Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter

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 o...

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Main Authors: Sheng-Dong Shih, 施盛棟
Other Authors: Yu-Yuan Wo
Format: Others
Language:zh-TW
Published: 2004
Online Access:http://ndltd.ncl.edu.tw/handle/92765769882275016612
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spelling ndltd-TW-094NCYU51110042015-10-13T16:31:55Z http://ndltd.ncl.edu.tw/handle/92765769882275016612 Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter 以同質基因重組技術建構一含高效率百日咳毒素基因啟動子之突變株 Sheng-Dong Shih 施盛棟 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 . Yu-Yuan Wo 吳游源 2004 學位論文 ; thesis 86 zh-TW
collection NDLTD
language zh-TW
format Others
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description 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 .
author2 Yu-Yuan Wo
author_facet Yu-Yuan Wo
Sheng-Dong Shih
施盛棟
author Sheng-Dong Shih
施盛棟
spellingShingle Sheng-Dong Shih
施盛棟
Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
author_sort Sheng-Dong Shih
title Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
title_short Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
title_full Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
title_fullStr Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
title_full_unstemmed Homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
title_sort homologous recombination study for the construction of a mutant pertussis strain composed of a strong pertussis toxin promoter
publishDate 2004
url http://ndltd.ncl.edu.tw/handle/92765769882275016612
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