CN114316035B - Universal foot-and-mouth disease virus structural protein antibody, preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a general-purpose foot-and-mouth disease virus structural protein antibody, a preparation method and an application. In the present invention, the O-type foot-and-mouth disease virus 146s is used as an immunogen to immunize Balb/c mice. After cell fusion, two positive hybridoma cell lines that secrete monoclonal antibodies efficiently are obtained by screening, and two mouse monoclonal antibodies m33 and m77. Western blotting and indirect immunofluorescence tests showed that the monoclonal antibodies m33 and m77 had broad-spectrum reactivity to O-type different lineage viruses and A-type different lineage viruses. The m33 and m77 antibodies specifically bind to the conserved antigenic epitopes between types in the FMDV structural protein. Compared with existing antibodies, the sensitivity and specificity of the detection results are significantly improved, and they are suitable for O and A type FMDV infection or inactivation The detection of structural protein antibodies after vaccine immunization is a new method for monitoring the infection status of FMD non-immune animal groups.

Description

A kind of universal foot-and-mouth disease virus structural protein antibody, preparation method and application thereof

technical field

The invention relates to the field of biotechnology, in particular to a general-purpose foot-and-mouth disease virus structural protein antibody, a preparation method and an application.

Background technique

Foot-and-mouth disease is an infectious disease common to cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV) infection. FMDV is a member of the genus aphthavirus of the family picornaviridae. FMDV currently has seven serotypes: O, A, C, SAT1, SAT2, SAT3 (namely, South African foot-and-mouth disease virus types 1, 2, and 3) and Asia1 (Asian type 1). There is almost no immune protection between the various types, and animals infected with one type of FMD virus can still be infected with another type of FMD virus. The disease has a serious impact on the breeding industry and international trade in livestock products, so countries attach great importance to the prevention and control of the disease. The key to preventing and controlling FMD is the application of efficient vaccines and accurate diagnostic methods.

Because the antigenicity of different subtypes within the same serotype is different in different degrees, the degree of serological cross-reaction is also different, which makes the diagnosis and control of foot-and-mouth disease very difficult. Monoclonal antibodies have the ability to recognize a single antigenic site, have strong binding specificity to the antigen, have high homogeneity, single biological activity, are easy to standardize, and can be mass-produced, and are widely used in the field of biomedicine. Currently, type O and type A FMD are mainly prevalent in China. Therefore, the development of general-purpose monoclonal antibodies for O-type and A-type FMD is of great significance for the diagnosis and prevention of O-type and A-type FMD. However, due to the complex antigenic structure of FMDV, the antigenic sites and epitopes of different serotypes, genotypes and isolates are different, there are extensive antigenic variations, and there are also conservative antigenic structures that determine the characteristics of the virus species. To develop a broad-spectrum FMDV antibody detection kit, it is necessary to screen a broad-spectrum specific monoclonal antibody that recognizes a conserved epitope between types. The method presents certain obstacles.

Contents of the invention

For above-mentioned technical problem, the object of the present invention is to provide a kind of monoclonal antibody, preparation method and application that have broad-spectrum neutralizing effect to O-type and A-type foot-and-mouth disease virus different pedigree strain structural proteins, specifically comprise the following contents:

In the first aspect, the present invention provides a monoclonal antibody of a general foot-and-mouth disease virus structural protein, the monoclonal antibody is m33 or m77, and the amino acid sequence of the heavy chain variable region of the monoclonal antibody m33 is as SEQ ID As shown in NO.1, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is shown in SEQ ID NO.5, and the light chain can be The amino acid sequence of the variable region is shown in SEQ ID NO.6.

In the second aspect, the present invention provides a gene fragment encoding the monoclonal antibody m33 described in the first aspect, the gene sequence encoding the heavy chain variable region of the monoclonal antibody m33 is shown in SEQ ID NO.3, encoding The gene sequence of the light chain variable region of the monoclonal antibody m33 is shown in SEQ ID NO.4.

In the third aspect, the present invention provides a gene fragment encoding the monoclonal antibody m77 described in the first aspect, the gene sequence encoding the heavy chain variable region of the monoclonal antibody m77 is shown in SEQ ID NO.7, encoding The gene sequence of the light chain variable region of the monoclonal antibody m77 is shown in SEQ ID NO.8.

In a fourth aspect, the present invention provides an expression vector, the expression vector contains the gene fragment described in the second aspect above, or the expression vector contains the gene fragment described in the third aspect above.

In the fifth aspect, the present invention provides a host cell containing the expression vector described in the fourth aspect above, or the gene fragment described in the second aspect or the third aspect integrated in its genome.

