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甲型流感病毒M2蛋白是一种具有离子通道功能的跨膜蛋白,其氨基酸序列非常保守,可用于流感通用疫苗的研究。为了构建可调控的稳定表达甲型流感病毒M2蛋白的哺乳动物细胞系,首先应用PCR方法从含有流感病毒PR8株第七节段全长基因的质粒中扩增得到M2基因。将该片段亚克隆到真核表达载体pcDNA5/FRT/TO上,用BamHⅠ和NotⅠ双酶切鉴定正确后将重组质粒与表达Flp重组酶的pOG44质粒共转染Flp-In T-REx-293细胞,使目的基因整合到宿主细胞染色体。筛选具有Hygromycin B抗性的细胞株。在该细胞的培养基中加入四环素以诱导目的基因表达,48 h后通过间接免疫荧光方法检测到M2蛋白的表达。共得到16株高表达M2蛋白的重组细胞株,这些细胞株在传10代后仍能稳定表达目的蛋白。未加四环素诱导的细胞没有检测到M2蛋白,说明四环素调控系统严格控制着目的基因的表达。今后,该细胞系可用于流感病毒M2蛋白的功能研究、流感候选疫苗的免疫学评价以及流感病毒减毒活疫苗的研制。
Influenza A virus M2 protein is a transmembrane protein with ion channel function. Its amino acid sequence is very conservative and can be used in the research of influenza universal vaccine. In order to construct a mammalian cell line that can regulate the stable expression of influenza A virus M2 protein, the M2 gene was amplified from the plasmid containing the full-length gene of the seventh segment of influenza virus PR8 strain by PCR. The fragment was subcloned into the eukaryotic expression vector pcDNA5 / FRT / TO, and the recombinant plasmids were co-transfected into pPFP44 plasmid expressing Flp recombinase into Flp-In T-REx-293 cells , So that the purpose of integration into the host cell chromosome genes. Screen cell lines with Hygromycin B resistance. Tetracycline was added to the cell culture medium to induce the expression of the target gene, and the expression of M2 protein was detected by indirect immunofluorescence 48 h later. A total of 16 highly expressed M2 protein recombinant cell lines, these cell lines in the passage of 10 generations after the stable expression of the target protein. No tetracycline-induced cells did not detect the M2 protein, indicating that the tetracycline regulatory system strictly controls the expression of the target gene. In the future, this cell line can be used for functional studies of the M2 protein of influenza virus, immunological evaluation of influenza candidate vaccines, and development of a live attenuated influenza vaccine.