CN112125962B - Antigen for identifying infection of mycoplasma bovis vaccine strain or wild strain and indirect ELISA detection kit - Google Patents

Antigen for identifying infection of mycoplasma bovis vaccine strain or wild strain and indirect ELISA detection kit Download PDF

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CN112125962B
CN112125962B CN202010700976.5A CN202010700976A CN112125962B CN 112125962 B CN112125962 B CN 112125962B CN 202010700976 A CN202010700976 A CN 202010700976A CN 112125962 B CN112125962 B CN 112125962B
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membrane protein
antigen
mycoplasma bovis
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贺笋
任立松
郝成武
潘毅平
李延涛
任郭子君
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Tiankang Pharmaceutical Co.,Ltd.
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Abstract

The invention relates to the field of vaccines, and particularly provides an antigen and an indirect ELISA detection kit for identifying infection of mycoplasma bovis vaccine strains or wild strains. The invention provides a group of antigens for identifying infection of mycoplasma bovis vaccine strains or wild strains, which comprise membrane protein fragments and leucyl aminopeptidase fragments. The inventor discovers that only the change of the membrane protein antibody and the leucyl aminopeptidase antibody shows certain regularity after the cattle is infected with different pathogens (vaccine strains and wild strains), and the content of the membrane protein antibody and the leucyl aminopeptidase antibody in the cattle body is detected by utilizing the membrane protein antigen and the leucyl aminopeptidase antigen, so that the differential diagnosis of the infection of the mycoplasma bovis vaccine strains or the wild strains can be realized, and a new thought and technical means are provided for the differential diagnosis of the vaccine strains and the wild strains after the vaccine strains are used.

Description

Antigen for identifying infection of mycoplasma bovis vaccine strain or wild strain and indirect ELISA detection kit
Technical Field
The invention relates to the field of vaccines, in particular to an antigen and an indirect ELISA detection kit for identifying infection of mycoplasma bovis vaccine strains or wild strains.
Background
The mycoplasma bovis is an important pathogenic mycoplasma, can cause bovine mastitis, bovine pneumonia, arthritis, genital tract inflammation, infertility and other diseases, and the bovine infected with the mycoplasma can be infected with other pathogens, so that the production capacity of the bovine is influenced, the healthy development of the livestock breeding industry is seriously damaged, and the mycoplasma bovis becomes one of important pathogens which endanger the cattle breeding industry at present.
Mycoplasma bovis pneumonia caused by Mycoplasma bovis is a bovine respiratory infectious disease mainly characterized by necrotizing pneumonia. The body temperature of sick cattle is raised to 42 ℃, the cattle is depressed, the appetite is reduced, the asthma and the severe cough are caused, severe cattle can die due to some secondary diarrhea, some secondary arthritis, arthrocele and the like, generally, the calf is seriously ill, and the fatality rate can reach 50%. Most of the medicines for treating mycoplasma bovis are antibiotics, but the death rate of calves caused by mycoplasma pneumonia and the diffusion and pollution of mycoplasma in a cattle farm are difficult to effectively reduce.
The vaccine is an effective measure for controlling mycoplasma bovis infection, and the current project researched in China mostly takes attenuated vaccine as a main part, and although the vaccine has good immunogenicity, the objective of cattle farm purification is difficult to achieve due to the fact that the vaccine belongs to live virus. Because the inactivated vaccine adopts a chemical method to inactivate the vaccine strain, the inactivated vaccine is a preferential choice in terms of protection effect and safety after being used. However, there is no accurate method for differential diagnosis of the virus after use and the wild virus.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a group of antigens for identifying infection of mycoplasma bovis vaccine strains or wild strains.
The second purpose of the invention is to provide the application of the antigen.
The third purpose of the invention is to provide an indirect ELISA detection kit for identifying infection of mycoplasma bovis vaccine strains or wild strains.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a set of antigens for identifying infection by a mycoplasma bovis vaccine strain or a wild strain, said antigens comprising a membrane protein fragment and a leucyl aminopeptidase fragment.
