丙肝病毒(HCV)及乙肝病毒(HBV)双表达载体的构建及表达

Constructs and expression of hepatitis C virus core gene combined hepatitis B virus core gene with two multiple cloning sites vector

目的 :为探索HCV/HBV高效联合基因免疫策略 ,构建具有 2套独立表达单元的HCV/HBV真核表达载体。方法 :分别将与HCV核心区基因互补的cDNA和HBV核心区基因克隆于具有 2套独立表达单元的真核表达载体pRSC的巨细胞病毒启动子和RSV启动子下游 ,称为pRSC HBV/HCV ,转染SP2 / 0细胞 ,通过免疫荧光和Western印迹法观测蛋白的表达 ,免疫Balb/c小鼠 ,用酶联免疫小鼠体液免疫应答。结果 :pRSC HBV/HCV转染SP2 / 0细胞可见HBcAg及HCV核蛋白染色阳性荧光细胞 ,SDS Page电泳显示在 14kD及 2 1kD处均可见蛋白条带 ,与HBcAg及HCV核蛋白的的理论预期值一致 ,Western印迹分析显示在 14kD及 2 1kD处可见特异性的蛋白条带。 5只免疫鼠中全部出现抗 HCV及抗 HBV抗体 ,而对照组全阴性。结论 :pRSC HBV/HCV可分别表达HBcAg及HCV核蛋白 ,免疫Balb/c小鼠后可诱导其体液免疫应答 ,为进一步开展HCV/HBV联合基因免疫奠定了实验基础。

Objective:To develop a HCV combined HBV DNA-based therapeutic vaccine.Methods:The HBV core gene and HCV core cDNA were inserted into the eukaryotic expression vector with two multiple cloning sites mammalian expression vector under the CMV promoter and RSC promoter respectively, named pRSC-HBV/HCV. Cellular expression of pRSC-HBV/HCV was assessed by Western blot and immunofluorescent study following transfection into SP2/0 cells. The Balb/c mice were immunized by multiple sites intramuscular injection with pRSC-HBV/HCV and the immune responses were detected.Results:The 21 kD and 14 kD core protein were observed. Both anti-HBc Ab and anti-HCV core Ab were detected in all immunized mice.Conclusion:The investigation demonstrated that pRSC-HBV/HCV could express HBcAg and HCV core protein, humoral immune response could be detected in mice immunized with pRSC-HBV/HCV.