霍乱肠毒素基因DNA疫苗的构建和体外表达

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。

Construction and expression of DNA vaccine of gene of Vibrio cholerae

Objective To construct recombinant plasmid pcDNA3.1ctxAB 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.