Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli

日本血吸虫原肌球蛋白cDNA的克隆及其在大肠杆菌中表达(英文)

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

克隆日本血吸虫原肌球蛋白编码基因 ,并在大肠杆菌中表达。方法 抽提日本血吸虫 (大陆株 )成虫总RNA ,经逆转录—聚合酶链反应 (RT PCR)获得编码日本血吸虫原肌球蛋白的cDNA片段 ,该片段与全序列比较 ,缺氨基端 1 4个氨基酸。该PCR产物克隆入T载体并对插入片段进行序列测定后 ,亚克隆入表达载体pbV2 2 0 ,经琼脂糖凝胶电泳、限制性酶切反应和PCR鉴定后 ,选择克隆用于温控表达。结果 RT PCR产物克隆入T载体 ,序列测定表明 ,在核苷酸水平和推断和氨基酸水平 ,与曼氏血吸虫分别有96 5%和 98 1的同源性。目的DNA片段亚克隆入原核表达载体pBV2 2 0 ,在大肠杆菌DH5α中诱导表达。经SDS PAGE和Westernblot分析表明 ,该非融合表达产物相对分子质量约为 3 2 0 0 0 ( 3 2kDa) ,日本血吸虫 (大陆株 )成虫天然原肌球蛋白免疫兔血清能特异识别该重组蛋白。