单纯疱疹病毒I型DNA疫苗的构建

摘要

Objective To construct a recombinant plasmid DNA containing herpes simplex virus type 1(HSV-1) glycoprotein D (gD) gene.Methods The HSV-1 gD gene was obtained by polymerase chain reaction (PCR) and inserted into TA cloning vector pGEM-T, then cloned into the eukaryotic expression vector pcDNA3.1 to generate pLy-D. The recombinant plasmid pLy-D, which was confirmed by partial sequencing and restriction endonuclease analysis, was transfected into Cos-7 cells and used to inoculate ICR mice via muscular injection. Immunohistochemistry and enzyme-linked immunoabsorbent assay (ELISA) were employed to test the gD expression in transfected cells and the specific anti-HSV-1 antibody in the serum of immunized mice, respectively.Results The gD eukaryotic expression plasmid pLy-D was constructed. Using the immunohistochemistry technique, the gD expression in pLy-D-transfected cells was detected. The ELISA demonstrated that specific anti-HSV-1 antibody could be induced in immunized mice after three times injection.Conclusions We constructed HSV-1 gD eukaryotic expression plasmid pLy-D which could express gD protein in transfected cells and could induce humoral immune response in mice. This observation will be helpful in designing HSV prophylactic vaccine.%目的构建表达单纯疱疹病毒1型糖蛋白D的DNA疫苗.方法采用聚合酶链式反应(PCR),从HSV-1基因组中扩增出gD基因,插入中间载体pGEM-T,然后克隆入真核表达载体pcDNA3.1, 构建重组质粒pLy-D.经部分测序和限制性内切酶分析证实.将pLy-D转染Cos-7细胞,并肌肉接种ICR小鼠.用免疫组化和ELISA方法分别检测gD蛋白表达及小鼠血清中的特异性抗体.结果转染的Cos-7细胞中有gD蛋白的表达;经三次肌肉免疫接种的小鼠血清中有特异性HSV-1抗体产生.结论本研究构建的重组质粒pLy-D能够在哺乳动物细胞中表达目的蛋白,免疫接种后可以诱导动物产生免疫反应.