乙肝基因疫苗系列质粒的构建及其在真核细胞中的表达

目的 构建一系列乙肝病毒表面抗原大、中、小蛋白的真核细胞表达质粒，并研究其在真核细胞中的表达情况。方法 采用常规PCR法扩增S、前S2－S、前S1－前S2－S片断，利用重叠PCR法扩增出前S1－S片断后，分别插入Rc/CMV及pSG5UTPL/Flag载体质粒中，并在其ATG起始密码前加入KOZAK序列后转染SP2／0细胞，Western-Blot杂交检测所表达蛋白，并用核酸自动分析仪分析所插入片断的核酸序列。结果 插入片断的核苷酸序列与相应的大、中、小蛋白和前S1－S蛋白的编码基因一致，Western-Blot杂交检测出了相应的目的蛋白。结论 获得了8个能高效表达乙肝病毒表面抗原大、中、小蛋白和前S1－S蛋白的真核细胞表达质粒。

Construction of recombinant plasmids containing genes of HBsAg and their expression in the eukaryotic cells]

OBJECTIVE: To construct recombinant plasmids expressing L, M, S and pre-S1-S protein of HBsAg. METHODS: Amplifying segments of S, pre-S2-S, pre-S1-pre-S2-S genes of HBV by PCR and amplifying segment of pre-S1-S by overlap extension PCR; inserting the segments into Rc/CMV and pSG5UTPL/Flag plasmids respectively and exploring their expressions by Western-Blot hybridization, identifying the inserting segments by sequencing. RESULTS: The sequences of the inserted segments were the same as the genes of S, pre-S2-S, pre-S1-pre-S2-S and pre-S1-S and the results of Western-Blot hybridization were positive for the aimed proteins. CONCLUSION: We have gained 8 recombinant plasmids expressing S, M, L and pre-S1-S proteins with high efficacy.