In a sixth aspect, the present invention provides an immunoconjugate comprising:

(i) monoclonal antibody m33 and/or monoclonal antibody m77 described in the first aspect above;

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

In the seventh aspect, the present invention provides the application of the monoclonal antibody m33 and/or the monoclonal antibody m77 described in the first aspect in the preparation of reagents for detecting foot-and-mouth disease virus, or test strips, or kits.

In the eighth aspect, the present invention provides a general-purpose monoclonal antibody composition of the structural protein of foot-and-mouth disease virus, including the monoclonal antibody m33 and the monoclonal antibody m77 described in the first aspect.

In the ninth aspect, the present invention provides an application of the monoclonal antibody composition described in the eighth aspect in the preparation of a reagent for detecting foot-and-mouth disease virus, or a test strip, or a kit.

In the tenth aspect, the present invention provides a solid-phase competitive ELISA detection kit for the structural protein of foot-and-mouth disease virus, said kit comprising the monoclonal antibody m33 and/or the monoclonal antibody m77 described in the first aspect above, or the above-mentioned The monoclonal antibody composition of the ninth aspect.

Preferably, the ELISA detection kit comprises the monoclonal antibody m33 and foot-and-mouth disease virus inactivated antigen enzyme plate coated with the above-mentioned first aspect, the monoclonal antibody m77 described in the above-mentioned first aspect, antibody diluent, Concentrated washing solution, chromogen, stop solution;

Or, the ELISA detection kit includes the monoclonal antibody m77 and foot-and-mouth disease virus inactivated antigen enzyme plate coated with the above-mentioned first aspect, the monoclonal antibody m33 described in the above-mentioned first aspect, antibody diluent, concentrated Washing solution, developer, stop solution.

Preferably, the inactivated antigen of foot-and-mouth disease virus is an inactivated antigen of type O foot-and-mouth disease virus or an inactivated antigen of type A foot-and-mouth disease virus.

Preferably, the ELISA detection kit further includes positive control serum and negative control serum.

Preferably, the working concentration of the monoclonal antibody m77 is 6 μg/mL.

Preferably, the coating concentration of the monoclonal antibody m33 is 1 μg/mL.

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

Preferably, the coating concentration of the type A foot-and-mouth disease virus inactivated antigen is 1:8.

Eleventh aspect, the present invention provides a kind of solid-phase competition ELISA detection method of general type foot-and-mouth disease virus structural protein, described method comprises the following steps:

Coat the microtiter plate with the above-mentioned first aspect monoclonal antibody m33, then add the foot-and-mouth disease virus inactivated antigen to the microtiter plate, add the serum to be tested and the monoclonal antibody m77, mix well, and incubate;

Or, use the above-mentioned first aspect monoclonal antibody m77 to coat the microtiter plate, then add the foot-and-mouth disease virus inactivated antigen to the microtiter plate, add the serum to be tested and the monoclonal antibody m33, mix well, and incubate;

After incubation, add goat anti-mouse secondary antibody, after incubation, add TMB to avoid light for color development, then add H ₂ SO ₄ solution to terminate the reaction, and read OD ₄₅₀ with a microplate reader;

Calculate the inhibition percentage (PI), with 40% PI as the critical value, when the PI of the serum to be tested is ≥ 40%, the test result is positive, and when the PI of the serum to be tested < 40%, the test result is negative.

Preferably, the method is:

(1) Dilute monoclonal antibody m33 with Na ₂ CO ₃ /NaHCO ₃ coating solution with pH = 9.6, coat the microtiter plate, overnight at 4°C, and the coating concentration of monoclonal antibody m33 is 1 μg/mL;

(2) Wash the plate with PBST and pat dry, dilute the inactivated antigen of type A foot-and-mouth disease virus with PBST 1:8, or dilute the inactivated antigen of type O foot-and-mouth disease virus 1:16, add it to the microtiter plate, and incubate at 37°C for 1 hour;

(3) Use PBST to wash the plate and pat dry, use PBST to dilute the serum to be tested, then add 6 μg/mL monoclonal antibody m77, shake gently to mix, and incubate at 37°C for 1 hour;

(4) Wash the plate with PBST and pat dry, add goat anti-mouse secondary antibody, and incubate at 37°C for 1 hour;

(5) Use PBST to wash the plate and pat it dry, add TMB, 37 ° C in the dark for 15 minutes, add 2M H ₂ SO ₄ solution to terminate the reaction, read OD ₄₅₀ with a microplate reader;

(6) Calculate the inhibition percentage PI, when the PI of the serum to be tested is ≥ 40%, the serum to be tested is positive, and when the serum to be tested is <40%, the serum to be tested is negative.

