CN114316037A - Antibody m19 of O-type foot-and-mouth disease virus structural protein, preparation method and application - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to an antibody of O-type foot-and-mouth disease virus structural protein, a preparation method and application. The invention takes O-type foot-and-mouth disease virus 146s as immunogen to immunize Balb/c mice, and obtains a hybridoma cell line which efficiently secretes monoclonal antibody through cell fusion, and obtains a mouse monoclonal antibody m 19. An indirect immunofluorescence test shows that the monoclonal antibody m19 has good reactivity to O-type different lineage strains, but does not react with A-type different lineage strains. The monoclonal antibody m19 and the O-type structural protein rabbit antiserum are used for establishing the O-type foot-and-mouth disease virus structural protein solid-phase competition ELISA detection kit, compared with the existing antibody, the sensitivity and the specificity of a detection result are obviously improved, the kit is suitable for detecting the structural protein antibody after O-type foot-and-mouth disease infection or inactivated vaccine immunization, and meanwhile, the kit is a novel method for monitoring the infection condition of the foot-and-mouth disease non-immune area.

Description

Antibody m19 of O-type foot-and-mouth disease virus structural protein, preparation method and application

Technical Field

The invention relates to the technical field of biology, and in particular relates to an antibody m19 of an O-type foot-and-mouth disease virus structural protein, a preparation method and application.

Background

Foot-and-mouth disease is an infectious disease of artiodactyl co-morbid animals caused by infection with Foot-and-mouth disease virus (FMDV). FMDV belongs to members of the genus foot-and-mouth disease virus (aphthviruses) of the picornaviridae family (picornaviridae), and currently there are O, A, C, SAT1, SAT2, SAT3 (i.e., south african foot-and-mouth disease virus types 1, 2, 3) and Asia1 (Asia type 1) 7 serotypes. There is little immune protection among the types, and animals infected with one type of foot-and-mouth disease can still be infected with another type of foot-and-mouth disease virus to cause diseases.

Because the antigenicity of different subtypes in the same serotype are different in degree, and the serological cross reaction degree is different, the diagnosis and control of the foot-and-mouth disease are very difficult. The monoclonal antibody has the characteristics of capability of identifying a single antigen site, strong specificity of combining with an antigen, high homogeneity, single biological activity, easiness in standardization and capability of being produced in batches, and is widely applied to the field of biomedicine. At present, FMD of type O, Asial and A are mainly prevalent in China, so that the research and development of monoclonal antibodies of type O foot-and-mouth disease virus are of great significance for the diagnosis and prevention of the disease.

Currently, there are 3 serological foot-and-mouth disease detection methods: the Virus Neutralization Test (VNT), the liquid phase blocking ELISA (LPB-ELISA) and the solid phase competition ELISA (SPC-ELISA) have the traditional advantages of the liquid phase blocking ELISA, have better specificity and simpler operation, and are established as the latest recommended method for foot-and-mouth disease serological investigation by OIE in 2004. However, because the FMDV antigen has a complex structure, the development of a broad-spectrum and specific detection kit for an O-type foot-and-mouth disease virus antibody requires the screening of a specific monoclonal antibody against a recognized internal conserved epitope, but the specific monoclonal antibodies for the detection of the O-type foot-and-mouth disease virus are rare at present, which brings certain obstacles for the establishment of a broad-spectrum and specific detection method for the O-type FMDV antibody.

Disclosure of Invention

In view of the above technical problems, the present invention aims to provide an antibody against structural protein of foot-and-mouth disease virus type O, a preparation method and an application thereof, specifically comprising the following contents:

in a first aspect, the invention provides a monoclonal antibody m19 of a structural protein of a foot-and-mouth disease virus type O, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody m19 is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2.

Preferably, the monoclonal antibody m19 is an IgG2b type monoclonal antibody.

In a second aspect, the present invention provides a gene fragment encoding the monoclonal antibody m19 described in the first aspect, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody m19 is shown as SEQ ID NO.3, and the gene sequence encoding the light chain variable region of the monoclonal antibody m19 is shown as SEQ ID NO. 4.

In a third aspect, the present invention provides an expression vector comprising the gene fragment of the second aspect.

