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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
Objective To construct a DNA vaccine expressing herpes simplex virus type 1 glycoprotein D. Methods The gD gene was amplified from HSV 1 by polymerase chain reaction (PCR) and inserted into the intermediate vector pGEM T, then cloned into the eukaryotic expression vector pcDNA3. 1 to construct the recombinant plasmid pLy D. The partial sequencing and restriction Endonuclease analysis confirmed. PLy D was transfected into Cos 7 cells and ICR mice were intramuscularly inoculated. Immunohistochemistry and ELISA were used to detect the expression of gD protein and the specific antibodies in the serum of mice. Results The expression of gD protein was found in the transfected Cos 7 cells. The specific HSV 1 antibody was produced in the sera of mice immunized with the three muscular immunizations. Conclusion The recombinant plasmid pLy D constructed in this study can express the target protein in mammalian cells and induce immune response after immunization