Preferably, the method is:

(1) Dilute the monoclonal antibody m77 with Na ₂ CO ₃ /NaHCO ₃ coating solution with pH=9.6, coat the microtiter plate, overnight at 4°C, and the coating concentration of monoclonal antibody m77 is 6 μg/mL;

(2) Wash the plate with PBST and pat dry, dilute the inactivated antigen of type A foot-and-mouth disease virus with PBST 1:8, or dilute the inactivated antigen of type O foot-and-mouth disease virus 1:16, add it to the microtiter plate, and incubate at 37°C for 1 hour;

(3) Use PBST to wash the plate and pat dry, use PBST to dilute the serum to be tested, then add 1 μg/mL monoclonal antibody m33, shake gently to mix, and incubate at 37°C for 1 hour;

(4) Wash the plate with PBST and pat dry, add goat anti-mouse secondary antibody, and incubate at 37°C for 1 hour;

(5) Use PBST to wash the plate and pat it dry, add TMB, 37 ° C in the dark for 15 minutes, add 2M H ₂ SO ₄ solution to terminate the reaction, read OD ₄₅₀ with a microplate reader;

(6) Calculate the inhibition percentage PI, when the PI of the serum to be tested is ≥ 40%, the serum to be tested is positive, and when the serum to be tested is <40%, the serum to be tested is negative.

The beneficial effect of the present invention is: 1. the present invention provides a kind of universal foot-and-mouth disease virus structural protein antibody m33 or m77, and described antibody m33 and m77 are IgG2b type monoclonal antibody; 2. immunoblotting test shows that described monoclonal antibody m33 and m77 Representative strains of different lineages of m77 and type O foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93) and representative strains of different lineages of type A foot-and-mouth disease (A/AF72, A/GDMM/2013, A /WH/CHA/09) all have good reactivity, and are general-purpose foot-and-mouth disease virus structural protein antibodies; ③ the present invention utilizes said monoclonal antibody m33 and m77 to establish a general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA detection The kit and detection method, compared with the existing universal antibody detection method, the method significantly improves the sensitivity and specificity of the detection results, and is suitable for broad-spectrum detection of structural protein antibodies after foot-and-mouth disease infection or inactivated vaccine immunization, At the same time, it is also a new method to monitor the infection of FMD in non-immune areas.

Description of drawings

Figure 1WB detects the reactivity of the general-purpose monoclonal antibody m33 to the representative strains of different lineages of O-type and A-type FMDV;

Figure 2WB detects the reactivity of the general-purpose monoclonal antibody m77 to the representative strains of different lineages of O-type and A-type FMDV;

Fig. 3 indirect immunofluorescence test detects the reactivity of general type monoclonal antibody m33 to O type and A type FMDV different lineage representative strains;

Fig. 4 indirect immunofluorescence test detects the reactivity of the universal monoclonal antibody m77 to representative strains of different lineages of type O and type A FMDV.

Detailed ways

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

The preparation of embodiment 1 monoclonal antibody m33 and m77

1. Preparation of hybridoma cells

1.1 Immunization of mice

5-6 week-old Balb/c mice were immunized with type O foot-and-mouth disease virus (O/Mya98) 146s as the antigen. The immunization method was multi-point subcutaneous injection on the back, and the injection volume was 100 μg per mouse. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations. The interval between each immunization was two weeks. The mouse serum was collected one week after the third immunization to detect the antibody titer. When the antibody titer is not low At 1:1440, impulse immunization was performed, that is, the antigen was injected intraperitoneally, and three days after the impulse immunization, the spleen of the mouse was taken for fusion with myeloma cells.

1.2 Cell Fusion

Spleen cells of immunized mice were aseptically obtained, mixed with myeloma cells at a ratio of 10:1, at 37°C, 1 mL of PEG 1500 was added within 1 minute, and after 1 minute of action, 1 mL of 37°C preheated DMEM was added The culture solution terminates the reaction, add it within 1 minute, and act for 1 minute. Add 5 times in this way (the fusion tube needs to be shaken during the whole fusion process), add preheated DMEM culture solution to 20mL, act at 37°C for 15 minutes, and centrifuge at 800rpm 7min; discard the supernatant and resuspend with fetal bovine serum culture medium (plus HAT selection medium); add the above cells into a 96-well plate, 50 μL per well; place the culture plate in a 37°C, 5% CO ₂ incubator cultivated in. On the 3rd and 6th days, half-change the medium with 20% fetal calf serum culture medium (adding HAT selection medium), observe that the hybridoma cells grow to about 1/5 of the bottom of the well, and take the supernatant to detect the specific antibody.

1.3 Screening hybridoma cells

Using ELISA test, the positive hybridoma cells were made into cell suspension with 20% fetal bovine serum culture medium (plus HT selection medium, Sigma), counted, and the cells were adjusted to 3-5 cells/mL. Add 100 μL of diluted cells, then add 100 μl of HT selection medium to each well, and place in a 37°C, 5% CO ₂ incubator; perform a half-change on the 3rd and 6th day with 20% fetal bovine serum culture solution (Add HT selection medium, Sigma), observe the formation of cell clones, and detect antibody activity in time. After subcloning positive monoclonal cells, subculture and expand them, and store them in liquid nitrogen at 2×10 ⁶ cells/branch.

