日本血吸虫32kDa蛋白质基因在真核表达载体中的亚克隆

设计和合成特定寡核苷酸引物,TRIZOL提取日本血吸虫成虫RNA,RT-PCR法扩增日本血吸虫32kDa蛋白质(Sj32)基因编码序列,将扩增产物连接pGEM-T克隆载体,再亚克隆到真核表达载体pBKCMV中.结果RT-PCR法特异性扩增出Sj32编码基因片段,其大小约为1270bp,经双酶切、PCR鉴定表明所构建的质粒pGEM-Sj32和pBK-Sj32中含有目的基因.pBK-Sj32重组质粒的成功构建,为进一步表达Sj32及其在血吸虫病免疫诊断、免疫预防中的作用研究提供了条件.

Subcloning of 32kDa proteins gene of Schistosoma japonicum in the eukaryocyte expression vector

Objective: 32kDa proteins gene of Schistosoma japonicum(Sj32) was subcloed into eukaryocyte expression vector pBK CMV,in order to its study as nucleic acids vaccine and diagnostic antigens.Methods: The design and synthesis of specific oligonucleotide primers.Toal RNA was isolated from adult worms of S.japonicum using TRIZOL reagent.Coding region gene of Sj32 was amplified by RT-PCR technique.The products from PCR were firstly cloned into pGEM-T vector and subcloned into eukaryocyte expression vector pBK CMV via BamH1 and Xba1 sites.The result was determined by restriction analysis and PCR methods.Results: The coding region gene of Sj32 was specifically amplified by PCR,the size of fragment was 1270 base pairs.The cloning plasmid constructed(pGEM-Sj32)and expression plasmid(pBK-Sj32)contained the amplified fragment.Conclusion: The results demonstrated that was pBK-Sj32 successfully constructed and provided the basis for further study on sj32 expression and its immunological diagnosis and prevention.