尼帕病毒受体结合糖蛋白在哺乳动物细胞中的可溶性表达和纯化及小鼠抗血清制备

Soluble expression and purification of Nipah virus receptor-binding G glyprotein in mammalian cells and preparation of mouse antiserum

参照GenBank中尼帕病毒受体结合蛋白(G蛋白)编码序列,使用DNAStar软件对各基因型尼帕病毒G蛋白进行核苷酸、氨基酸同源性分析,并绘制进化树,选择代表性毒株(NCBI登录号:AF212302.2),截取合成G蛋白头部结构域(sG)的核苷酸序列,连接至pcDNA3.1(+)真核表达载体上,构建为重组质粒pcDNA-sG,将测序正确的重组质粒在Expi293F细胞中进行表达,利用蛋白免疫印迹(Western blot)进行鉴定。通过Strep-Tactin XT亲和层析纯化目的蛋白,薄层扫描分析蛋白纯度。用获得的sG免疫小鼠制备抗sG蛋白的多克隆抗体血清,并通过间接ELISA鉴定结合活性。结果显示,尼帕病毒G蛋白在Expi293F细胞中获得可溶性表达,纯化得到的目的蛋白大小正确,纯度大于99%;制备的抗血清能特异性结合目的蛋白。

The homology analysis of nucleotide and amino acid of each genotype Nipah virus G protein was performed using DNAStar software with reference to the coding sequence of Nipah virus receptor binding G glyprotein in GenBank,the evolutionary tree was drawn,and representative strains were selected.The nucleotide sequence of the soluble G protein head domain(sG)was selected and synthesized,the sequence was ligated to pcDNA3.1(+)eukaryotic expression vector,yielding pcDNA-sG.sG protein was expressed in mammalian cells and was purified by Strep-Tactin XT column affinity chromatography.Western blot was used to verify the expression of foreign genes in cells.The mice was immunized with sG protein to prepare polyclonal antibody serum against sG protein,and the binding activity was identified by indirect ELISA.The results showed that Nipah virus G protein was soluble in mammalian cells,and the purified protein was correct in size and the prepared antiserum could specifically bind to the target protein.The above research provided technical support for the evaluation of Nipah virus vaccine.