2. Preparation of monoclonal antibodies m33 and m77

2.1 Preparation of monoclonal antibodies m33 and m77 ascites

Sensitize 7-8 week-old Balb/c mice with sterile liquid paraffin, 0.5 mL/mouse, and inject 0.5 mL (about 3.0×10 ⁶ cells/mL) of hybridoma cells into the peritoneal cavity within 10 days to 18 days. After the injection, the abdomen of the mice was rubbed lightly every day, and the ascites was extracted when the abdomen of the mice was obviously enlarged laterally. Centrifuge the ascitic fluid at 10,000 rpm for 10 min, take the supernatant, aliquot and store at -20°C.

2.2 Purification of monoclonal antibodies m33 and m77

The ascites of hybridoma cells was purified by affinity chromatography, and the specific operation was as follows: thaw the frozen ascites sample, centrifuge at 10,000 rpm at 4°C for 10 min, absorb the clear liquid, and use 3 times the column volume of sample buffer (Bindingbuffer formula : 20mM Na ₂ HPO ₄ , 0.15M NaCl, pH8.0) after dilution, carry out Protein A affinity chromatography column purification, column chromatography eluent is pH2.5, 0.1M glycine buffer, the eluted The antibody needs to be immediately adjusted to neutral pH with neutralization buffer (1MTris-HCl, pH8.5), and then subjected to SDS-PAGE gel analysis and protein content determination.

The results showed that the protein contents of the purified antibodies m33 and m77 were 5.2mg/mL and 4.3mg/mL, respectively, which could meet the needs of clinical application.

2.3 Subtype identification and sequencing of monoclonal antibodies m33 and m77

Subtype identification: According to the instructions of the mouse monoclonal antibody subtype identification kit (purchased from Sigma Company), the Ig subtype of the monoclonal antibody was identified, and the subtypes of the monoclonal antibodies m33 and m77 were both IgG2b. The specific antibody IgG2b variable region sequence was determined as follows:

(1) Extraction of total RNA: take ¹⁰⁶ cells, lyse with Trizol, add chloroform to layer to obtain RNA, precipitate with isopropanol, wash with ethanol, dry, and dissolve RNA with DEPC water;

(2) Reverse transcription and PCR amplification: Take 500ng RNA, add Oligo(dT), Random, dNTP and 5×RTbuffer, reverse transcriptase (Takara company), 37°C for 25min, 85°C for 5sec, and 4°C to terminate the reaction to obtain cDNA, followed by PCR amplification;

(3) Sequencing and analysis: clone the amplified heavy chain and light chain variable regions into pMD18-T, send them to the company for sequencing, and use the IMGT/V-QUEST database for data comparison and analysis.

After sequencing, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody m33 is: caggtgcagctgaaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctcacctgcactgtcactggctactcaatcaccagtgattatgcctggaactggatccggcagtttccaggaaacaaactggagtggatgggctacatattctacagaggtag cactacccacaacccatctctcagaagtcgaatctctatcactcgagacacatccaagaaccagttcttcctgcagttgaattctgtgactactgaggacacagccacatattattgtgcaagggggctccacggtagtagcccgtttgcttactggggccaagggactctggtcactgtctcgagc (shown in SEQ ID NO. 3);

The coding DNA sequence of the light chain variable region of the monoclonal antibody m33 is as follows: gacattgtgatgacccagtctcaaaaactcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcacaatgtgtatagtaatgtagtctggtatcaagagaaaccagggcaatctcctaatgcactgatttattcggcatcccaccggtacagtgggagt ccctgatcgcttcacaggcagtggacctgggacagatttcactctcaccatcagcaatgtgcagtctgaagacttggcaaagtctttctgtcagcaatgtaacagctatcctattcacgttcggctcgggcacaaagttggaaatcaa (shown in SEQ ID NO.4);

According to the codon coding rules, it can be known that:

The amino acid sequence of the heavy chain variable region of the monoclonal antibody m33 is as follows: QVQLKESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIFYRGSTTHNPSLRSRISITRDTSKNQFFLQLNSVTTEDTATYYCARGLHGSSPFAYWGQGTLVTVSS (shown in SEQ ID NO.1);

The amino acid sequence of the light chain variable region of the monoclonal antibody m33 is as follows: DIVMTQSQKLMSTSVGDRVSVTCKASHNVYSNVVWYQEKPGQSPNALIYSASHRYSGVPDRFTGSGPGTDFLTLTISNVQSEDLAKSFCQQCNSYPIHVRLGHKVGNQ (shown in SEQ ID NO.2);

