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目的建立稳定表达叶酸受体α(FRα)的C26细胞,用于FRα基因疫苗的后续研究。方法采用脂质体转染法,将前期构建的重组真核表达质粒pc DNA3.1-FRα基因导入C26细胞,用G418加压进行筛选,并通过单克隆操作,建立稳定转染叶酸受体α基因的单克隆细胞株。利用反转录PCR及免疫荧光细胞化学染色检测稳定转染细胞中叶酸受体α基因的表达情况,并利用反转录PCR检测传代细胞中FRα基因表达的稳定性。结果经转染、G418筛选以及单克隆化操作,得到单克隆FRα基因稳定转染细胞株,所得单克隆稳定转染细胞株可表达FRαmRNA及蛋白,并能够在多次传代后保持表达的稳定性。结论成功构建了FRα稳定转染细胞株。
Objective To establish C26 cells stably expressing folate receptor α (FRα) for the subsequent study of FRα gene vaccine. Methods The recombinant plasmid pcDNA3.1-FRα was subcloned into C26 cells by lipofection method. The recombinant plasmid pcDNA3.1-FRα was transfected into C26 cells by pressurized G418. The stable transfected folate receptor α Genes of monoclonal cell lines. The expression of folate receptor α gene in stable transfected cells was detected by reverse transcription PCR and immunofluorescent cytochemical staining. The stability of FRα gene expression in the passaged cells was detected by reverse transcription PCR. Results After transfection, G418 selection and cloning, the monoclonal antibody was successfully transfected into the cell line expressing FRα gene. The stable transfected cell line could express FRα mRNA and protein and maintain its stability after many passages . Conclusion The stable FRα-transfected cell line was successfully constructed.