| Summary: | 碩士 === 國立嘉義大學 === 生物科技研究所 === 91 === To effectively and safely prevent whooping cough, acellualr pertussis vaccines (ACVs) have been developed to replace whole cell pertussis vaccines (WCVs). In spite of the remarkable efficiency for disease prevention, WCVs exhibits various adversed effects. Based on several previous studies and the analysis of commercialized vaccines, pertussis toxin (PT) has been demonstrated to be the essential protective antigen and major receipt in ACVs. However, due to the weak gene promoter, PT was generally obtained only in a very low yield (low than 8 mg/L), making a high cost in vaccine production. Although many experiments have been conducted to obtain an optimal condition for improving the yield of PT, little success has been achieved. The efforts made to modulate the efficiency of wild-type PT promoter by site-direction mutagenesis also result in no improvement. Previous studies also demonstrated that all subunits of PT could be produced in large quantities by molecular recombinant technology in E. coli or yeast expression system, but a immuno active PT however could not be produced. Therefore, at present, the PT that was used for vaccine production are purified strictly from pertussis fermentation, suffering a unavoidable low yield.
In this study, we propose a novel approach that may be used to promote a higher production rate of PT, searching for a strong promoter sequence form pertussis genome. We isolated and purified eleven high-yield proteins from pertussis. Following the determination of their N-terminal amino acid sequences, we have obtained deduced their 5’-upstream sequence, and their promoter activation have been analyzed by b-galactosidase enzyme activity. From the results of transient transformation of E. coli, we found the construct vectors exhibited diverged promoter activity. The b-galactosidase enzyme activity of construct 07 is about twenty folds as compared with that of wild-type PT promoter, and construct 11’ is about sixty fold efficiency as compared with that of wild-type PT promoter. We further analyze that promoter activities of all constructs in B. pertussis, the reporter activity of construct 07 is about five fold than wild-type PT promoter, and the construct 11’ is about seven fold than wild-type PT promoter.
The results of this study would provide not only an insight into pertussis gene regulation, but also the obtained promoter may be used to construct a mutant pertussis strain, which may produce great amount of pertussis toxin. It is believed that the study should be valuable both academically and commercially.
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