After sequencing, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody m77 is: gatgtgcagctggtggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgaagcctctggattcactttcagtagctataccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtactggaggtag tttcacctactatctagacagtgcgaagggccgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtattactgctcaaaagaggggagaattctatggtaactacggggattactatgctatggactactggggtcaaggaacctcagtcaccgtctcgagc (SEQ ID NO .7);

The coding DNA sequence of the light chain variable region of the monoclonal antibody m77 is as follows: gacatccagatgacccagtctcctgcttccttagctgtatctctggggcagagggccaccatctcatacagggccagcaaaagtgtcagtacatctggctatagttatatgcactggaaccaacagaaaccaggacagccaccagactcctcatctatcttgtatccaacctagaatctgggg tccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacattagggagcttacacgttcggaggggggaccagactggaaatcaaac (shown in SEQ ID NO.8);

According to the codon coding rules, it can be known that:

The amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is as follows: DVQLVESGGGLVKPGGSLKLSCEASGFTFSSYTMSWVRQTPEKRLEWVATISTGGSFTYYLDSAKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCSKEGEFYGNYGDYYAMDYWGQGTSVTVSS (shown in SEQ ID NO.5);

The amino acid sequence of the light chain variable region of the monoclonal antibody m77 is as follows: DIQMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPDWKSN (shown in SEQ ID NO. 6).

Broad-spectrum reactivity assay of embodiment 2 monoclonal antibodies m33 and m77

1. Western blot test

Representative strains of different lineages of type O foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93 strains) and representative strains of different lineages of type A foot-and-mouth disease (A/AF72, A/GDMM/ 2013 and A/WH/CHA/09) (both strains are preserved by the National Reference Laboratory for Foot-and-Mouth Disease) infected BHK-21 cells, collected cells 6 hours after infection, prepared samples for WB test, and prepared monoclonal antibodies m33 and m77 Western blot was performed as primary antibody. The specific experimental process is as follows:

(1) Electrophoresis: Add the denatured O-type and A-type FMDV antigens to 12% prefabricated gel holes;

(2) Load the sample at 10 μL/well, conduct electrophoresis at a constant voltage of 90V for the stacking gel, and perform electrophoresis at a constant voltage of 120V for the separating gel;

(3) Membrane transfer: Assemble the membrane transfer device, place it in the ice-water mixture, set 200mA constant current to transfer the membrane for 2 hours, and transfer the electrophoresis-separated samples from the gel to the PVDF membrane;

(4) Sealing: use 5% skimmed milk powder prepared by TBST solution as the blocking solution, and seal at room temperature for 1 hour on a shaking table;

(5) Incubate the primary antibody: dilute the monoclonal antibodies m33 and m77 to be tested to a final concentration of 2 μg/mL with 5% skimmed milk powder prepared in TBST solution, and incubate overnight at 4°C on a shaker;

(6) Membrane washing: wash the membrane three times by oscillating with TBST solution, and wash for ten minutes each time;

(7) Secondary antibody incubation: dilute goat anti-mouse antibody (1:5000) with 5% skimmed milk powder prepared in TBST solution, and incubate at room temperature for 1 hour on a shaker;

(8) Membrane washing: wash the membrane three times by oscillating with TBST solution, and wash for ten minutes each time;

(9) Development: Gently mix liquid A and liquid B in the ECL luminescent liquid according to the ratio of 1:1, and evenly drop them on the PVDF membrane, react for 3 minutes, and then scan the membrane.

Detecting result as shown in Figure 1 and 2, monoclonal antibody m33 of the present invention and m77 and O type foot-and-mouth disease different pedigree representative strains (O/ZK/93, O/Mya/98 and O/HK/93) and Representative strains of different lineages of type A foot-and-mouth disease (A/AF72, A/GDMM/2013, A/WH/CHA/09) all had good reactivity. It shows that the monoclonal antibodies m33 and m77 of the present invention are general-purpose antibodies against the structural protein of foot-and-mouth disease virus.

2. Indirect immunofluorescence test:

Representative strains of different lineages of type O foot-and-mouth disease (O/ZK/93, O/Mya/98 and O/HK/93 strains) and representative strains of different lineages of type A foot-and-mouth disease (A/AF72, A/GDMM/ 2013 and A/WH/CHA/09) (both strains are preserved by the National Reference Laboratory for Foot-and-Mouth Disease) infected BHK-21 cells, and the reaction of the prepared monoclonal antibodies m33 and m77 with the FMDV antigen was determined by the fluorescence intensity under the fluorescence microscope situation, the specific steps are as follows:

(1) Cell culture: spread BHK-21 cells evenly in a 12-well cell culture plate, and inoculate the cells after the cells grow to about 70%;

(2) Inoculation: inoculate 3 lineages of type O virus strains and 3 lineages of type A virus strains in 12-well plates at the ratio of MOI=1, and set up uninfected normal cells as negative controls at the same time. The inoculated cells were incubated in a 37°C incubator, and the cell supernatant was inactivated when the cell lesion reached about 50%;