In a fourth aspect, the present invention provides a host cell comprising the expression vector of the third aspect or a genome of the host cell into which the gene fragment of the second aspect has been integrated.

In a fifth aspect, the invention provides an immunoconjugate comprising:

(i) the monoclonal antibody m19 of the first aspect described above;

(ii) and a coupling moiety selected from the group consisting of: a detectable label, a drug, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.

In a sixth aspect, the invention provides an application of the antibody m19 of the first aspect in preparing a reagent, or a test strip, or a kit for detecting foot-and-mouth disease virus type O.

In a seventh aspect, the invention provides a solid phase competition ELISA detection kit for structural protein of type O foot-and-mouth disease virus, comprising the monoclonal antibody m19 described in the first aspect.

Preferably, the kit comprises an ELISA plate, an O-type foot-and-mouth disease virus inactivated antigen, a monoclonal antibody m19, an antibody diluent, a concentrated washing solution, a color developing agent and a stop solution.

Preferably, the ELISA plate is coated with O type foot-and-mouth disease virus structural protein rabbit antiserum and O type foot-and-mouth disease virus inactivated antigen.

Preferably, the kit further comprises a positive control serum and a negative control serum.

Preferably, the concentration of the O type foot-and-mouth disease structural protein rabbit antiserum is 1: 2000.

Preferably, the coating concentration of the O type foot-and-mouth disease virus inactivated antigen is 1: 16.

Preferably, the working concentration of the monoclonal antibody m19 is 7 μ g/mL.

In an eighth aspect, the invention provides a solid-phase competitive ELISA detection method for structural proteins of foot-and-mouth disease virus type O, which comprises the following steps:

coating an enzyme label plate with O-type foot-and-mouth disease virus structural protein rabbit antiserum; adding the O-type foot-and-mouth disease inactivated antigen into an enzyme label plate for incubation;

adding a serum to be detected and the monoclonal antibody m19 of the first aspect, and incubating;

adding goat anti-mouse secondary antibody, incubating, adding TMB, developing in dark, and adding H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

Calculating an inhibition percentage PI, taking 50% of PI as a critical value, and if the PI of the serum to be detected is more than or equal to 50%, determining that the PI is positive; if the serum to be detected is less than 50%, the result is negative.

Preferably, the method is:

(1) na with pH 9.6 was used₂CO₃/NaHCO₃Coating solution 1:2000 diluting O-type structural protein rabbit antiserum, coating an enzyme label plate, and standing overnight at 4 ℃;

(2) washing a plate by using PBST and drying the plate by beating, diluting the O-type inactivated antigen by using PBST 1:16, adding the diluted O-type inactivated antigen into the ELISA plate in the step (1), and incubating for 1h at 37 ℃;

(3) washing a plate by using PBST and drying the plate, diluting the serum to be detected by using the PBST, adding 7 mu g/mL of the antibody m19 of the first aspect, shaking and uniformly mixing the mixture, and incubating the mixture for 1h at 37 ℃;

(4) washing the plate with PBST, patting dry, adding goat anti-mouse secondary antibody, and incubating for 1h at 37 ℃;

(5) washing the plate with PBST, drying, adding TMB, developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

(6) And (3) calculating the inhibition percentage PI, wherein the serum to be detected is positive when the PI of the serum to be detected is more than or equal to 50%, and the serum to be detected is negative when the PI of the serum to be detected is less than 50%.

The invention has the beneficial effects that: the invention provides a monoclonal antibody m19 of O type foot-and-mouth disease virus structural protein, wherein the monoclonal antibody m19 is an IgG2b type monoclonal antibody; indirect immunofluorescence test shows that the monoclonal antibody m19 does not react with A type spectral system representative strains (A/GDMM/2013, A/WH/CHA/09 and A/AF72), can react with O type spectral system representative strains (O/ZK/93, O/Mya/98 and O/HK/93) with broad spectrum specificity, and can be used for broad spectrum and specific detection and diagnosis of O type foot-and-mouth disease virus; the invention establishes the solid phase competition ELISA detection kit and the detection method for the structural protein of the foot-and-mouth disease virus of O type by utilizing the monoclonal antibody m19 and the structural protein rabbit antiserum of the foot-and-mouth disease of O type, compared with the existing antibody detection method, the method of the invention obviously improves the sensitivity and the specificity, is suitable for the specificity detection of the structural protein antibody after the infection of the foot-and-mouth disease of O type or the immunization of inactivated vaccine, and is a novel method for monitoring the infection condition of the non-immune areas of the foot-and-mouth disease.