Further, the sequence of the fragment of the membrane protein is shown as SEQ ID NO. 1.
Furthermore, the sequence of the fragment for coding the leucyl aminopeptidase is shown in SEQ ID NO. 2.
The antigen is applied to the preparation of a detection reagent for identifying the infection of the mycoplasma bovis vaccine strain or the wild strain.
An indirect ELISA detection kit for identifying mycoplasma bovis vaccine strain or wild strain infection, comprising an ELISA plate coated with the antigen;
the detection kit is used for respectively detecting the contents of the membrane protein antibody and the leucyl aminopeptidase antibody in a sample to be detected by using the antigen, and if the content ratio of the membrane protein antibody to the leucyl aminopeptidase antibody is not less than a threshold value, the infection of the mycoplasma bovis vaccine strain is judged.
Further, the threshold value is 1.8.
Further, the coating concentration of the membrane protein fragment is 40-60. mu.g/ml.
Further, the leucyl aminopeptidase fragment is coated at a concentration of 70 to 90. mu.g/ml.
Further, the kit also comprises at least one of a positive control, a negative control, an enzyme-labeled secondary antibody, a diluent, a developing solution, a washing solution and a stop solution.
Further, the vaccine strain is an inactivated vaccine;
preferably, the sample to be detected is bovine serum.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a group of antigens for identifying infection of mycoplasma bovis vaccine strains or wild strains, which comprise membrane protein fragments and leucyl aminopeptidase fragments. The inventor discovers that only the change of the membrane protein antibody and the leucyl aminopeptidase antibody shows certain regularity after the cattle is infected with different pathogens (vaccine strains and wild strains), and the content of the membrane protein antibody and the leucyl aminopeptidase antibody in the cattle body is detected by utilizing the membrane protein antigen and the leucyl aminopeptidase antigen, so that the differential diagnosis of the infection of the mycoplasma bovis vaccine strains or the wild strains can be realized, and a new thought and technical means are provided for the differential diagnosis of the vaccine strains and the wild strains after the vaccine strains are used.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 shows the result of PCR amplification of a target fragment in example 1, wherein M is marker; 1, CONTROL; 2: P275; 3, pepA.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
The inventor discovers that only the change of the membrane protein antibody and the leucyl aminopeptidase antibody shows certain regularity after the cattle is infected with different pathogens (vaccine strains and wild strains), and the content of the membrane protein antibody and the leucyl aminopeptidase antibody in the cattle body is detected by utilizing the membrane protein antigen and the leucyl aminopeptidase antigen, so that the differential diagnosis of the infection of the mycoplasma bovis vaccine strains or the wild strains can be realized, and a new thought and technical means are provided for the differential diagnosis of the vaccine strains and the wild strains after the vaccine strains are used.
In order to realize accurate detection of the contents of the membrane protein antibody and the leucyl aminopeptidase antibody, the inventor respectively analyzes, screens and optimizes the antigenic sites of the membrane protein antigen and the leucyl aminopeptidase, and provides an optimized membrane protein (p275) gene sequence shown in SEQ ID NO.1 and an optimized leucyl aminopeptidase (pepA) gene sequence shown in SEQ ID NO. 2. The content of the membrane protein antibody and the content of the leucyl aminopeptidase antibody can be accurately detected by respectively utilizing the two.
