Molecular Cloning of Helicobacter pylori Cytotoxin-Associated Gene A and Its Expression in Mammalian Cells

• Main Authors: Kai-li Tseng, 曾凱莉
• Other Authors: Hua-wen Fu
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/94095978050959971653

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 92 === Helicobacter pylori (H. pylori) is the major causative agent of gastritis, peptic ulcer diseases and even gastric cancer. This pathogen colonizes the human stomachs of at least half of the world’s population. Cytotoxin-associated gene A (cagA), one of the virulence factors generated by H. pylori, is considered to play an important role in the pathogenesis of these diseases. However, the molecular mechanism under the pathogenesis is still unclear and needs to be identified. It has been known that during cagA+-H. pylori infection, CagA is translocated into host epithelial cells via type IV secretion system. Translocated CagA is tyrosine phosphorylated on specific EPIYA sequence repeats by cellular Src family tyrosine kinases. Phosphorylation of CagA induces rearrangements of host-cell actin-cytoskeleton and cell scattering, resulting in the so-called “hummingbird phenotype”. On the other hand, CagA from different strains varies in sequences in the C-terminal region. Strain-specific genetic diversity of CagA has been proposed to be involved in the ability of different H. pylori strains to cause different diseases. There are also indications of significant geographic differences among strains. In order to study the effect of CagA in mammalian cells, in this study, I constructed pEGFP-N1 based cagA-expression vectors and expressed cagA in mammalian cells. I found that in AGS cells transfected with these cagA-expression vectors, some cells exhibited elongated and scattering phenotypes. However, the expression level of CagA was low. The result indicated that pEGFP-N1 based vectors might not be suitable for ectopically expressing cagA. In addition, I also performed total amino-acid sequence alignment of two H. pylori strains (26695 and NCTC11637) and sequence alignment of C-terminal region of five Taiwan strains with those of another East Asian strain (F32) and strain 26695. The result suggested that the presence of particular sequences might probably correlate with different activities of CagA. These sequences may serve as indications of different disease outcomes of different H. pylori infection.