Promoter activity of a mutated pertussis toxin promoter containing A-tracts

• Main Authors: Chi-Jui Lin, 林瑞珠
• Other Authors: Yu-Yuan Wo
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/68197493792048322882

碩士 === 國立嘉義大學 === 生物科技研究所 === 91 === Whooping cough is a highly infective acute disease on upper respiratory tract caused by Bordetella pertussis. To effectively, and safely, prevent whooping cough, acellular pertussis vaccines have been developed and used to replace the whole cell vaccines, which have been reported to associate with various adverse reactions. Based on several lines of evidence, pertussis toxin (PT) has been demonstrated to be the essential antigen for the acellular vaccine. However, due to its weak gene promoter, PT was generally only obtained in a rather low yield, making a considerably high cost in vaccine production. Although a lot of efforts have been made to improve the production yields through medium optimization, culture conditions, and gene manipulation of the B. pertussis stains, however a high yields of PT from bacterial culture still cannot be achieved. Consequently, the studies in promoting the yield of PT, and reducing the manufactory cost, apparently have become the critical projects in the vaccine development. In this study, based on the PT promoter of B. pertussis stains ATCC9340, we have constructed nine recombinant reporter plasmids containing either wild type or mutant PT promoters (including wild-type PT promoter, D4C mutated PT promoter, and mutated PT promoters containing A-T tract enhancer elements). Through the relative reporter gene（b-galactosidase activity） analysis of these promoter sequences, we attempt to find a highly efficient mutated PT promoter. According to the results from reporter genes studies in E. coli, PT promoter deleted four cytosine residues to bring the —35 and —10 element of PT promoter to a consensus 17 bp may significantly enhance promoter activity. We found all ∆4C serial mutated constructions exhibited higher b-galactosidase enzyme activity, range from one point five folds to forty-four folds. For example: the b-galactosidase activity of construct ∆4C Mut-A4 is about forty-four folds as compared with that of wild-type PT promoter, and construct ∆4C Mut-A9 is about fourteen folds as compared with that of wild-type PT promoter. In order to further analyze the promoter activities of all constructs in B. pertussis, we have transiently transfected the reporter constructs into B. pertussis cell by electroporation. As show our in results, the reporter activity of construct WtPT-A tracts is about three point nine folds as compared with that of wild-type PT promoter, and construct ∆4C Mut-A4 is about three point six folds as compared with that of wild-type PT promoter. This indicated, as our preliminary demonstration, the space between -35 region and -10 region might not be change to 17 bp (consensus space in high efficient bacterial promoters) for a batter transcription efficiency in B. pertussis. From the results of this study, it would provide not only the deeper insight into pertussis gene regulation, but also the possible strong promoter that may be used to construct a mutant pertussis strain, which in turn may produce great amount of pertussis toxin. It is believed that the results of current study should be valuable both academically and commercially.