GGAATATTAGCGCCCGTCTTAGCTATTCCTTTAGTAGCTGCTAGTT GCAATAATGAATCTAAACTCAAAGATTTACAAACAAAGTATGAAAACA ACAGAAAATCAGTTATAGATTTTTTAAATTCAGAAGAAAAATATAGCTT TTTAAAAACATATGTTGATATTAAAAATGCTCTAAATGCTAAAGTTGAT ATTAAAAGTAGCAAAGAAATCAATAATTGAATAAAAAACACAAATGAT GCAATTTCTTCATATAAATCATTTAAAAATAGCATTGTTACAGTTAATGA AGATAAAGAAAAAAATACATTTTCTGCTTTTGATAACTTACTAGTTTTA TCATCTAAAGTATCTGAAAAGGGAAAAGGCTTTTTCCCTAAAACTATT ATAAATCAGCTTAAAAGTAAAAATTCATTTGCAGAACAAGTTAAATTA CTAAATTCCTTTTTAGAATCAAGCTTATTAAAAGTTAATGAAGAATCAT TGAAAGATATTTCAATAGATTTTGAAAACTCTGTCCCAAATGATTTTAT TAATTCAGTCAATGATGGGTTAACACTAACTTTTGTAATGAACAAAAA TGGAACTAAGCATTATGCACCCATAACATGAAAAAATATTGGTTTAAAT AATGTTGAAAAAGCTGATGATGTTCACAGCGATGAAATTGATTTATCA AAAGTAACTGAAAATGATAAAAATCATTATCTAGATGATAGCTTTATTA TTGGAAAAATATTTAAAAATGCAGCTTTTGGCACAGAACAAACTGCTG ATAAAATCTTTGAAAAAATAAATTCTATCGTTTCCCAAAGTAACGGTAA ATTAGACACAAATGAATTTAAGAAGAAAGCTGAAGAATTGAATAAAC TTTTTGCATTTGAAACAAATCTTGAAAATAACCCTAGTGTTTCATATTC ATTAATTGGCTCACATGCGCATTCAACTAAGGAATACCATTTTTACTTA ACAAAATATGTAAATAACGCTAAGGATTCAAAAATCAGTTTCATTGTTC ACAATCAAAAAAACAAT(SEQ ID NO.1)。
TTTGCTGATGAAACTAAATGCAATTTTTTAATTAAAAAAGACAAC AGCATAACTGAATATCATGAAAAGAATGAAGCATTAGTATATTTTAGTA AGAAAGAATCATTATCATTTTCTGACTTAGAAGGCTTTTTTAAAGGCTT AGCAGTAAATGCCAACAGAAATTATCAAGTTGATTTAGCTTCGTTTGC AACTGAAAAAGTTGAAATAGTTAAAGTTATTGATGCATTTGTTAGAGC AGTTTATTTTGCAAAGGGCGAAATATTTTCAGCTAGAAAAAAAGATGA AAAGGAAGAAATTGAATTAGTTCCATTTATTGAAACTATTTCTGAACA AGCTAACGCACAATTTAATAAATCGCTTATCTTAGCCAAAGCAACAAA TTTTGCTCGTGATTTACAAATTATGCCCCCAAATATTTGCAACTCTGAA TTTTTAGCTCAAAAAGTTGCTGAAGATTTAGAACAATACAAAAACTTG AAAGTTACTGTTTTAAAGAAAAAAGAAATCGAAGAGTTGAAGATGGG TCTTTTACTTTCAGTAAACAAAGGAAGTGTTTATGAACCTAGAGTTGT TGTTATTGAATACAATGGGGACAAAGATTCAAGTGAAAAGACTGTAAT GATTGGTAAAGGTATTACTTTTGATTCAGGTGGATACTCATTAAAACCT TCTAGATCAATGGTTTCAATGAAATTTGATATGTCTGGTTCAGCTATTG TTGCTGCTACAATGAAAGCTATTGCACAATTAAAACCAAAGAAAAATG TTTCTGCAATAATGTGCATTACTGATAACAGAGTTAACGGTGATGCTTC ACTTCCTGATTCAGTATGGGTAGCTATGAATGGCAAAAGTGTTGAAAT TAATAATACTGATGCTGAAGGAAGATTGGTTATGGCTGATGGCTTAGTT TACGGAGCAAAAGTGTTGAATGCCACTAGATTAATTGACGTTGCAACT TTAACTGGTGCTATGGTTGTTGCACTTGGACAGACATACACAGGCACA TGGGCAACTAGTGATAAAGCTTGAGAAGACATAAAGAAAGCTGCTGA AAATGCTAACGAATTAGTTTGAAGAATGCCGCTTGATAAAGCATTTGC AAAAAACATAAAATCTTCAAAAGTAGCCGATTTAAAGAATACTGACTT TTCAGGAAATGCAGGCTCATGTTCAGCAGCAATGTTTTTAGAAGAATT TACAGAAGGTGTTGAACATATTCATCTTGATGTAGCTGGAACTGCTGA AATCAGTGAAGTGCCACAAGGAATTATGGTTAAAACTTTAACTGAATT AAGTTTACTT(SEQ ID NO.2)。
In some embodiments, the membrane protein fragment encoded by SEQ ID NO.1 is obtained by: PCR amplification is carried out by taking SEQ ID NO.3 and SEQ ID NO.4 as primers and mycoplasma bovis strain genome DNA as a template to obtain a target gene fragment, the target gene fragment is connected with plasmid pET-30a (+) by double enzyme digestion to obtain recombinant plasmid P275-pET-30a (+) and the recombinant plasmid P275-pET-30a (+) is transformed into escherichia coli BL21 to be subjected to induced expression and purification to obtain a membrane protein fragment.
