Functional studies of acid-inducible HP0232 protein in Helicobacter pylori

• Main Authors: Hsin-Yuan Wang, 王信元
• Other Authors: Haimei Huang
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/y49efr

碩士 === 國立清華大學 === 生物科技研究所 === 92 === Helicobacter pylori is a common pathogen in humans, which can adapt to acid environments and survive in the human stomach. HP0232, one of the H. pylori genes, was predicted as a secreted protein involved in flagellar motility by The Institute for Genomic Research (TIGR). DNA microarray-based study indicated that HP0232 gene can be acid-induced at pH 5.5 in the agar plate culture. Another functional genomic research showed that HP0232 gene is needed for gastric colonization. Since the cloning and expression of the HP0232 gene in E. coli was successful for anti-HP0232 antiserum production in our lab, continuous functional studies of recombinant HP0232 protein were carried out in this study. After Western analysis in using anti-HP0232 rabbit serum, approximate 2-fold HP0232 protein expression was found from samples cultured for 48 h on Brucella agar plates at pH 5.5, compared to samples at pH 7.2. This acid-induced expression can be prolonged for 72 h and 96 h in acid exposure. Similar amounts of expressed HP0232 protein were found from suspension samples harvested from agar slant cultures or Brucella broth cultures at pH 7.2 and 5.5. HP0232 protein, presented in supernatant of 24-48 h bacteria culture, appeared as secreted protein. In the presence of 50-800 µg/ml of HP0232 protein, 106 bacteria per 10 µl-spot on agar plates at pH 5.5 would form larger growth zone than those at pH 7.2. Further, 400 µg/ml of rec-HP0232 protein can enhance the bacteria growth zone (represented as the bacteria motility) to the maximum. The results from urease activity measurement (represented as the adhesion rate assay) showed that 50 µg/ml of HP0232 protein will increase the H. pylori adhesion to AGS cells. However, no extra sub-G1 apoptosis was found in AGS cells after 24-h culture containing 100 µg/ml of HP0232 protein. To investigate the alteration of functional activity when the signal peptide of HP0232 protein was truncated, the N-truncated HP0232 protein (NT-HP0232), that was cloned, expressed, and purified by the E. coli expression system, was used in these functional analyses. Theoretically, it is more conformed to the real situation, if the functional studies were resolved by using the NT-HP0232 protein which is similar to that secreted by H. pylori.