Drawings

FIG. 1 Indirect immunofluorescence assay to examine the reactivity of monoclonal antibody m19 against different lineage representative strains of type O and type A FMDV.

Detailed Description

The following embodiments of the present invention are described in detail, and it should be noted that the following embodiments are exemplary only, and are not to be construed as limiting the present invention. In addition, all reagents used in the following examples are commercially available or can be synthesized according to texts or known methods, and are readily available to those skilled in the art for reaction conditions not listed, if not explicitly stated.

EXAMPLE 1 preparation of monoclonal antibody m19

1. Preparation of hybridoma cells

1.1 immunization of mice

A5-6-week-old Balb/c mouse is immunized by taking O-type foot-and-mouth disease virus (O/Mya98)146s as an antigen in a mode of back subcutaneous multipoint injection, wherein the injection amount of each mouse is 100 mu g. The first immunization uses a Freund complete adjuvant, then the boosting immunization uses a Freund incomplete adjuvant, the immunization time interval is two weeks each time, the serum of the mouse is collected 1 week after the third immunization, the antibody titer is detected, when the antibody titer is not less than 1:1440, the impact immunization is carried out, namely the antigen is injected in the abdominal cavity, three days after the impact immunization, the spleen of the mouse is taken to be fused with myeloma cells.

1.2 cell fusion

Aseptically obtaining splenocytes of the immunized mice, mixing the splenocytes with myeloma cells according to the proportion of 10:1, adding 1mL of PEG 1500 within 1 minute at 37 ℃, after 1 minute of action, adding 1mL of DMEM culture solution preheated at 37 ℃ to stop the reaction, finishing the reaction within 1 minute, and after 1 minute of action, adding the solution for 5 times in total (the whole fusion process needs to shake a fusion tube), supplementing the preheated DMEM culture solution to 20mL, and after 15 minutes of action at 37 ℃, centrifuging at 800rpm for 7 minutes; discarding the supernatant, and resuspending with fetal bovine serum culture medium (adding HAT selection medium); adding the cells into a 96-well plate, wherein each well contains 50 mu L of the cells; place the plates at 37 ℃ 5% CO₂Culturing in an incubator. Day 3 and day 6 with 20% fetal bovine serum culture medium (plus HAT)Selection medium) was half-exchanged and the supernatant was taken to detect specific antibodies when hybridoma cells were observed to grow to about 1/5 f at the bottom of the well.

1.3 selection of hybridoma cells

Using ELISA test to prepare cell suspension of the hybridoma cells which are detected to be positive by using 20% fetal bovine serum culture solution (added with HT selection medium, Sigma), counting, adjusting the cells to be 3-5 cells/mL, dripping 100 mu L of diluted cells into each hole, then adding 100 mu L of HT selection medium into each hole, placing at 37 ℃ and 5% CO₂An incubator; half-change of 20% fetal bovine serum culture medium (supplemented with HT selection medium, Sigma) was performed on days 3 and 6, and the formation of cell clones was observed to detect antibody activity in time. After subcloning to obtain positive monoclonal cells, they were subcultured on an expansion scale at 2X 10⁶Individual cells/branch were stored in liquid nitrogen.

2. Preparation of monoclonal antibody m19

2.1 preparation of monoclonal antibody m19 ascites

0.5 mL/mouse sensitized with sterilized liquid paraffin for 7-8 weeks old Balb/c is injected into the abdominal cavity within 10-18 days by 0.5mL (about 3.0 multiplied by 10)⁶Individual cells/mL). Gently Rou the abdomen of the mouse every day after injection, and extract ascites when the abdomen of the mouse has significantly risen laterally. Centrifuging ascites at 10000rpm for 10min, collecting supernatant, packaging, and storing at-20 deg.C.

