甲型流感病毒M2e及CtB基因融合表达及其免疫特性初步探究.docVIP

甲型流感病毒M2e及CtB基因融合表达及其免疫特性初步探究 　 作者：杨晓芳, 江滟, 李婉宜, 邝玉, 蒋忠华, 王丰平, 李明远 【摘要】 　　目的: 构建真核表达重组质粒pcDNA3.1a(+)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pcDNA3.1a(+)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 TRANS Ⅲ阳离子聚合物将重组质粒pcDNA3.1a(+)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pcDNA3.1a(+)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pcDNA3.1a(+)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。 【关键词】 甲型流感病毒 M2e 黏膜免疫佐剂 霍乱毒素B亚单位 融合蛋白 　　[Abstract] 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 Ⅲ and Xho Ⅰ, PCR, and sequencing. The NIH3T3 cells were transfected with pcDNA3.1a(+)M2e/CtB by positive ion polymer. The product of M2e/CtB gene with stable expression was analysed by immunofluorescence, RTPCR sequencing, and Western blot. RESULTS: The digested pcDNA3.1a (+)M2e/CtB contains correct M2e and CtB gene. Blastn results showed the genetic homongenicity of M2e and CtB were 100%, respectively. The pcDNA3.1a(+)M2e/CtB efficiently expressed M2e/CTB protein in the eukaryotic NIH3T3 cells. The cell lysis and supernant both contained the M2e/CTB protein, which had the immunity and reactivity to both antiM2e and antiCTB. The Mr of M2e/CTB protein was about 18 000 analysed by Western blot. CONCLUSION: A new recombinant eukaryotic plasmid pcDNA3.1a(+) M2e/CtB has been succ