甲型流感病毒M2e与CtB基因融合表达及其免疫特性初步研究

Expression and immunity of fused protein H1N1 M2e and cholera toxin B

目的:构建真核表达重组质粒pcDNA 3.1a(+)-M2e/CtB,并初步研究其在NIH3T3细胞中的表达特性及免疫特性。方法:重叠PCR法扩增甲型流感病毒M2e基因和霍乱毒素CtB基因,将扩增得到的融合基因片段M2e-CtB定向克隆入真核表达载体pcDNA 3.1a(+)中,再转化E.coli JM109。将酶切鉴定、PCR扩增及序列鉴定正确的重组质粒命名为pcDNA3.1a(+)-M2e/CtB。用KEGEN TRANSⅢ阳离子聚合物将重组质粒pcDNA 3.1a(+)-M2e/CtB转染NIH3T3细胞,经免疫荧光、RT-PCR产物序列分析、Western blot检测其稳定表达产物。结果:重组质粒pcDNA 3.1a(+)-M2e/CtB含完整的M2e和CtB基因,与相对应基因的序列同源性分别为100%。重组质粒转染NIH3T3细胞后获得了有效表达,并且稳筛株细胞裂解物和上清均能用抗霍乱毒素抗体和抗流感病毒抗体检测到Mr约18000的蛋白条带。结论:成功构建了真核表达重组质粒pcDNA 3.1a(+)-M2e/CtB,初步证明其在体外表达的重组蛋白可分泌至胞外,且有M2e和CTB的双特异反应原性和免疫原性,为流感病毒核酸疫苗的进一步研究奠定了坚实基础。

AIM:To construct the eukaryotic recombinant plasmid pcDNA3.1a(+)-M2e/CtB which contains H1N1 M2e gene and cholera toxin B subunit gene(CtB)and to study the expression and immunity of recombinant protein M2e/CTB in NIH3T3 cells.METHODS:M2e/CtB gene was cloned by PCR and digested with Hind Ⅲ and Xho Ⅰ.M2e/CtB was linked into pcDNA3.1a(+)to build eukaryotic expression plasmid pcDNA3.1a(+)-M2e/CtB.The pcDNA3.1a(+)-M2e/CtB was transfered into competent E.coli JM109.The transformed colonies were verified by Hind ...