霍乱弧菌ctxB基因真核重组质粒的构建及表达

Construction of Eukaryotic Recombinant Plasmid of ctxB Gene of Vibrio cholerae and Detection of Its Expression in NIH3T3 Cell

目的构建霍乱孤菌ctxB基因真核表达重组质粒,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-ctxB上切下ctxB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-ctxB。用脂质体法将重组质粒pcDNA3·1-ctxB转染NIH3T3细胞,采用免疫荧光法对pcDNA3·1-ctxB的瞬时表达产物进行鉴定。结果约380bp的ctxB被克隆到pcDNA3·1(+)真核表达载体中,经测序无误后,用阳离子脂质体转染的方法,检测到重组质粒pcDNA3·1-ctxB在NIH3T3细胞的胞浆和胞膜上得到了表达。结论ctxB真核表达的成功构建及表达为进一步从分子水平研究霍乱肠毒素B亚单位的免疫原性及其作为佐剂的应用价值提供研究基础。

Objective To construct recombinant plasmid pcDNA3.1-ctxB and to detect its expression in NIH3T3 cell. Method The ctxB gene was obtained from pET32a-ctxB by restriction endonuclease digestion and subcloned into eukaryotic expressed vector pcDNA3. 1 （ ＋ ）. The recombinant plasmid named pcDNA3.1-ctxB was identified by restriction analysis,PCR and DNA sequencing analysis. NIH3T3 cell was transfected by recombinant plasmid pcDNA3. 1-ctxB with Lipofection strategy. Transient products of ctxB gene was detected by immunofluorescence. Result A 380bp ctxB gene was subcloned into eukaryotic expressed vector pcDNA3.1 （ ＋ ） and after identified by sequence analysis, the recombinam plasmid pcDNA3.1-ctxB was expressed in cytoplasm and cytomembrane of NIH3T3 cell with Lipofection method. Conclusion The recombinant plasmid pcDNA3.1-ctxB was constructed and realized expression of ctxB gene in NIH3T3 cell successfully, which will provide the basis for future research on the immunogenicity and adjuvanficity from molecular level.