甲型流感病毒M2基因真核表达载体的构建及序列分析

Construction and sequence analysis of the eukaryotic expression vector for M2 gene from influenza A virus

目的构建含有甲型流感病毒M2基因的真核表达载体并分析插入的M2基因序列。方法从接种人流感病毒株A/PR/8/34(H1N1)的鸡胚尿囊液中提取病毒RNA,用特异引物进行RT-PCR,扩增M2基因。通过分子克隆技术将所扩增片段克隆入真核表达质粒载体pcDNA3.1(+)。经双酶切、PCR鉴定后挑选阳性克隆测序鉴定并对插入的M2基因序列进行分析。结果经双酶切、PCR及测序鉴定证实M2基因的真核表达载体构建成功。序列分析有4个氨基酸出现变异,提交Genbank进行Blast比对显示同源性97%(Genbank/NCBI AY768951)。结论M2四聚体蛋白具有H+通道功能,能够协助病毒与宿主细胞膜融合后释放RNP复合体,还能稳定HA蛋白的结构。M2蛋白的高度保守性和具有交叉保护能力特性使之成为新型的通用流感疫苗的突破口。该结果将为甲型流感病毒基因工程疫苗,通用疫苗和核酸疫苗的研究打下基础。

To construct the eukaryotic expression vector for M2 gene from influenza A virus and to analyze its sequence, the viral RNA was extracted from allantoic fluid of chicken embryos infected with influenza virus A/PR8/34（HIN1）, and M2 gene was amplified by RT-PCR with specific primer, The amplified gene fragment was then cloned into the eukaryotic expression vector pcD-NA3.1（＋）, the selected positive clones were subjected to sequence analysis of the inserted M2 gene after double enzymes digestion and PCR identification. It was found that the construction of the eukaryotic expression vector for M2 gene was successful after restrictive enzyme and PCR identification and sequence analysis. Sequence analysis demonstrated 4 amino acid variations and 97 96 of homology with influenza virus A/PR8/34（ GerLBank/NCBI AY768951 ）. The resultsof the present investigation could provide a foundation for the further study on the development of DNA vaccine and broad-spectrum vaccine for influenza A virus infection.