P275-F:5’-GG GGTACC GGAATATTAGCGCCCGTCTTAG-3’(SEQ ID NO.3)。
P275-R:5’-CCGC GGATCC ATTGTTTTTTTTGATTGTGAAC-3’ (SEQ ID NO.4)。
In some embodiments, the leucyl aminopeptidase fragment encoded by SEQ ID No.2 is obtained by: PCR amplification is carried out by taking SEQ ID NO.5 and SEQ ID NO.6 as primers and mycoplasma bovis strain genome DNA as a template to obtain a target gene fragment, the target gene fragment is connected with plasmid pET-30a (+) by double enzyme digestion to obtain a recombinant plasmid pepA-pET-30a (+) and the recombinant plasmid is transformed into escherichia coli BL21 to be subjected to induced expression and purification to obtain the leucyl aminopeptidase fragment.
pepA-F:5’-GG GGTACC TTTGCTGATGAAACTAAATGC-3’(SEQ ID NO.5)。
pepA-R:5’-CCGC GGATCCAAGTAAACTTAATTCAGTT-3’(SEQ ID NO.6)。
The antigen is preferably used for detecting the content of the membrane protein antibody and the leucyl aminopeptidase antibody in a sample to be detected by an indirect ELISA detection mode. The invention thus provides an indirect ELISA detection kit for identifying mycoplasma bovis vaccine strain or wild strain infection, wherein the kit comprises an ELISA plate coated with the antigen provided by the invention, the kit is used for respectively detecting the contents of a membrane protein antibody and a leucyl aminopeptidase antibody in a sample to be detected by using the antigen, and if the content ratio of the membrane protein antibody to the leucyl aminopeptidase antibody is not less than a threshold value, the mycoplasma bovis vaccine strain infection is judged. The membrane protein antigen and the leucyl aminopeptidase antigen are coated in different wells of the microplate, respectively, and are not coated in a mixed manner.
In the research process of the inventor, the contents of the membrane protein antibody and the leucyl aminopeptidase antibody in the cattle body after the mycoplasma bovis vaccine strain and the wild strain are infected have certain rules, but the rules of the membrane protein antibody and the leucyl aminopeptidase antibody are not the same, so that the infection type of the cattle can be judged by comparing the difference between the two rules. Detection of the membrane protein (P275) antibody OD in bovine serum, for example, by indirect ELISA detection450nmValue and Bright Aminoacylaminopeptidase (pepA) antibody OD450nmWhen the ratio of the two is more than or equal to 1.8, the inactivated vaccine is judged to be the bovine inoculation mycoplasma vaccine; when the ratio of the two is less than 1.8, the cattle is judged to be infected by the mycoplasma wild strain. It will be appreciated that the wavelength of the light absorbed for the amount detection may vary depending on the chromogenic substrate, and thus different or similar thresholds may be obtained, although such diagnostic criteria are within the scope of the present application.
