| Summary: | 碩士 === 國立嘉義大學 === 生物科技研究所 === 94 === Pertussis toxin is an essential antigen against whooping cough . It is also a required component of acellular pertussis vaccine (DPaT) . Probably due to an inappropriate complex formation , the recombinant pertussis toxin subunits （i.e. S1-S5) produced in other organisms has yet successfully be assembled to exert a protective immunity . In addition , the intrinsically weak promoter of pertussis toxin gene ( ptx operon ) has resulted in the fact that the yield of the purified toxin obtained from pertussis culture was unavoidably low ( 2–5 mg/L ) . This appears to be a bottleneck for pertussis vaccine development .
To improve the expression rate of pertussis toxin , we have previously constructed two mutant ptx promoters and expressed in vitro . With an E. coli rrnb gene upstream element fused to the upstream of -35 box of ptx promoter , the construct revealed a 44-fold and 3.6-fold reporter activity in E. coli and B. pertussis respectively as compared with wild type promoter construct . In addition , a strong pertussis promoter derived from the regulatory region of the gene of a high-production-rate pertussis protein , was found to confer a 60-fold and 7.5-fold increase in an in vitro reporter gene expression .
We have then attempted to make a mutant pertussis strain by the replacement of wild type ptx promoter with these two strong promoters . Following homologous recombination , we have isolated a mutant strain and demonstrated it by PCR . The production rate of pertussis toxin of this strain will be further evaluated by cultivation in the future .
Presumably , the pertussis toxin gene transcription driven by these artificial promoters should be more reliably as compared with the previous studies using transient expression . It is expected that they may confer a much higher promoter activity as compared with that revealed in the transient assay . In addition , we believe the obtained mutant strain would be a very valuable tool in pertussis vaccine development .
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