2.2 purification of antibody m19

The purification of the ascites of the hybridoma mouse is carried out by adopting affinity chromatography, and the specific operation is as follows: thawing frozen ascites sample, centrifuging at 10000rpm and 4 deg.C for 10min, sucking clear liquid, and loading with 3 column volumes of loading buffer (Binding buffer formula: 20mM Na)₂HPO₄0.15M NaCl, pH8.0), purifying with Protein A affinity chromatography column, eluting with 0.1M glycine buffer solution (pH 2.5) to obtain antibody, adjusting pH to neutral with neutralizing buffer solution (1M Tris-HCl, pH8.5), analyzing with SDS-PAGE gel, and determining Protein content,

the results show that: the purified monoclonal antibody m19 protein content is 4.8mg/mL, and the requirement of clinical application can be met.

2.3 subtype identification and sequencing of monoclonal antibody m19

Subtype identification: the subtype of the monoclonal antibody Ig was identified according to the instructions of the murine monoclonal antibody subtype identification kit (purchased from Sigma) and was determined to be IgG2 b. The sequences of the variable regions of antibody IgG2b were determined as follows:

(1) total RNA extraction: get 10⁶Performing Trizol lysis on each cell, adding chloroform for layering to obtain RNA, washing with ethanol after isopropanol precipitation, drying, and dissolving the RNA with DEPC water;

(2) reverse transcription and PCR amplification: taking 500ng RNA, adding oligo (dT), Random, dNTP and 5 × RT buffer, reverse transcriptase (Takara company), terminating the reaction at 37 ℃ for 25min, 85 ℃ for 5sec and 4 ℃ to obtain cDNA, and then carrying out PCR amplification;

(3) sequencing and analysis: the amplified heavy and light chain variable regions were cloned into pMD18-T, sent to the company for sequencing, and analyzed for data alignment using the IMGT/V-QUEST database.

The DNA sequence of the variable region of the heavy chain of the monoclonal antibody m19 is sequenced as follows: caggtccaactccagcagcctgggtctgagctggtgaggcctggagcttcagtgaacctgtcctgcaaggcttctggctacacattccccagctactggatgcactgggtgaagcagaggcctggacaaggccttgagtggattggaaatatttttcctgatggtggttttactaactacgatgagaactttaagagcagggccacactgactgtagacacatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctattactgtacaacaatttatgattacgtcgtcggagtcggtgctatggactactggggtcaaggaacctcagtcaccgtctcgagc (shown in SEQ ID NO. 3);

the DNA sequence encoding the light chain variable region of monoclonal antibody m19 is as follows: gacattgtgatgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccataacctgcagtgccagctcaagtataagttatatgtactggttccagcagaagacaggcacttctcccaaactctggatttataaaacatccgacctggcttctggagtcccggctcgcttcagtggcagtggatctgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcaaaggaatagttacacgttcggaggggggaccaagctggagctgaaa (shown in SEQ ID NO. 4);

according to the codon encoding rule, it is known that:

the amino acid sequence of the heavy chain variable region of the monoclonal antibody m19 is shown as follows: QVQLQQPGSELVRPGASVNLSCKASGYTFPSYWMHWVKQRPGQGLEWIGNIFPDGGFTNYDENFKSRATLTVDTSSSTAYMQLSSLTSEDSAVYYCTTIYDYVVGVGAMDYWGQGTSVTVSS (shown in SEQ ID NO. 1);

the amino acid sequence of the variable region in the light chain of the monoclonal antibody m19 is shown as follows: DIVMTQSPAIMSASPGEKVTITCSASSSISYMYWFQQKTGTSPKLWIYKTSDLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRNSYTFGGGTKLELK (shown in SEQ ID NO. 2).

EXAMPLE 2 monoclonal antibody m19 reactive with different pedigree foot-and-mouth disease Virus antigens

1. Indirect immunofluorescence assay:

respectively infecting BHK-21 cells with different pedigree representative strains of O-type foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93 strains) and A-type foot-and-mouth disease (A/AF72, A/GDMM/2013 and A/WH/CHA/09) (the strains are preserved by a foot-and-mouth disease national reference laboratory), and judging the reaction condition of the prepared monoclonal antibody m19 and FMDV antigen by the fluorescence intensity under a fluorescence microscope, wherein the specific operation steps are as follows:

(1) culturing the cells: uniformly spreading BHK-21 cells into a 12-hole cell culture plate, and inoculating virus when the cells grow to about 70%;