(3) Fixation: add paraformaldehyde to each well for fixation for 15 minutes, and wash with PBS for 3 times;

(4) Permeabilization: Prepare a permeabilization solution containing 0.5% Triton X-100 with PBS solution, permeabilize the cells for 10 min, and wash with PBS 3 times;

(5) Incubate the primary antibody: wash 3 times with PBS, dilute the monoclonal antibodies m33 and m77 to be tested with PBS to a final concentration of 10 μg/mL, add 500 μL/well, and use a known positive mouse antibody as a positive control, at 37 °C Incubate for 1h;

(6) Secondary antibody incubation: wash with PBS 3 times, add goat anti-mouse IgG-FITC fluorescent antibody (diluted 1:1000), add 200 μL/well, incubate at 37°C for 1 hour;

(7) Wash with PBS for 5 times, add PBS at 500 μL/well to ensure the cell surface is wet, and put it under a fluorescence microscope for fluorescence observation.

As shown in Figures 3 and 4, the test results show that the monoclonal antibodies m33 and m77 of the present invention have good reactivity with three lineages of type A strains and three lineages of type O strains.

The establishment of embodiment 3 universal foot-and-mouth disease virus structural protein solid-phase competition ELISA detection method

1. Determination of the optimal reaction conditions for the solid-phase competitive ELISA detection method for the general-purpose foot-and-mouth disease virus structural protein

1.1 Determination of the optimal concentration of general-purpose structural protein monoclonal antibody m33, O-type and A-type inactivated antigen:

The optimum concentration of monoclonal antibody m33, O-type and A-type inactivated antigen (preserved in our laboratory) was determined by square array titration. First, use the Na ₂ CO ₃ /NaHCO ₃ coating solution (pH=9.6) to serially dilute the universal monoclonal antibody m33 (horizontal) from 8 μg/mL in the dilution plate, and transfer it to the microtiter plate (50 μL/well), overnight at 4°C; discard the liquid in the microtiter plate, wash 3 times with PBST, and pat dry on absorbent paper; use PBST to serially dilute type O and type A foot-and-mouth disease virus from 1:2 Inactivated antigen (longitudinal) (50 μL/well), incubate at 37°C for 1 hour; wash the plate and pat dry (same as above); add guinea pig antiserum working solution (preserved in our laboratory) (50 μL/well), incubate at 37°C for 1 hour; wash the plate Pat dry (same as above); add goat anti-guinea pig secondary antibody (50 μL/well), incubate at 37°C for 1 h; wash plate and pat dry (same as above); add TMB (50 μL/well), develop color at 37°C in the dark for 15 min, add 2M H ₂ SO ₄ solution to terminate the reaction (50 μL/well), read the OD ₄₅₀ value with a microplate reader, and take the highest dilution factor corresponding to the universal antibody m33, O-type and A-type inactivated antigen when the OD ₄₅₀ value is in the range of 1.6-2.0 is the optimum concentration of both.

The results showed that the optimal coating concentration of monoclonal antibody m33 was 1 μg/mL, the optimal concentration of A-type inactivated antigen was 1:8, and the optimal concentration of O-type inactivated antigen was 1:16.

1.2 Determination of the optimal concentration of monoclonal antibody m77:

Using the optimal concentrations of monoclonal antibody m33, O-type and A-type inactivated antigens determined above, use the Na ₂ CO ₃ /NaHCO ₃ coating solution (pH=9.6) to coat the optimal concentration of m33 (50 μL/ well), overnight at 4°C; wash the plate and pat dry (same as above); add O-type and A-type inactivated antigens at the optimal concentration to the microtiter plate (50 μL/well), incubate at 37°C for 1 hour; wash the plate and pat dry (same as above ); Use PBST to serially dilute monoclonal m77 (horizontal) from 1:100 (50 μL/well), incubate at 37°C for 1 hour; wash the plate and pat dry (same as above); add goat anti-mouse secondary antibody (50 μL/well) , incubate at 37°C for 1 hour; wash the plate and pat dry (same as above); add TMB (50 μL/well), develop color in the dark at 37°C for 15 minutes, add 2M H ₂ SO ₄ solution to terminate the reaction (50 μL/well), and read with a microplate reader Take the OD ₄₅₀ value, and take the highest dilution factor corresponding to the monoclonal antibody m77 when the OD ₄₅₀ value is 1.6 as its optimal working concentration.

The results showed that the optimal concentration of the monoclonal antibody m77 was 6 μg/mL.

