霍乱肠毒素基因DNA疫苗的构建和体外表达  

Construction and expression of DNA vaccine of gene of Vibrio cholerae

作　　者：张莉[1] 张雷[1] 陈建平[2] 王涛[2] 杨志伟[2] 李金福[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]四川大学华西医学中心基础医学与法医学院寄生虫学教研室,四川成都610041

出　　处：《西部医学》2007年第5期775-777,780,共4页Medical Journal of West China

基　　金：国家自然科学基金资助(NO:30300302)

摘　　要：目的构建霍乱弧菌ctxAB融合基因分子佐剂DNA疫苗,并在NIH3T3细胞中进行表达。方法用限制性核酸内切酶从原核表达重组质粒pET32a-ctxAB上切下ctxAB基因,导入真核表达载体pcDNA3.1(+),重组子经限制性酶切分析、PCR鉴定正确后,命名为pcDNA3.1-ctxAB。用脂质体法将重组质粒pcDNA3.1-ctxAB转染NIH3T3细胞,采用免疫荧光法和Western blot对pcDNA3.1-ctxAB的瞬时表达产物和稳定表达产物进行鉴定。结果真核表达重组质粒pcDNA3.1-ctxAB成功转入NIH3T3细胞,利用免疫荧光技术检测到其在细胞膜和细胞浆中获得瞬时表达,对稳定转染细胞用Western blot分析,发现在约40.5kDa处有阳性杂交信号。结论成功构建霍乱弧菌ctxAB基因分子佐剂DNA疫苗,并在NIH3T3细胞中获得表达。Objective To construct recombinant plasmid pcDNA3.1-ctxAB and detect its expression in NIH3T3 cells. Methods The ctxAB gene was obtained from pET32a-ctxAB by restriction endonuclease digestion and was subcloned into eukaryotic expressed vector pcDNA3. 1（＋）. The recombinant plasmid, which was named pcDNA3. 1- ctxAB, was identified by restriction analysis, PCR and DNA sequencing analysis. NIH3T3 cell was transfected by recombinant plasmid pcDNA3. 1-ctxAB with lipofection strategy. Transient and stable products of ctxAB gene were detected by immunofluorescence and Western blot. Results The recombinant plasmid pcDNA3. 1-ctxAB was transfected into NIH3T3 cell successfully, and its transient product was detected in membrane and cytoplasm of NIH3T3 cell with immunofluorescence, There was 40. 5 kDa protein in stable transfection NIH3T3 cells by Western blot analysis. Conclusion Molecular adjuvant DNA vaccine of ctxAB gene has been constructed and realized its expression in NIH3T3 cell successfully.

关 键 词：霍乱肠毒素 DNA疫苗 

分 类 号：R387[医药卫生—医学寄生虫学]