In some embodiments, the membrane protein fragment is coated at a concentration of 40-60. mu.g/ml, preferably 50. mu.g/ml. The leucyl aminopeptidase fragment is coated at a concentration of 70-90. mu.g/ml, preferably 80. mu.g/ml. Besides the enzyme label plate coated with the antigen, the kit can also contain at least one of positive control, negative control, enzyme-labeled secondary antibody, diluent, developing solution, washing solution and stop solution. Wherein:
the coating diluent can be PBS solution with pH7.0;
5% protamine can be selected as the confining liquid;
the wash solution may be PBST (pH 7.4): NaCl 8.0g, KH2PO4 0.2g,NaHPO4·12H22.9g of O, 0.2g of KCl and 200.5 ml of Tween200, adding deionized water to 1000ml, and adjusting the pH value to 7.4;
sample diluent: 1 × PBS1000mL, Tween 205 mL, (pH 10.8);
the enzyme-labeled secondary antibody can be rabbit anti-bovine IgG horse radish peroxidase;
substrate color developing solution: 1ml of citric acid solution 20. mu.L of TMB (3,3',5,5' -tetramethylbenzidine) solution and 1.2. mu. L H were added2O2TMB water solution 15 mg/ml; the citric acid is 0.1mol/L and the pH value is 4.0;
the stop solution is: 2mol/L H2SO4And (3) solution.
The indirect ELISA detection kit provided by the invention is used for detecting the contents of the membrane protein antibody and the leucyl aminopeptidase antibody in a bovine serum sample, and the OD of the membrane protein (P275) antibody is judged450nmValue and Bright Aminoacylaminopeptidase (pepA) antibody OD450nmThe ratio of the values is changed to judge whether the infectious agent is a mycoplasma inactivated vaccine strain or a wild strain. Specifically, when the ratio of the two is more than or equal to 1.8, the inactivated vaccine is judged to be the bovine inoculation mycoplasma vaccine; when the ratio of the two is less than 1.8, the cattle is judged to be infected with mycoplasma wild virus. The specific detection procedure is preferably as follows:
1. collecting blood from neck of cattle 5ml, centrifuging at 37 deg.C for 2h and 1000g for 20min, collecting supernatant, and storing at 4 deg.C;
2. coating: respectively diluting the concentration of P275 expression protein to 50 mu g/ml and the concentration of pepA protein to 80 mu g/ml by using coating diluent (PBS pH7.0), respectively adding the diluted solutions into different micropores of an ELISA plate, wherein each micropore is 100 mu l, and the temperature is 4 ℃ for 24 hours;
3. and (3) sealing: discarding the coating solution, adding 250 μ l of 5% protamine into each well, and keeping the temperature at 37 deg.C for 1 h;
4. washing the plate: removing confining liquid, adding washing liquid to fill the pores, slightly vibrating for 5S, removing the washing liquid, washing for 3 times, and drying;
5. sample adding: serum sample dilution 1: 100, adding 100 mul of the mixture into each hole, and keeping the temperature at 37 ℃ for 1 h;
6. washing the plate: discarding the sample, adding washing liquid to fill the hole, slightly vibrating for 5S, discarding the washing liquid, washing for 3 times, and patting dry;
7. adding an enzyme-labeled secondary antibody: dilution of rabbit anti-bovine IgG horseradish peroxidase 1: 5000, adding 100 mul of the mixture into each hole, and keeping the temperature at 37 ℃ for 1 h;
8. washing the plate: discarding the sample, adding washing liquid to fill the hole, slightly vibrating for 5S, discarding the washing liquid, washing for 3 times, and patting dry;
9. substrate addition (ready mix): adding 100 μ l of substrate color development solution into each well, and standing at 37 deg.C in dark for 20 min;
10. and (3) terminating the reaction: add 50. mu.l of stop buffer and detect OD450nmThe value is obtained.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
The membrane protein fragment and the leucyl aminopeptidase fragment in the embodiment are obtained by respectively expressing the sequences of SEQ ID NO.1 and SEQ ID NO.2, and primers used for obtaining the fragments of SEQ ID NO.1 and SEQ ID NO.2 through PCR amplification are respectively primers shown in SEQ ID NO. 3-6.
