摘要
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1 2 70bp ,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。
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.
出处
《生物学杂志》
CAS
CSCD
2001年第3期25-26,28,共3页
Journal of Biology
基金
安徽省自然科学基金 (编号 9741 0 0 4 2 )
安徽省教委科研基金 (编号 98JL0 62 )
安徽省卫生厅科研基金资助
