocking ELISA antibody detection kit established by the standard method recommended by the World Organization for Animal Health (WOAH) and the international trade designated method for detecting foot-and-mouth disease virus antibodies. The results show that the coincidence rate of the A-type foot-and-mouth disease virus antibody reagent kit established by the enzyme-linked immunosorbent method and the magnetic particle chemiluminescence immunoassay method reaches 95.33% (Table 4) and 97.16% (Table 5) respectively compared with the A-type foot-and-mouth disease virus liquid-phase blocking ELISA antibody detection kit produced by Lanzhou Shunyan Biotechnology Co., Ltd.

[0155] Table 4: Comparison results of the A-type foot-and-mouth disease virus antibody detection kit established by the enzyme-linked immunosorbent method and the A-type foot-and-mouth disease virus liquid-phase blocking ELISA antibody detection kit of Lanzhou Shunyan Biotechnology Co., Ltd. on 238 serum clinical samples:

[0156]

[0157] Table 5: Comparison results of the A-type foot-and-mouth disease virus antibody magnetic particle chemiluminescence detection kit established by the magnetic particle chemiluminescence immunoassay method and the A-type foot-and-mouth disease virus liquid-phase blocking ELISA antibody detection kit of Lanzhou Shunyan Biotechnology Co., Ltd. on 238 serum clinical samples:

[0158]

[0159] As can be seen from Table 4, the coincidence rate of the A-type foot-and-mouth disease virus antibody detection kit established by the enzyme-linked immunosorbent method and the foot-and-mouth disease A liquid-phase blocking antibody detection kit of Lanzhou Shunyan Biotechnology Co., Ltd. is 95.33%; as can be seen from Table 5, the coincidence rate of the A-type foot-and-mouth disease virus antibody magnetic particle chemiluminescence detection kit established by the magnetic particle chemiluminescence immunoassay method and the foot-and-mouth disease A liquid-phase blocking antibody detection kit of Lanzhou Shunyan Biotechnology Co., Ltd. is 97.16%. Both can significantly improve the detection sensitivity of the A-type foot-and-mouth disease virus antibody.

[0160] The application first provides an A-type foot-and-mouth disease virus VP1 recombinant nanoparticle protein and an A-type foot-and-mouth disease virus polyclonal antibody. A technical platform is provided for A-type foot-and-mouth disease immune antibody level detection, epidemic situation monitoring and epidemiological investigation.

[0161] A kit for detecting A-type foot-and-mouth disease virus antibody based on enzyme-linked immunosorbent assay is established by using competitive enzyme-linked immunosorbent technology.

[0162] The kit has the following advantages:

[0163] (1) The kit has the advantages of high specificity and good stability, and has a coincidence rate of 95.33% compared with the LPB-ELISA detection kit produced by Lanzhou Animal Research Biotechnology Co., Ltd. It can be used for detection of serum antibodies of foot-and-mouth disease virus type A.

[0164] (2) The recombinant nanometer protein of foot-and-mouth disease virus type A VP1 is used as antigen, which has high sensitivity and can cover all genotypes of foot-and-mouth disease virus type A, avoiding missed detection.

[0165] (3) The detection time is short, the operation is simple, the cost is low, and it is suitable for large-scale rapid detection, and has good popularization value in the monitoring and diagnosis of foot-and-mouth disease.

[0166] A kit for detecting foot-and-mouth disease virus type A antibody based on magnetic particle chemiluminescence immunoassay is established by using magnetic particle chemiluminescence immunoassay technology combined with biotin labeling technology and acridinium ester labeling technology. The kit can quickly and accurately detect FMDV-A serum antibodies in large quantities, and provides new technical support for rapid diagnosis of FMDV-A.

[0167] The kit has the following advantages:

[0168] (1) The acridinium ester reaction system is used for instantaneous luminescence, and the reaction time is short and the stability is good.

[0169] (2) The specific binding process of antigen and antibody uses competition method, one-step reaction, short time and simple operation.

[0170] (3) The multi-strain foot-and-mouth disease virus type A VP1 multi-epitope recombinant nanometer protein is used as coating and labeled antibody, which has high specificity.

[0171] (4) The chemiluminescence immunoassay technology is used, which has good sensitivity, and has a coincidence rate of 97.16% compared with the foot-and-mouth disease virus type A liquid phase blocking ELISA antibody detection kit produced by Lanzhou Animal Research Biotechnology Co., Ltd.

[0172] At the same time, the automatic chemiluminescence immunoassay instrument and standard curve are matched, which can realize precise quantitative and fully automatic detection with high sensitivity and high specificity, and can be used for screening of large number of samples of foot-and-mouth disease virus type A antibody in clinic.

[0173] The above specific embodiments are only several optional embodiments of the present application, and based on the technical solutions of the present application and the related inspirations of the above embodiments, those skilled in the art can make various alternative improvements and combinations on the above specific embodiments.

Claims

1. A recombinant multi-epitope nanoprotein of Foot-and-Mouth Disease Virus Type A, characterized in that, The amino acid sequence of the A-type foot-and-mouth disease virus polyepitope recombinant nanoprotein is shown as SEQ ID NO.

14.

2. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the coding gene of the A-type foot-and-mouth disease virus polyepitope recombinant nanoprotein according to claim 1.

3. A kit for detecting antibodies against foot-and-mouth disease virus type A based on enzyme-linked immunosorbent assay, characterized in that, It comprises a labeled antibody and an A-type foot-and-mouth disease virus antigen; The labeled antibody is an A-type foot-and-mouth disease virus polyclonal antibody prepared by using the polyepitope recombinant nanoprotein according to claim 1 as an immunogen and labeled by HRP; The A-type foot-and-mouth disease virus antigen is the A-type foot-and-mouth disease virus polyepitope recombinant nanoprotein according to claim 1.

4. A kit for detecting antibodies against foot-and-mouth disease virus type A based on magnetic microparticle chemiluminescent immunoassay, characterized in that, It comprises a labeled polyclonal antibody and a labeled antigen; The labeled polyclonal antibody is an A-type foot-and-mouth disease virus polyclonal antibody prepared by using the polyepitope recombinant nanoprotein according to claim 1 as an immunogen and labeled by acridinester; The labeled antigen is the A-type foot-and-mouth disease virus polyepitope recombinant nanoprotein according to claim 1 labeled by biotin.

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