含preS2免疫表位的HBsAg基因质粒的构建及表达

Plasmid construction and expression of Hepatitis B surface antigen gene containing the preS2 immune epitope

目的 :应用表位设计构建高效表达乙型肝炎表面抗原含preS2免疫表位基因的真核表达质粒。方法 :PCR法从慢性乙型肝炎患者血清中扩增和分离preS2 12 0 - 146表位和S基因片段 ,将两者融合并置于载体pcDNA3 1的巨细胞病 (CMV)启动子作用之下 ,构建真核表达质粒pcDNA -S2S。序列分析和脂质体转染COS - 7细胞瞬时表达鉴定。结果 :序列测定结果表明插入序列与乙型肝炎病毒中国株全基因参照序列 (adr亚型 )一致。COS - 7细胞瞬时表达鉴定实验中 ,ELISA检测到preS2 -Ag和HBsAg的OD450 分别为 0 4 6 9和 0 4 2 6。结论 :应用表位设计成功地构建了含preS2免疫表位基因的重组质粒pcDNA -S2S所构建质粒能高效表达和分泌目的的蛋白。

Objective: To construct the plasmid of high effect expression the Hepatitis B surface middle protein applying for epitope designing.Methods: Isolated the preS2 120-146 epitope and S gene from the serum of chronic Hepatitis B patient by PCR.S gene fused with the preS2 epitope were placed downstream of CMV promoter of the pcDNA3.1 vector,we succeeded in constructing the eukaryotic expression plsmid pcDNA-S2S.The plasmid was identified by sequence analysis and transient transfection into COS-7 cell line by lipoid mediation.Results: Sequence analysis suggested that the inserted gene belongs to adr subtype.ELISA detected the transient expression of the preS2-Ag and HBsAg in the media of COS-7 cells tyranfected with plsmid pcDNA-S2S,the OD 450 values were 0.469 and 0.426 respectively.Conclusion:The plasmid pcDNA-S2S which can express and secret the target protein high effectively was constructed.