(2) and (3) virus inoculation: respectively inoculating O-type strains of 3 lineages and A-type strains of 3 lineages into a 12-well plate according to the proportion of MOI (maximum oxygen index) 1, simultaneously establishing non-toxic normal cells as negative control, incubating the toxic cells in an incubator at 37 ℃, and inactivating cell supernatant when cytopathic effect reaches about 50%;

(3) fixing: adding paraformaldehyde into each well, fixing for 15min, and washing with PBS for 3 times;

(4) permeability: preparing a permeation solution containing 0.5% Triton X-100 with PBS solution, allowing cells to permeate for 10min, and washing with PBS for 3 times;

(5) incubating the primary antibody: washing with PBS for 3 times, diluting monoclonal antibody m19 to be detected with PBS to final concentration of 10 μ g/mL, adding 500 μ L/well, using known positive murine antibody as positive control, and incubating at 37 deg.C for 1 h;

(6) incubation of secondary antibody: washing with PBS for 3 times, adding goat anti-mouse IgG-FITC fluorescent antibody (diluted 1: 1000), adding at 200 μ L/well, and incubating at 37 deg.C for 1 h;

(7) washed 5 times with PBS, 500. mu.L/well PBS was added to ensure cell surface wetting, and fluorescence was observed under a fluorescence microscope.

As shown in FIG. 1, the monoclonal antibody m19 of the present invention has good reactivity with O-type strains of 3 lineages, but does not react with A-type strains of three lineages.

Example 3 establishment and application of solid phase competitive ELISA detection method for structural protein of type O foot-and-mouth disease virus

Determination of optimal reaction conditions of O-type foot-and-mouth disease virus structural protein solid-phase competition ELISA detection method

1.1 determination of optimal use concentration of O type foot-and-mouth disease virus structural protein rabbit antiserum and O type foot-and-mouth disease virus inactivated antigen:

the optimal use concentration of O type foot-and-mouth disease virus structural protein rabbit antiserum and O type foot-and-mouth disease virus inactivated antigen (stored in the laboratory) is determined by a square matrix titration method. First, Na was used₂CO₃/NaHCO₃Diluting O type foot-and-mouth disease virus structural protein rabbit antiserum (transverse) by the coating solution (pH is 9.6) in a dilution plate from 1:250 in a continuous double ratio, correspondingly transferring the antiserum into an enzyme label plate (50 mu L/hole), and standing at 4 ℃ overnight; discarding the liquid in the enzyme label plate, washing for 3 times by using PBST, and patting dry on absorbent paper; serially diluting O-type foot-and-mouth disease virus inactivated antigen (longitudinal) (50 uL/well) in a doubling ratio from 1:2 by using PBST, and incubating for 1h at 37 ℃; plate washing and drying (same as above); adding guinea pig antiserum working solution (stored in the laboratory) (50 μ L/well), and incubating at 37 deg.C for 1 h; plate washing and drying (same as above); adding goat anti-guinea pig secondary antibody (50 μ L/well), and incubating at 37 deg.C for 1 h; plate washing and drying (same as above); adding TMB (50 μ L/well), developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution (50. mu.L/well) and OD read with a microplate reader₄₅₀Value, taking OD₄₅₀When the value is in the range of 1.6-2.0, the highest dilution times corresponding to the O-type structural protein rabbit antiserum and the O-type inactivated antigen are the optimal use concentrations of the O-type structural protein rabbit antiserum and the O-type inactivated antigen.

The result shows that the optimal coating concentration of the O-type foot-and-mouth disease virus structural protein rabbit antiserum is 1:2000, and the use concentration of the O-type foot-and-mouth disease virus inactivated antigen is 1: 16.