2. The operation steps of the general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA detection method

(1) Use the Na ₂ CO ₃ /NaHCO ₃ coating solution (pH=9.6) to dilute the monoclonal antibody m33 to the optimal concentration, coat the microtiter plate (50 μL/well), and overnight at 4°C;

(2) Wash the plate 5 times with PBST and pat dry, dilute type O and type A inactivated antigens with PBST to the working concentration and add to the microtiter plate (50 μL/well), incubate at 37°C for 1 hour;

(3) Wash the plate and pat dry (same as above), use PBST to dilute the positive serum and the serum to be tested (1:4 times diluted to 1:512), and the negative serum (1:4 times diluted to 1:32) (50μL /well), and then add monoclonal antibody m77 diluted to the working concentration to each well, shake gently to mix, and incubate at 37°C for 1h;

(4) Wash the plate and pat dry (same as above), add goat anti-mouse secondary antibody (50 μL/well), and incubate at 37°C for 1 hour;

(5) Wash the plate and pat dry (same as above); add TMB (50 μL/well), develop color in the dark at 37°C for 15 minutes, add 2M H ₂ SO ₄ solution to terminate the reaction (50 μL/well), and read OD ₄₅₀ with a microplate reader ;

(6) Through the detection of serum, calculate the percentage of inhibition (PI), take 40% of PI as the critical value, if the PI of the tested serum is ≥ 40%, the highest dilution of positive serum is the antibody titer of the serum, that is, the titer , it is positive. If the tested serum is less than 40%, it is judged as negative.

3. The specificity of the general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA method

Use the general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA method that the present invention establishes to measure O-type foot-and-mouth disease, A-type foot-and-mouth disease, porcine vesicular disease, Seneca and swine fever positive reference serum, the results show that the general-purpose foot-and-mouth disease virus structural protein The solid-phase competitive ELISA method reacted specifically with reference positive sera for FMD types O and A, but not with positive reference sera for porcine vesicular disease, Seneca, and CSF (Table 1).

Table 1 The solid-phase competitive ELISA method for the structural protein of general-purpose foot-and-mouth disease virus to determine the antibody titer of O-type foot-and-mouth disease, A-type foot-and-mouth disease, porcine vesicular disease, Seneca and swine fever positive reference serum

4. Sensitivity of the general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA method

Use the solid-phase competition ELISA method of general-purpose foot-and-mouth disease virus structural protein established by the present invention to 75 parts of pig negative sera, 50 parts of pig O-type positive sera, 40 parts of pig A-type positive sera, 30 parts of bovine negative sera, and 16 parts of bovine A-type Positive sera, and 18 parts of bovine O-type positive sera are measured, and the results show that the general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA method has good sensitivity and specificity (seeing table 2)

Table 2 Determination of general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA method to different properties of serum

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences

<120> A general-purpose foot-and-mouth disease virus structural protein antibody, preparation method and application thereof

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Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Phe Tyr Arg Gly Ser Thr Thr His Asn Pro Ser Leu

50 55 60

Arg Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

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

85 90 95

Ala Arg Gly Leu His Gly Ser Ser Pro Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 2

<211> 107

<212> PRT

<213> Artificial Sequence

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Asp Ile Val Met Thr Gln Ser Gln Lys Leu Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Val Thr Cys Lys Ala Ser His Asn Val Tyr Ser Asn

20 25 30

Val Val Trp Tyr Gln Glu Lys Pro Gly Gln Ser Pro Asn Ala Leu Ile

35 40 45

Tyr Ser Ala Ser His Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Lys Ser Phe Cys Gln Gln Cys Asn Ser Tyr Pro Ile

85 90 95

His Val Arg Leu Gly His Lys Val Gly Asn Gln

100 105

<210> 3

<211> 357

<212>DNA

<213> Artificial Sequence

<400> 3

caggtgcagc tgaaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60

acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120

tttccaggaa acaaactgga gtggatgggc tacatattct acagaggtag cactacccac 180

aacccatctc tcagaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240

ctgcagttga attctgtgac tactgaggac acagccacat attattgtgc aagggggctc 300

cacggtagta gcccgtttgc ttactggggc caagggactc tggtcactgt ctcgagc 357

<210> 4

<211> 321

<212>DNA

<213> Artificial Sequence

<400> 4

gacattgtga tgacccagtc tcaaaaactc atgtccacat cagtaggaga cagggtcagc 60

gtcacctgca aggccagtca caatgtgtat agtaatgtag tctggtatca agagaaacca 120

gggcaatctc ctaatgcact gatttattcg gcatcccacc ggtacagtgg agtccctgat 180

cgcttcacag gcagtggacc tgggacagat ttcactctca ccatcagcaa tgtgcagtct 240

gaagacttgg caaagtcttt ctgtcagcaa tgtaacagct atcctattca cgttcggctc 300

gggcacaaag ttggaaatca a 321

<210> 5

<211> 125

<212> PRT

<213> Artificial Sequence

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Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Thr Gly Gly Ser Phe Thr Tyr Tyr Leu Asp Ser Ala