Example 1 PCR amplification of Membrane protein (P275) Gene, leucine aminopeptidase (pepA) Gene fragment
1. Culturing the mycoplasma bovis strain: mycoplasma separated from sick cattle by the company is inoculated into PPLO liquid culture medium, and the temperature is 37 ℃ and the CO content is 5 percent2Culturing in an incubator;
2. extracting genome DNA: extracting a genome according to a method provided by a mycoplasma genome DNA extraction kit;
3. amplification of a target gene: the amplification system was a 50 μ L system: taq enzyme (1000u/mL) 2. mu.L, 10 XBuffer 5. mu.L, dNTP (10mM) 2. mu.L, forward primer (10. mu.M) 2. mu.L, reverse primer (10. mu.M) 2. mu.L, template 10. mu.L, ddH2O27. mu.L. And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 15min at 72 ℃; storing at 4 deg.C.
4. Purifying a target gene: the PCR product was subjected to gel electrophoresis, and the target band was observed by a gel imaging system, and the target gene was purified by a gel purification kit, and the results are shown in FIG. 1.
Example 2 ligation and transformation
1. Carrying out KpnI and BamHI double enzyme digestion on plasmid pET-30a (+), membrane protein (P275) gene and leucinamidopeptidase (pepA) gene;
1.1 carrying out 1% agarose gel electrophoresis on the enzyme digestion product, and recovering and purifying a target band by gel;
1.2 ligation of plasmid and p275, pepA genes according to the procedure provided by the kit for ligation of T4;
1.3 kanamycin (100ml/L) solid medium screening P275-pET-30a (+), pepA-pET-30a (+) transformed into Escherichia coli BL21 positive strain;
1.4 double enzyme digestion identification of P275-pET-30a (+), pepA-pET-30a (+) recombinant prokaryotic expression vector.
2. Prokaryotic expression and purification of P275-pET-30a (+), pepA-pET-30a (+) proteins
2.1 picking out Escherichia coli BL21 containing P275-pET-30a (+), pepA-pET-30a (+) expression vectors, culturing a single colony in LB solid medium (containing 100mg/L kanamycin) overnight in 20mL LB liquid medium (containing 100mg/L kanamycin);
2.2 taking 5ml of the culture broth, inoculating the culture broth into 500ml of LB liquid medium (containing 100mg/L kanamycin), and culturing to OD600When the temperature reaches 0.6, adding IPTG (0.4mmol/L) solution, and inducing for 4 hours at 28 ℃ and 150 rpm;
2.36000 rpm, and centrifuging for 15min to precipitate thallus;
2.4 adding 10mL of lysate to resuspend the thalli;
2.5 ultrasonic disruption: 300W, 10S/10S, 20 min;
2.6 purification of expressed proteins according to the steps provided in the Ni column purification instructions;
2.7 protein purity was checked according to the SDS-PAGE protocol and the concentration of expressed protein was checked according to the Bradford protocol.
Example 3ELISA detection of bovine serum
Randomly selecting mycoplasma negative cattle farm, and inactivating20 cattle in a vaccine immunity experiment cattle farm and a mycoplasma detection positive cattle farm respectively. Detecting the P275 protein and pepA protein antibody OD in the serum according to the indirect ELISA method step450nmValues (results are shown in table 1).
