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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 an efficient HCV / HBV combination strategy for gene vaccination, an HCV / HBV eukaryotic expression vector with two independent expression units was constructed. METHODS: cDNA and HBV core region genes complementary to HCV core region genes were cloned downstream of the cytomegalovirus promoter and RSV promoter of two eukaryotic expression vectors pRSC with two independent expression units, respectively, and named as pRSC HBV / HCV. The SP2 / 0 cells were transfected, and the expression of the protein was detected by immunofluorescence and Western blotting. The Balb / c mice were immunized and the humoral immune response was also detected by ELISA. Results: The positive cells of HBcAg and HCV nucleoprotein staining were found in SP2 / 0 cells transfected with pRSC HBV / HCV. SDS PAGE electrophoresis showed that the protein bands were visible at both 14kD and 21kD, which was consistent with the expected value of HBcAg and HCV nucleoprotein Consistently, Western blot analysis revealed specific protein bands at 14 kD and 21 kD. All five immunized mice showed anti-HCV and anti-HBV antibodies, while the control group was all negative. CONCLUSION: HBV / HCV can express HBcAg and HCV nucleoprotein, respectively. Balb / c mice immunized with pRSC can induce their humoral immune response, which lays the foundation for further HCV / HBV combination gene immunization.