稳定表达流感病毒基质蛋白2的哺乳动物细胞系的建立

Establishment of Mammalian Cell Lines for Constitutive Expression of Influenza Virus Matrix Protein 2

本研究构建了稳定表达甲型流感病毒基质蛋白2(M2)的哺乳动物细胞系。应用PCR方法扩增A/PR/8/34(H1N1)株流感病毒M2基因,将其克隆至真核表达载体pcDNA5/FRT(pDF)上,构建出pDF-M2重组质粒。将鉴定正确的pDF-M2与表达Flp重组酶的pOG44质粒共转染Flp-In-CHO细胞,通过体内同源重组使目的基因整合到宿主细胞染色体上。筛选具有Hygromycin B抗性的重组细胞株命名为CHO-M2。以间接免疫荧光法(IFA)和Western blot法检测M2的表达,共获得15株高表达M2蛋白的重组细胞株。这些重组细胞株在连续培养10代后,PCR方法仍可检测到M2基因的存在,IFA也检测到蛋白的稳定表达。本研究成功获得了稳定表达甲型流感病毒M2的哺乳动物细胞系,为M2蛋白的功能研究和非复制型流感病毒疫苗的研制提供了新的工具。

To establish a mammalian celI line for stable expression of the matrix protein 2 （M2） of influenza virus type A. M2 gene was amplified by PCR from the influenza virus strain A/PR/8/34. The PCR prod- uct was cloned into eukaryotic expression vector pcDNAS/FRT. After identification with restriction enzyme digestion, the plasmid was co-transfected with plasmid pOG44 which expressed Flp in Flp-In-CHO cells. The target gene was integrated into chromosome of CHO cells by homologous recombination in vivo. Recombinant CHO-M2 cell lines were selected for hygromycin B resistance. A total of 15 recombinant cell strains with high expression of M2 protein were screened by hygromycin, and the expression of M2 protein was determined by IFA and Western blot. After subculturing for l0 passages, the presence of M2 gene in the CHO-M2 cells was confirmed by PCR, and the expression of M2 protein were proved by IFA and Western blot. We successfully constructed a mammalian cell line which stably expressed M2 protein of in- fluenza virus type A. The cell line will be useful for studies on function of M2 protein and provide tools for novel influenza virus vaccine development.