乙肝病毒S_2S蛋白表达质粒NV-HB/s_2s长期稳定转染细胞系(SP2/0-S_2S)的建立

Establishment of a SP2/0 Cell Line Transfected with Plasmid NV-HB/s_2s Stably Expressing HBV S_2S Protion

目的 :建立乙肝病毒S2 S蛋白表达质粒NV -HB/s2 s转染小鼠骨髓瘤细胞SP2 /0的长期稳定转染细胞系 ,为后期检测乙肝核酸疫苗的细胞免疫效果提供靶细胞。方法 :利用改良磷酸钙法将NV -HB/s2 s转染入SP2 /0细胞 ,用G418抗性筛选粗筛出 16株单克隆细胞株 ,分别用ELISA、Western -Blot、斑点杂交、细胞免疫组化、细胞原位杂交等方法检测目的抗原蛋白的表达及NV -HB/s2 s在细胞中是否存在及定位。结果 :获得一株NV -HB/s2 s长期稳定转染的单克隆细胞株SP2 /0 -S2 S ,在细胞中检出了乙肝病毒HBsAg、preS2 Ag的表达 ,并在细胞核中检出了NV -HB/s2 s。结论 :建立了乙肝病毒S2 S蛋白表达质粒NV -HB/s2 s转染小鼠骨髓瘤细胞SP2 / 0的长期稳定转染细胞系。

Objective:To establish a monoclone SP2/0 cell line long -term transfected with Plasmid NV-HB/s 2s,which is capable of stably expressing HBV Protion S 2S,and analyze the transfected gene.The stably transfected monoclone cell will be used as the target cell for CTL assessment in the study of HBV DNA vaccine.Methods:SP2/0 cells(BALB/c mice myeloma cell)were transfected with NV-HB/s 2s,then selected by G418.Every selected monoclone cell was detected for it's HBsAg and preS 2Ag expression by ELISA,Western-Blot and immunohistochemistry.The existence of NV-HB/s 2s was detected by dot-blot hybridization and in situ hybridization.Results:Through G418 selection,16 long-term transfected monoclone cell lines were gained.Four of the sixteen cell lines were demonstrated by ELISA and Western-Blot to express HBsAg and preS2Ag,and demonstrated by dot-blot hybridization to have NV-HB/s 2s in nuclear.The results were confirmed by cell immunohistochemestry and in suit hybridization.The best one of the four cell lines was choosed.Conclusion:A SP2/0 cell line transfected with NV-HB/s 2s was obtained,which could stably express the HBsAg and preS2Ag in cells.