日本血吸虫tetraspanins胞外环2编码基因的克隆和表达  

Cloning and Expression of Gene Encoding Extracellular Loop 2 of Tetraspanins of Schistosoma japonicum

作　　者：付译节[1] 杨莉[1] 刘康[1] 袁创[1] 陈县城[1] 

机构地区：[1]四川大学华西医院肿瘤生物治疗实验室,四川成都610041

出　　处：《寄生虫病与感染性疾病》2008年第1期5-8,共4页Parasitoses and Infectious Diseases

摘　　要：目的克隆和表达日本血吸虫(中国大陆株)tetraspanins胞外环2(EC-2)编码基因(Sj-tsp-2)。方法在日本血吸虫基因组中筛选获得与曼氏血吸虫tetraspanins基因胞外环2结构域序列同源的片段,经过一定修饰,采用全基因合成方式获得目的基因片段,构建重组质粒pET-32a-Sj-tsp-2,并转化至BL21(DE3)感受态细胞。经IPTG诱导表达,在非变性条件下亲和纯化融合蛋白。结果通过核苷酸测序证实重组表达质粒构建成功。SDS-PAGE结果表明,融合蛋白和目的肽段与预计大小基本一致。Western blotting结果说明,该融合蛋白具有良好的免疫原性。结论本实验成功表达了日本血吸虫Sj-tsp-2基因,并纯化获得其融合蛋白,为进行动物保护性实验奠定了基础。Objective To clone and express the gene encoding extracellular loop 2 （EC-2） of tetraspanins （TSPs） of Schistosoma japonicum （ Chinese strain）. Method An unknown ortholog of S. mansoni TSP-2 was screened from the ESTs of S. japonicum,and the corresponding gene of which was finally called Sj-tsp-2 after modifications. The interest gene synthesized was then directionally cloned into prokaryotic expression vector pET-32a. The recombinant plasmid pET-32a-Sj-tsp-2 was transformed into competent E. coli BL21 （DE3） and expressed in the presence of IPTG . Fusion protein was purified from E. coli lysates under nondenaturing conditions. Result DNA of recombinant plasmid pET- 32a-Sj-tsp-2 was sequenced in its own right. SDS-PAGE analysis showed that the expressed fusion protein was around 26 kDa. More importantly, the results of Western blotting revealed that the interest protein Sj-tsp-2 could be specifically recognized by sera from mice vaccinated with recombinant protein Trx-Sj-tsp-2 . Conclusion The successful expression and purification of recombinant protein Trx-Sj-tsp-2 make it possible for further research on animal protection experiments.

关 键 词：日本血吸虫 TETRASPANINS 克隆 表达 融合蛋白 

分 类 号：R352.1[医药卫生—基础医学]