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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 long-term stable transfected cell line of mouse myeloma SP2 / 0 transfected with the S2-S protein expression plasmid NV -HB / s2s of hepatitis B virus and provide target cells for the later detection of the cellular immunity effect of the hepatitis B nucleic acid vaccine. METHODS: NV-HBs / s2s were transfected into SP2 / 0 cells by modified calcium phosphate method. Sixteen monoclonal strains were screened out by G418 resistance screening. ELISA, Western-blot, dot blot, The expression of target antigen protein and the presence and location of NV-HBs / s2 in cells were detected by immunohistochemistry, histochemistry and in situ hybridization. Results: A monoclonal cell line SP2 / 0-S2S stably transfected with NV-HBs / s2 s was obtained. The expression of HBsAg and preS2 Ag was detected in the cells and NV -HB / s2 s. Conclusion: The long-term stable transfected cell line of mouse myeloma cell line SP2 / 0 transfected with hepatitis B virus S2 S protein expression plasmid NV-HB / s2 s was established.