霍乱肠毒素ctxA基因真核表达重组质粒的构建及表达  

Construction and expression of the eukaryotic recombinant plasmid of ctxA gene of Vibrio cholerae

作　　者：张莉[1] 张雷[1] 陈建平[2] 王涛[2] 

机构地区：[1]大理学院基础医学院病原生物学综合实验室,云南大理671000 [2]四川大学基础医学与法医学院寄生虫学教研室,四川成都610041

出　　处：《中国病原生物学杂志》2006年第5期324-326,F0003,共4页Journal of Pathogen Biology

基　　金：国家自然科学基金资助项目(No.30300302)

摘　　要：目的构建霍乱肠毒素A亚单位基因(ctxA)真核表达重组质粒,并在NIH3T3细胞中进行表达。方法用限制性核酸内切酶从重组质粒pET32a-ctxA上切下ctxA基因,导入真核表达载体pcDNA3.1(+),重组子pcDNA3.1-ctxA经限制性酶切分析、PCR鉴定和序列测定正确后,用脂质体法将重组质粒pcDNA3.1-ctxA转染NIH3T3细胞,采用免疫荧光法和Western blot对pcDNA3.1-ctxA的瞬时表达产物和稳定表达产物进行鉴定。结果重组质粒pcD-NA3.1-ctxA成功转入NIH3T3细胞,利用免疫荧光技术在NIH3T3细胞膜和细胞浆中检测到了瞬时表达,用Westernblot检测到转染的阳性细胞克隆稳定表达出约29 ku的蛋白。结论成功构建霍乱肠毒素A亚单位基因真核表达重组质粒pcDNA3.1-ctxA,并在NIH3T3细胞中表达出29 ku的CTA蛋白。Objective To construct recombinant plasmid pcDNA3, 1-ctxA and detect the expression of pcDNA3, 1-ctxA in NIH3T3 cell, Methods The ctxA gene was obtained from pET32a-ctxA by restriction endonuclease digestion and was subcloned into eukaryotic expressed vector pcDNA3.1 （＋）. The recombinant plasmid, which was named pcDNA3, 1- ctxA, was identified by restriction analysis, PCR and DNA sequencing analysis, NIH3T3 cell was transfected by recombinant plasmid pcDNA3.1-ctxA with Lipofection strategy. Transient and stable products of ctxA gene was detected by immunofluorescence and Western blot. Results The recombinant plasmid pcDNA3. 1-ctxA was detected its expression in NIH3T3 cell membrane and cytoplasm with immunofluorescence. There was the 29 ku protein in stable transfection NIH3T3 cells by Western blot analysis. Conclusion The recombinant plasmid pcDNA3.1-ctxA was successfully constructed and 29 ku CTA protein is expressed in NIH3T3 cells.

关 键 词：霍乱肠毒素 ctxA基因 转染 体外表达 

分 类 号：R378.3[医药卫生—病原生物学]