1.2 determination of the optimal concentration of monoclonal antibody m 19:

the optimal use concentration of the O type foot-and-mouth disease virus structural protein rabbit antiserum and the O type foot-and-mouth disease virus inactivated antigen determined above is used, and Na is used₂CO₃/NaHCO₃Coating the optimal use concentration (50 mu L/hole) of O-type foot-and-mouth disease virus structural protein rabbit antiserum with a coating solution (pH 9.6) at 4 ℃ overnight; plate washing and drying (same as above); adding the O-type foot-and-mouth disease virus inactivated antigen with the optimal use concentration into an enzyme label plate (50 mu L/hole), and incubating for 1h at 37 ℃; plate washing and drying (same as above); monoclonal antibody m19 (transverse) (50. mu.L/well) was serially diluted in duplicate starting from 1:200 using PBST and incubated at 37 ℃ for 1 h; plate washing and drying (same as above); adding goat anti-mouse secondary antibody (50 μ L/well), and incubating at 37 deg.C for 1 h; plate washing and drying (same as above); adding TMB (50 μ L/well), developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution (50. mu.L/well) and OD read with a microplate reader₄₅₀Value, taking OD₄₅₀The value at 1.6 corresponds to the highest dilution of antibody m19 at its optimal working concentration.

The results show that the optimal concentration of monoclonal antibody m19 used is 7. mu.g/mL.

Operation steps of O type foot-and-mouth disease virus structural protein solid phase competition ELISA detection method

(1) Using Na₂CO₃/NaHCO₃Diluting O-type foot-and-mouth disease virus structural protein rabbit antiserum to the optimal use concentration by using a coating solution (pH is 9.6), coating an ELISA plate (50 mu L/hole), and standing overnight at 4 ℃;

(2) washing the plate for 5 times by using PBST, drying by beating, diluting the O type foot-and-mouth disease virus inactivated antigen by using PBST to working concentration, adding the diluted O type foot-and-mouth disease virus inactivated antigen into an enzyme label plate (50 mu L/hole), and incubating for 1h at 37 ℃;

(3) washing and drying the plate (same as above), using PBST to dilute the positive serum and the serum to be detected (1:4 times to 1:512) and the negative serum (1:4 times to 1:32) (50 mu L/hole), adding the monoclonal antibody m19 diluted to the working concentration into each hole, gently shaking and uniformly mixing, and incubating for 1h at 37 ℃;

(4) washing the plate and beating the plate dry (same as above), adding goat anti-mouse secondary antibody (50 mu L/hole), and incubating for 1h at 37 ℃;

(5) plate washing and drying (same as above); adding TMB (50 μ L/well), developing at 37 deg.C in dark for 15min, adding 2M H₂SO₄The reaction was stopped in solution (50. mu.L/well) and OD read with a microplate reader₄₅₀；

(6) The Percentage Inhibition (PI) is calculated by detecting the serum, the PI of 50% is taken as a critical value, if the PI of the detected serum is more than or equal to 50%, the highest dilution of the positive serum is the antibody titer of the serum, namely the titer, and the positive serum is determined. If the tested serum is less than 50%, the result is negative.

Specificity of O type foot-and-mouth disease virus structural protein solid phase competition ELISA method

The solid-phase competition ELISA method for the structural protein of the foot-and-mouth disease virus type O is used for measuring the foot-and-mouth disease type O, the foot-and-mouth disease type A, the swine vesicular disease, the seneca and the swine fever positive reference serum, and the result shows that the solid-phase competition ELISA method for the structural protein of the foot-and-mouth disease type O only reacts with the O type foot-and-mouth disease reference positive serum specifically but not with the A type foot-and-mouth disease, the swine vesicular disease, the seneca and the swine fever positive reference serum (Table 1).

TABLE 1 solid-phase competitive ELISA method for O-type foot-and-mouth disease virus structural protein to determine the antibody titer of positive reference serum of O-type foot-and-mouth disease, A-type foot-and-mouth disease, swine vesicular disease, seneca and swine fever

Sensitivity of O type foot-and-mouth disease virus structural protein solid phase competition ELISA method

The solid phase competition ELISA method for the structural protein of the foot-and-mouth disease virus O established by the invention is used for measuring 80 parts of pig negative serum, 48 parts of pig O type positive serum, 40 parts of pig A type positive serum, 30 parts of cattle negative serum, 16 parts of cattle A type positive serum and 17 parts of cattle O type positive serum, and the result shows that the solid phase competition ELISA method for the structural protein of the foot-and-mouth disease virus O type has good sensitivity and specificity for detecting the foot-and-mouth disease virus O type (see table 2)

TABLE 2 determination of different-property serum by solid-phase competitive ELISA method for O type foot-and-mouth disease virus structural protein

