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Mycoplasma bovis immune related protein, detection kit containing same and application thereof to detection of mycoplasma bovis antibody

A technology for detection of mycoplasma bovis and antibodies, applied in the biological field, can solve problems such as difficulties in disease prevention and control

Active Publication Date: 2018-03-06
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no internationally recognized specific diagnostic method for M.bovis, which brings difficulties to the prevention and control of the disease

Method used

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  • Mycoplasma bovis immune related protein, detection kit containing same and application thereof to detection of mycoplasma bovis antibody
  • Mycoplasma bovis immune related protein, detection kit containing same and application thereof to detection of mycoplasma bovis antibody
  • Mycoplasma bovis immune related protein, detection kit containing same and application thereof to detection of mycoplasma bovis antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning and expression of the gene encoding the Mycoplasma bovis protein P28 protein

[0041] 1 material

[0042] 1.1 Strain, plasmid

[0043] M.bovis Hubei-1 isolate was isolated, identified and preserved in this experiment (Xin Jiuqing et al., 2008); pET-28aVector was purchased from Invitrogen. The whole genome sequence analysis of M.bovis Hubei-1 isolate was completed by our laboratory. Huashun DNA Column Gel Recovery Kit was purchased from Shanghai Huashun Biotechnology Co., Ltd. Competent cells DH5α and BL21(DE3) were preserved by our laboratory.

[0044] 1.2 Main reagents

[0045] TMB substrate chromogenic solution was purchased from Tianhe Biochemical Technology Co., Ltd.; restriction endonucleases BamH I, SalI, dNTP, and rTaq DNA polymerase were purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0046] 1.3 Main solution and preparation

[0047] Mycoplasma liquid medium: PPLO broth 21g, inactivated horse serum 200mL, fresh yeast extrac...

Embodiment 2

[0092] Example 2 Establishment of M.bovis antibody indirect ELISA method

[0093] Using the P28 recombinant protein prepared in Example 1 as an antigen, an ELISA method for M. bovis serum antibody was established. By optimizing the reaction conditions, determine the best conditions such as antigen coating concentration, primary antibody dilution, optimal blocking conditions, primary antibody action time, secondary antibody action time, color development time, and judgment of critical values. This test laid a theoretical foundation for the research of the indirect ELISA kit for the detection of Mycoplasma bovis antibody, and also provided a reference for the improvement of the field diagnosis method of M.bovis.

[0094] 1 material

[0095] 1.1 Main reagents

[0096] Fish gelatin was purchased from Sigma Company; Ammonium sulfate was purchased from Tianjin Chemical Reagent No. 1 Factory; horseradish peroxidase-labeled rabbit anti-bovine IgG antibody was purchased from Sigma Co...

Embodiment 3

[0172] Example 3 Performance evaluation of the MbH kit of the present invention and its comparison with other commercial kits

[0173] In order to speed up the industrialization of the MbH kit, the present invention evaluates the performance of the kit such as sensitivity, specificity, accuracy of detecting clinical samples, duration of host antibody and storage period of the kit. In addition, the MbH kit is compared with commercial kits to provide data reference for the application of the MbH kit.

[0174] 1 material

[0175] 1.1 ELISA kit

[0176] The "MbH M.bovis ELISA antibody detection kit" ("MbH kit" for short) of the present invention was prepared according to the method in Example 2, and the batches were: 201001, 201002, and 201003, with 20 kits in each batch.

[0177] "Mycoplasma bovis Antibody Test Kit (ELISA)" ("Kit 1" for short) was purchased from Biovet, Canada; "BIO-X MYCOPLASMA BOVIS ELISA KIT" ("Kit 2" for short) was purchased from Bio-X Diagnostics, Belgium....

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Abstract

The invention discloses a mycoplasma bovis immune related protein, a detection kit containing the same and application thereof to detection of a mycoplasma bovis antibody. The mycoplasma bovis immunerelated protein is named p28 protein, and the amino acid sequence of the protein is shown in SEQ ID NO.2. A sensitivity test proves that the mycoplasma bovis serum antibody ELISA detection kit (MbH kit) provided by the invention can detect positive serum with the dilution multiple of 1:2560 at the minimum; and a specificity test proves that the kit has the specificity of 97.8% and has no specificreaction with the positive serums of contagious bovine pleuropneumonia (CBPP), foot-and-mouth disease (FMD), bovine tuberculosis (MB), bovine viral diarrhea (BVDV) and infectious bovine rhinotracheitis (IBRV), and has good stability and high accuracy. The invention provides a new technical means for detecting a mycoplasma bovis antibody.

Description

technical field [0001] The invention relates to a mycoplasma bovis immune-related protein, its encoding nucleotide sequence and application, and also relates to a detection kit containing the protein and its application in the detection of mycoplasma bovis. The invention belongs to the field of biotechnology. Background technique [0002] Mycoplasma bovis (Mycoplasma bovis) is an important pathogenic pathogen that infects cattle. It mainly causes bovine pneumonia, arthritis, mastitis, keratoconjunctivitis and other diseases, and has caused serious economic losses to the world's cattle industry. M.bovis pneumonia was first reported in my country in 2008, and this pathogen has increasingly become an important factor that endangers my country's cattle industry. At present, there is no internationally recognized specific diagnostic method for M.bovis, which brings difficulties to the prevention and control of the disease. It is of far-reaching significance to study the M.bovis ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/30C12N15/31G01N33/535G01N33/544G01N33/569
CPCG01N33/535G01N33/544G01N33/56933C07K14/30G01N2333/30
Inventor 辛九庆李媛刘洋
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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