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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 eukaryotic expression recombinant plasmid of cholera ctxB gene and express it in NIH3T3 cells. Methods The ctxB gene was cut from the recombinant plasmid pET32a-ctxB by restriction endonuclease and inserted into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid was confirmed by restriction enzyme digestion and named as pcDNA3. 1-ctxB. The recombinant plasmid pcDNA3.1-ctxB was transfected into NIH3T3 cells by lipofectamine. The transient expression product of pcDNA3.1-ctxB was identified by immunofluorescence. Results About 380bp ctxB was cloned into pcDNA3.1 (+) eukaryotic expression vector. After correct sequencing, the recombinant plasmid pcDNA3.1-ctxB was detected in the cytoplasm of NIH3T3 cells by cationic liposome transfection And the cell membrane was expressed. Conclusion The successful construction and expression of ctxB eukaryotic expression provide the basis for further research on the immunogenicity of cholera enterotoxin B subunit at the molecular level and its value as an adjuvant.