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Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli
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作者 曹建平 刘述先 +1 位作者 宋光承 徐馀信 《Chinese Medical Journal》 SCIE CAS CSCD 2002年第10期25-29,144,共6页
To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli Methods SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, ... To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli Methods SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from adult worms of S japonicum The RT PCR product was cloned into T vector and sequenced The SjcTM cDNA, derived from the constructed TA clone pGEM SjcTM, was then subcloned into the expressing vector pBV220 After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature dependent condition Results The RT PCR product, cloned into a T vector, was sequenced and shown to be 96 5% identical at the nuclei acid level and 98 1% identical in deduced amino acid sequence to that of S mansoni tropomyosin The target DNA fragment was then subcloned into a prokaryotic vector pBV220 Induced expression in E coli DH5α cells resulted in a constant level of recombinant protein production The results of SDS PAGE and Western blot revealed that the molecular weight of non fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S japonicum tropomyosin (SjcTM) Conclusion The engineering of the cDNA encoding S japonicum tropomyosin and its bacterial expression was successfully made 展开更多
关键词 Schistosoma japonicum · tropomyosi n · cloning · expression
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