【摘要】 目的克隆日本血吸虫组织蛋白酶B基因,构建真核表达重组质粒,转染真核细胞,观察其表达情况。方法用PCR技术从已构建好的重组质粒pBC SK+/Sjcb2中扩增出Sjcb2基因片段,重组入克隆载体pUCm-T。再将Sjcb2基因片段亚克隆入真核表达载体pcDNA3.1(+)。进行PCR、双酶切和DNA序列鉴定后,运用电穿孔技术将重组体pcDNA3.1(+)/Sjcb2转染HeLa细胞,间接免疫荧光技术观察目的基因的表达。结果成功扩增出Sjcb2基因片段并构建DNA疫苗;重组质粒在HeLa细胞中获得表达。结论Sjcb2基因能够在体外真核细胞中表达。更多还原

【Abstract】 Objective To clone and construct the recombinant plasmid containing cathepsin B endopeptidase of Schistosoma japonicum(Sjcb2) and transfer it into mammalian cells to express Cathepsin B endopeptidase protein. Methods By polymerase chain reaction(PCR) tenique,Sjcb2 was amplified from the constructed recombinant plasmid pBC SK+/Sjcb2.Followly Sjcb2 was inserted into cloning vector pUCm-T.Then Sjcb2 was subcloned to the eukaryotic expression vector pcDNA3.1(+) by linking reactions.After identifying it by PCR,restrictive enzymes digestion and DNA sequencing,the recombinant plamid was transfected into HeLa cells using electroporation,and analyze the expression of the recombinant protein by indirect immunofluorescence assay. Results The specific gene fragment about 1047bp was successfully amplified.The DNA vaccine of Sjcb2 was successfully constructed.Indirect immunofluorescence assay showed that Sjcb2 was expressed in HeLa cells and located in cytoplasm. Conclusion Sjcb2 gene can be expressed in eukaryotic system,which lay the foundation for studying the pathogenic mechanism and the development of the Sjcb2 DNA vaccine against Schitosomiasis.更多还原