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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。
Objective: To construct eukaryotic expression plasmid pcDNA3.1a (+) - M2e / CtB and study its expression and immunological properties in NIH3T3 cells. Methods: M2e gene and cholera toxin CtB gene were amplified by overlap PCR. M2e-CtB gene fragment was cloned into eukaryotic expression vector pcDNA 3.1a (+) and transformed into E.coli JM109. Restriction enzyme digestion, PCR amplification and sequence identification of the correct recombinant plasmid named pcDNA3.1a (+) - M2e / CtB. The recombinant plasmid pcDNA3.1a (+) - M2e / CtB was transfected into NIH3T3 cells with KEGEN TRANS III cationic polymer. The products of stable expression were detected by immunofluorescence, RT-PCR product sequence analysis and Western blot. Results: The recombinant plasmid pcDNA3.1a (+) - M2e / CtB contained the complete M2e and CtB genes with 100% homology with the corresponding genes respectively. The recombinant plasmids were transfected into NIH3T3 cells, and the expression of the recombinant plasmids was validated. The protein bands of Mr about 18000 were detected by anti-cholera toxin and anti-influenza virus antibodies in the cell lysates and supernatant of the stable cell lines. CONCLUSION: The eukaryotic recombinant plasmid pcDNA3.1a (+) - M2e / CtB has been successfully constructed. The recombinant protein can be secreted extracellularly and has the biologic specificity of M2e and CTB and immunogen Which laid a solid foundation for the further study of influenza virus nucleic acid vaccine.