1. Collecting blood from neck of cattle 5ml, centrifuging at 37 deg.C for 2h and 1000g for 20min, collecting supernatant, and storing at 4 deg.C;
2. coating: respectively diluting the concentration of P275 expression protein to 50 mu g/ml and the concentration of pepA protein to 80 mu g/ml by using coating diluent (PBS pH7.0), respectively adding the diluted solutions into different micropores of an ELISA plate, wherein each micropore is 100 mu l, and the temperature is 4 ℃ for 24 hours;
3. and (3) sealing: discarding the coating solution, adding 250 μ l of 5% protamine into each well, and keeping the temperature at 37 deg.C for 1 h;
4. washing the plate: removing confining liquid, adding washing liquid to fill the pores, slightly vibrating for 5S, removing the washing liquid, washing for 3 times, and drying;
5. sample adding: serum sample dilution 1: 100, adding 100 mul of the mixture into each hole, and keeping the temperature at 37 ℃ for 1 h;
6. washing the plate: discarding the sample, adding washing liquid to fill the hole, slightly vibrating for 5S, discarding the washing liquid, washing for 3 times, and patting dry;
7. adding an enzyme-labeled secondary antibody: dilution of rabbit anti-bovine IgG horseradish peroxidase 1: 5000, adding 100 mul of the mixture into each hole, and keeping the temperature at 37 ℃ for 1 h;
8. washing the plate: discarding the sample, adding washing liquid to fill the hole, slightly vibrating for 5S, discarding the washing liquid, washing for 3 times, and patting dry;
9. substrate addition (ready mix): adding 100 μ l of substrate color development solution into each well, and standing at 37 deg.C in dark for 20 min;
10. and (3) terminating the reaction: add 50. mu.l of stop buffer and detect OD450nmThe value is obtained.
TABLE 1 ELISA test results for sera of negative cattle farm, immune experimental cattle farm and positive cattle farm
Figure BDA0002591821630000111
Figure BDA0002591821630000121
By passingStatistical analysis is performed on the data in the table to obtain the standard for judging infection of the mycoplasma bovis vaccine strain and the wild strain: membrane protein (P275) antibody OD450nmValue and leucyl aminopeptidase (pepA) antibody OD450nmWhen the ratio of the two is more than or equal to 1.8, the inactivated vaccine is judged to be the bovine inoculation mycoplasma vaccine; when the ratio of the two is less than 1.8, the cattle is judged to be infected by the mycoplasma wild strain.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
SEQUENCE LISTING
<110> Tiankang biological products Ltd
<120> antigen for identifying infection of mycoplasma bovis vaccine strain or wild strain and indirect ELISA detection kit
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<170> PatentIn version 3.5
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Claims (6)

1. A set of antigens for identifying infection by a mycoplasma bovis vaccine strain or a wild strain, wherein said antigens comprise a membrane protein fragment and a leucyl aminopeptidase fragment;
the sequence of the fragment of the membrane protein is shown as SEQ ID NO. 1;
the sequence of the fragment for coding the leucyl aminopeptidase is shown as SEQ ID NO. 2.
2. Use of the antigen of claim 1 for the preparation of a detection reagent for identifying infection by a mycoplasma bovis vaccine strain or a wild strain.
3. An indirect ELISA detection kit for identifying infection of a mycoplasma bovis vaccine strain or a wild strain, comprising an ELISA microplate coated with the antigen of claim 1.
4. The indirect ELISA detection kit of claim 3 wherein the membrane protein fragment is coated at a concentration of 40-60 μ g/ml.
5. The indirect ELISA detection kit of claim 3 wherein the leucylaminopeptidase fragment is coated at a concentration of 70-90 μ g/ml.
6. The indirect ELISA detection kit of claim 3 further comprising at least one of a positive control, a negative control, an enzyme-labeled secondary antibody, a diluent, a developing solution, a washing solution, and a stop solution.
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