Studies on molecular cloning and expression of the virulence factors from helicobacter pylori

• Main Author: 沈品均
• Other Authors: 連一洋
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/94785325599598279116

碩士 === 國立屏東科技大學 === 獸醫學系 === 92 === Helicobacter pylori is the major pathogen that causes human gastric ulcer, duodenal ulcer, gastric cancer and mucosa associated lymphoid tissue lymphoma. In Taiwan, there are more and more gastric patients who infected this pathogen. The virulence factors of H. pylori are flagella, urease, vacuolating cytotoxin A( VacA ), cytotoxin-associated antigen ( CagA ), adhesion and nutrophil-activating protein ( NAP ). In this study, we focus on the subunit A and B of urease (ureA and ureB ) which is most important virulence factors of H. pylori. We design the specific primers for the amplification of ureA and ureB gene by using polymerase chain reaction ( PCR ). The amplicons were 714 bp and 1707 bp and were used as an inserted DNA respectively. After the inserted DNA fragment and vector, pGEX6P-1, were digested with Eco RI and Xho I respectively, and then T4 ligase was employed for the ligation and the construction of recombinant plasmid was carried out. The recombinant plasmid was transferred to E. coli DH5α for expression. The purified expressed recombinant fusion protein was obtained by using GST affinity column and was strongly reactive to the specific antibody arisen from rabbit by using Western blotting assay in terms of antigenicity being confiremed. By the way, the expressed purified ureA( 54 kDa) and ureB( 90 kDa) were also strongly reactive to the IgY obtained from the chickens immunized with the lysate of H. pylori. However, the antigencity of the expressed ureA and ureB are quite stable when they reacted to IgY of chickens as well. Thus, it is feasible for the further study on the inhibition of H. pylori by using the specific IgY put in the drinks for the patients suffering from infection with H. pylori. Further studies need to be verified in the future.