乙肝基因疫苗系列质粒的构建及其在真核细胞中的表达

Construction of Recombinant Plasmids Containing Genes of HBsAg and Their Expression in the Eukaryotic Cells

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 p SG5 U TPL/ 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。

Objective To construct recombinant plasmids expressing L,M,S and pre S 1 S protein of HBsAg. Methods Amplifying segments of S, pre S 2 S, pre S 1 pre S 2 S genes of HBV by PCR and amplifying segment of pre S 1 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 S 2 S, pre S 1 pre S 2 S and pre S 1 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 S 1 S proteins with high efficacy.