引入CpG基序的汉滩病毒G2糖蛋白基因的克隆及表达

构建汉滩病毒G2糖蛋白的真核表达载体,并加入可增强小鼠免疫刺激作用的CpG基序,检测其可否在真核细胞中表达。参照Genebank中汉滩病毒M的全基因序列设计引物,引物两端引入可增强小鼠免疫刺激作用的CpG基序及双酶切位点,通过聚合酶链反应(PCR)获得含CpG基序的G2片段,并将其与T载体pMD18-T相连,测序后克隆至真核表达载体pcDNA3.1 上,将此真核表达载体以脂质体法转染至真核细胞Vero-E6中,利用间接免疫荧光法(IFA)检测发现转染后的Vero-E6中出现特异性的绿色荧光。结果表明本实验成功构建了汉滩病毒包膜糖蛋白G2的重组体。

Clone and Expression of Glycoprotein Gene G2 Added with Hantaan Virus' CpG Motifs

The eukaryotic expressing vector of glycoprotein was constructed and added with CpG motifs from Hantaan virus that could strengthen mouse immune stimulation,in order to detect whether or not it could express in the eukaryotic cell.A primer was designed referring to M holo-gene sequence of Hantaan virus in the GenBank,CpG motifs that could strengthen mouse immune stimulation and double enzyme cut sites were added at the both ends of the primer.Then G2 fragment that contains CpG motifs was obtained through PCR,and ligated it to T-vector of pMD18-T, and then cloned into eukaryotic expressing vector pcDNA3.1 after sequencing.And transfected the eukaryotic expressing vector into eukaryotic cells Vero-E6 with liposome method.It was found that specific green fluorescence appeared in the Vero-E6 after the transfection using indirect immunofluorescence assay(IFA) detection.The results showed that this experiment successfully constructed Hantaan virus en-membraned glucoprotein G2 recombinant.