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Expression and Purification of ESAT-6 and MPT83 Proteins of Bovine Tuberculosis and Their Primary Application in ELISA Detection

Author: XuLiYi
Tutor: HuangJiong；RanDuoLiang
School: Xinjiang Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Mycobacterium bovis ESAT-6 protein MPT83 protein The prokaryotic expression Immunogenicity Enzyme - linked immunosorbent assay
CLC: S858.23
Type: Master's thesis
Year: 2010
Downloads: 41
Quote: 0
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Abstract


Bovine tuberculosis is caused by Mycobacterium bovis (Mycobacterium bovis) a cattle chronic wasting diseases. The spread of bovine tuberculosis epidemic will not only seriously affect the healthy and sustainable development of animal husbandry, but also a serious threat to human health. In view of the above problems, a new type of vaccine for bovine tuberculosis and specificity of rapid diagnostic reagents is important to study. Secreted proteins is currently studied in Mycobacterium tuberculosis-specific antigens ESAT-6 and MPT83 protein early filtrate proteins secreted by Mycobacterium bovis, has an important role in the serological diagnosis of bovine tuberculosis and vaccine applications. ESAT-6 and MPT83 gene sequences provided by the GenBank own primers designed to the standard strain of Mycobacterium bovis DNA genome as a template, the target gene was amplified by PCR method. ESAT-6 gene fragment was cloned into the prokaryotic expression vector pET22b, the MPT83 gene fragment was cloned into the prokaryotic expression vector pET28a build weight plasmid pET22b-ESAT-6 and pET28a-MPT83. Both were transformed to competent cells DH5a (DE3), positive clones were screened by restriction enzyme digestion and PCR amplification and sequencing. Then were transformed into E. coli BL21 (DE3) for expression. After digestion, PCR and sequencing, the selection of a single engineered bacteria positive clones containing the recombinant plasmid was induced by IPTG. Western-Blot analysis of the expression product immunogenicity. The results show that the purpose of this experiment prokaryotic expression protein has strong immunogenicity, thereby indirect ELISA assay for detection of bovine tuberculosis subunit vaccine candidate proteins. The obtained two has a strong immunogenicity of the ESAT-6 and MPT83 protein molecule through the His-Bind affinity column purification, and semipermeable membrane dialysis, and were used as antigen, the establishment of Mycobacterium bovis specific antibody indirect ELISA assay method. This study on the basis of previous studies, established ELISA method of antigen concentration and serum dilution, the enzyme-labeled secondary antibody concentration yin and yang Judgeded and substrate reaction time to explore and improve. And two indirect ELISA method using established 285 parts PPD positive bovine serum, respectively, 74 parts PPD negative bovine serum, 79 parts of bovine γ-interferon (IFN-γ) detected positive sera were detected. ESAT-6-ELISA assay coincidence rate was 62.1%, 98.64%, 83.54%; of MPT83-ELISA assay coincidence rate of 57.19%, 100%, and 78.48%. Showed that the ELISA method established in this study can be used for Census and timely monitoring of bovine tuberculosis. Successfully cloned, expressed and purified ESAT-6 and MPT83 recombinant protein, and Mycobacterium bovis ELISA antibody detection methods with its desire to develop into a diagnostic kit to provide technical support for the prevention and control of bovine tuberculosis in China.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Cow
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