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> antibody m19 of O-type foot-and-mouth disease virus structural protein, preparation method and application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 122

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Val Gln Leu Gln Gln Pro Gly Ser Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Pro Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Phe Pro Asp Gly Gly Phe Thr Asn Tyr Asp Glu Asn Phe

50 55 60

Lys Ser Arg Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Thr Ile Tyr Asp Tyr Val Val Gly Val Gly Ala Met Asp Tyr Trp

100 105 110

Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 2

<211> 104

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Asp Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met

20 25 30

Tyr Trp Phe Gln Gln Lys Thr Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Lys Thr Ser Asp Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Asn Ser Tyr Thr Phe Gly

85 90 95

Gly Gly Thr Lys Leu Glu Leu Lys

100

<210> 3

<211> 366

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caggtccaac tccagcagcc tgggtctgag ctggtgaggc ctggagcttc agtgaacctg 60

tcctgcaagg cttctggcta cacattcccc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggaaat atttttcctg atggtggttt tactaactac 180

gatgagaact ttaagagcag ggccacactg actgtagaca catcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtac aacaatttat 300

gattacgtcg tcggagtcgg tgctatggac tactggggtc aaggaacctc agtcaccgtc 360

tcgagc 366

<210> 4

<211> 312

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gacattgtga tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60

ataacctgca gtgccagctc aagtataagt tatatgtact ggttccagca gaagacaggc 120

acttctccca aactctggat ttataaaaca tccgacctgg cttctggagt cccggctcgc 180

ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagccgaat ggaggctgaa 240

gatgctgcca cttattactg ccagcaaagg aatagttaca cgttcggagg ggggaccaag 300

ctggagctga aa 312

Claims (10)

1. A monoclonal antibody m19 of O type foot-and-mouth disease virus structural protein is characterized in that the amino acid sequence of the variable region of the m19 heavy chain of the monoclonal antibody is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2.

2. A gene fragment for encoding the monoclonal antibody m19 of claim 1, wherein the gene sequence for encoding the heavy chain variable region of the monoclonal antibody m19 is shown as SEQ ID NO.3, and the gene sequence for encoding the light chain variable region of the monoclonal antibody m19 is shown as SEQ ID NO. 4.

3. An expression vector comprising the gene fragment of claim 2.

4. A host cell comprising the expression vector of claim 3, or a genome of the host cell having the gene segment of claim 3 integrated therein.

5. An immunoconjugate, wherein the immunoconjugate comprises:

(i) the monoclonal antibody m19 of claim 1;

(ii) and a coupling moiety selected from the group consisting of: a detectable label, a drug, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.

6. The use of the monoclonal antibody m19 of claim 1 in the preparation of a reagent, or a test strip, or a kit for detecting foot-and-mouth disease virus type O.

7. A kit for detecting the structural protein solid-phase competition ELISA of the foot-and-mouth disease virus type O, which is characterized by comprising the monoclonal antibody m19 of claim 1.

8. The ELISA detection kit of claim 7 wherein said kit comprises an ELISA plate, an O-type foot-and-mouth disease virus inactivated antigen, a monoclonal antibody m19, an antibody diluent, a concentrated washing solution, a color-developing agent, and a stop solution.

9. The ELISA detection kit of claim 8 wherein said ELISA plate is coated with the structural protein of type O foot-and-mouth disease Virus, rabbit antiserum, and inactivated antigen of type O foot-and-mouth disease Virus.

10. A solid phase competition ELISA detection method for O type foot-and-mouth disease virus structural protein is characterized by comprising the following steps:

coating the ELISA plate with O-type foot-and-mouth disease virus structural protein rabbit antiserum, and adding O-type foot-and-mouth disease inactivated antigen into the ELISA plate for incubation;

adding the serum to be detected and the monoclonal antibody m19 of claim 1, and incubating;

adding goat anti-mouse secondary antibody, incubating, adding TMB, developing in dark, and adding H₂SO₄The reaction was stopped in solution and OD was read with a microplate reader₄₅₀；

Calculating an inhibition percentage PI, taking 50% of PI as a critical value, and if the PI of the serum to be detected is more than or equal to 50%, determining that the PI is positive; if the serum to be detected is less than 50%, the result is negative.

Images