Characterization of the ipaB gene of Taiwan Shigella flexneri isolates and its protein product

• Main Authors: Pei-Shih Yen, 顏珮詩
• Other Authors: 陳建華
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/26058183198369217247

碩士 === 國立中興大學 === 分子生物學研究所 === 95 === Many Shigella flexneri isolates were collected previously by our laboratory during a shigellosis outbreak in Nantou country form 1996 to 2000. These isolates were sub-typed by pulsed-field gel electrophoresis (PFGE), and 26 isolates in 1996 and 47 isolates form 1997 to 2000 were found with the same PFGE types. They belonged to an epidemic strain. In this study, 7 of 26 epidemic isolates in 1996, and 8 of the 47 of the epidemic isolates form 1997 to 2000 were found having the ipaB gene stably maintained in the first- and tenth-day subcultures. Plasmid pET21b-ipaB803 carrying the first 803 bps of the ipaB gene under the T7 promoter was used to over-express the partial IpaB protein. The protein was injected into mice for antibody production. By western hybridization with the IpaB protein stably maintained in the first- and tenth-day subcultures. An epidemic isolate(SH2308) form 1996 was randomly picked and the spontaneous mutation rate was compared with that of SH3160. The result indicates both isolates had the same mutation rate. The promoter activities of the ipaB genes in SH2308 and SH3160 were analyzed by using the β-gala gene as the reporter gene. About 50﹪ higher promoter activity was found in SH3160. A SH2308 mutant called SH2308-10A was previously isolated form the tenth-day subculture of SH2308 and proved to carry a deletion of the virF gene. The virF gene form SH2308 was cloned into a plasmid and transformed into SH2308-10A. Western analysis shows that SH2308-10A could not produce IpaB protein, but the transformant could. The partial IpaB protein was over-produced and purified by induction of BL21(DE3) carrying the plasmid pET21b-ipaB803. The purified protein was used to infect the humand U937 cells for different time spans and the cells were analyzed by flow cytometry. It was found that apoptosis of the U936 cells increased as the infection time increased.