Antigenicity evaluation of bovine tuberculin recombinant purified protein derivative

• Main Authors: Man-Kei Ian, 殷敏琦
• Other Authors: Chih-Jung Kuo
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/8e5p68

碩士 === 國立中興大學 === 獸醫學系暨研究所 === 106 === Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important disease in cattle and other ruminants throughout the world. Bovine purified protein derivative (PPD-B) that used in intradermal tuberculin test (ITT) is commonly used for diagnosis of bTB in cattle. Since certain proteins of PPD-B might share sequence homology with other mycobacteria, especially Mycobacterium avium subsp. paratuberculosis (MAP), the specificity and cross-reactive response should be further considered. In this study, Mycobacterium bovis strain AN5 genes that encode 25 proteins of PPD-B were amplified and ligated into the expression vector pET16b or pGEX4T-2. Recombinant proteins were expressed in E. coli and purified by affinity chromatography columns and analysis by SDS-PAGE. Furthermore, 7 recombinant proteins (MAP2950c, Mb1961c, Mb2898, Mb2900, Mb3904, Mb3905 and Mb3904/3905) were evaluated for their antigenicity against bTB serum samples (bTB positive, n=98 and bTB negative, n=88) by using enzyme-linked immunosorbent assay (ELISA). By using ITT and ELISA (commercial kit), serum were classified into 3 groups: Group 1, ITT (+) and ELISA (-); Group 2, ITT (+) and ELISA (+); Group 3, ITT (-) and ELISA (+). The result showed that MAP2950c and Mb1961c had low antigenicity in bTB positive serum samples; Mb2898, Mb3904, Mb3905, and Mb3904/3905 complex were mostly recognized by bTB positive serum in serum Group 1; Mb2900 was mostly recognized by bTB positive serum in serum Group 3. In conclusion, these results showed that different antigen can be detected in different serum group due to animals’ immune response and their antibody titre. Therefore, combination of antigen may serve a potential strategy to improve diagnosis of bTB using serological assays in the future.