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ser Lys Glu Gly Glu Phe Tyr Gly Asn Tyr Gly Asp Tyr Tyr Ala Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

115 120 125

<210> 6

<211> 110

<212> PRT

<213> Artificial Sequence

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

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Asp Trp Lys Ser Asn

100 105 110

<210> 7

<211> 375

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gatgtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60

tcctgtgaag cctctggatt cactttcagt agctatacca tgtcttgggt tcgccagact 120

ccggagaaga ggctggagtg ggtcgcaacc attagtactg gaggtagttt cacctactat 180

ctagacagtg cgaagggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240

ctgcaaatga gcagtctgaa gtctgaggac acagccatgt attackgctc aaaagaggga 300

gaattctatg gtaactacgg ggattactat gctatggact actggggtca aggaacctca 360

gtcaccgtct cgagc 375

<210> 8

<211> 330

<212>DNA

<213> Artificial Sequence

<400> 8

gacatccaga tgacccagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcataca gggccagcaa aagtgtcagt acatctggct atagttatat gcactggaac 120

caacagaaac caggacagcc acccagactc ctcatctatc ttgtatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc acattaggga gcttacacgt 300

tcggaggggg gaccagactg gaaatcaaac 330

Claims (9)

1. the monoclonal antibody of a general foot-and-mouth disease virus structural protein, is characterized in that, described monoclonal antibody is m33 or m77, and the aminoacid sequence of the heavy chain variable region of described monoclonal antibody m33 is as SEQ ID NO. 1, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is shown in SEQ ID NO.5, the light chain variable region The amino acid sequence of is shown in SEQ ID NO.6. 2. The monoclonal antibody according to claim 1, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody m33 is as shown in SEQ ID NO.3, encoding the light chain of the monoclonal antibody m33 The gene sequence of the variable region is shown in SEQ ID NO.4. 3. The monoclonal antibody according to claim 1, wherein the gene sequence encoding the heavy chain variable region of the monoclonal antibody m77 is as shown in SEQ ID NO.7, encoding the light chain of the monoclonal antibody m77 The gene sequence of the variable region is shown in SEQ ID NO.8. 4. a general monoclonal antibody composition of foot-and-mouth disease virus structural protein, is characterized in that, is made up of monoclonal antibody m33 and m77, and the aminoacid sequence of the heavy chain variable region of described monoclonal antibody m33 is as SEQ ID NO .1, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain variable region of the monoclonal antibody m77 is shown in SEQ ID NO.5, and the light chain variable region The amino acid sequence of the region is shown in SEQ ID NO.6. 5. monoclonal antibody as claimed in claim 1 or the monoclonal antibody composition described in claim 4 are in the reagent of preparation detecting foot-and-mouth disease virus, or test strip, or the application in kit. 6. A solid-phase competition ELISA detection kit for the structural protein of foot-and-mouth disease virus, characterized in that the kit comprises the monoclonal antibody according to claim 1 or the monoclonal antibody composition according to claim 4. 7. ELISA detection kit as claimed in claim 6, is characterized in that, described kit comprises monoclonal antibody m33 and foot-and-mouth disease virus inactivated antigen microtiter plate, monoclonal antibody m77, antibody diluent, concentrated Washing solution, chromogen, stop solution; or the ELISA detection kit includes monoclonal antibody m77 and foot-and-mouth disease virus inactivated antigen microtiter plate, monoclonal antibody m33, antibody diluent, concentrated washing solution, color development agent, stop solution. 8 . The ELISA detection kit according to claim 7 , wherein the inactivated antigen of foot-and-mouth disease virus is an inactivated antigen of type O foot-and-mouth disease virus or an inactivated antigen of type A foot-and-mouth disease virus. 9. a general-purpose foot-and-mouth disease virus structural protein solid-phase competition ELISA detection method, is characterized in that, described method comprises the following steps: Coat the microtiter plate with the monoclonal antibody m33 described in claim 1, then add the foot-and-mouth disease virus inactivated antigen in the microtiter plate, add the serum to be checked and the monoclonal antibody m77 described in claim 1, mix, Incubation; or coat the microplate with the monoclonal antibody m77 described in claim 1, then add the foot-and-mouth disease virus inactivated antigen in the microplate, add the serum to be tested and the monoclonal antibody m33 described in claim 1, mix, incubate; After incubation, add goat anti-mouse secondary antibody, after incubation, add TMB to avoid light for color development, then add H ₂ SO ₄ solution to terminate the reaction, and read OD ₄₅₀ with a microplate reader; Calculate the inhibition percentage (PI), with 40% PI as the critical value, when the PI of the serum to be tested is ≥ 40%, the test result is positive, and when the PI of the serum to be tested < 40%, the test result is negative.

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