【摘要】 目的探讨日本血吸虫(Schistosoma japonicum,Sj)中国大陆株(Chinese strain)组织蛋白酶B肽链内切酶基因(Cathepsin B endopeptidase)的原核表达载体pWR450-1的构建及表达情况。方法根据Genbank中Cathepsin B en-dopeptidase全序列(Genbank登录号为AY226984)设计特异性引物应用聚合酶链反应技术扩增基因全长,克隆入pUCm-T载体,酶切后亚克隆入原核表达载体pWR450-1并进行表达;利用SDS-PAGE及Western blotting观察日本血吸虫组织蛋白酶B肽链内切酶表达情况。结果成功构建了Sjcb2/pWR450-1原核表达重组质粒,该重组质粒在大肠杆菌中可经IPTG诱导表达分子量约为86kD的融合蛋白,Western blotting分析表明,该融合蛋白能被兔阳性血清所识别。结论日本血吸虫组织蛋白酶B肽链内切酶可在原核表达载体pWR450-1中表达,为研究其功能打下了必要的基础。更多还原

【Abstract】 Objective To discuss the construction of recombinant prokaryotic expression plasmid Sjcb2/pWR450-1 and expression cathepsin B endopeptidase of Schistosoma japonicum in E.coli DH5α. Methods The gene of cathepsin B endopeptidase of Schistosoma japonicum(Sjcb2) was ampified from the recombinant plasmid pBC SK~+/Sjcb2 by PCR,and cloned into cloning vector pUCm-T,cutting the objective domain by enzymes,then cloned into expression vector pWR450-1 to form the recombinant plasmid.The expression of the recombinant plasmid by SDS-PAGE and Western blotting was analyzed. Results The specific fragment of Sjcb2 was amplified and the recombinant prokaryotic expression plasmid Sjcb2/pWR450-1 was successfully constructed.After being induced by IPTG,the transformed E.coli DH5α expressed an about 86 kDa fusion protein.The results of Western blotting analysis showed that the fusion protein could be recognizd by sera of rabbits with Schistosomiasis japonica. Conclusion The recombinant prokaryotic expression plasmid Sjcb2/pWR450-1 was constructed and the Sjcb2 gene was successfully expressed in E.coli DH5α,which paved the way for further study on the function of Sjcb2.更多还原