丙型肝炎病毒CTL表位基因真核表达载体的构建及稳定转染细胞系的建立

Construction of eukaryotic expression vector of HCV CTL epitopes and establishment of stable transfected CHO cell line

目的构建含丙型肝炎病毒(HCV)复合多表位基因的真核表达载体,转染CHO细胞,建立稳定转染细胞系。方法通过生物信息学预测分析的方法得到涵盖HCV不同基因型与小鼠H2复合体的多个细胞毒性T淋巴细胞(CTL)表位,串联后人工合成基因序列,将其克隆入真核表达载体pEGFP-N3中,然后利用脂质体法转染CHO细胞,通过持续G418筛选建立稳定转染细胞株。最后通过荧光显微镜观察、RT-PCR和Western Blot等方法证实多表位基因的表达。结果所构建的含有HCV复合多表位基因的真核表达载体pEGFP-mEpi在CHO细胞中能稳定表达。结论真核表达载体的成功构建和稳定转染CHO细胞系的建立为进一步研究复合多表位疫苗的基因免疫奠定了基础。

Objective To construct eukaryotic expression vector of hepatitis C virus（HCV） cytotoxic T-lymphocyte（CTL） epitopes,and to establish stable transfected CHO cell-lines.Methods The HCV CTL epitopes of different genotypes and the mouse H2 complex were predicted by bioinformatics,then synthesized and inserted into eukaryotic expression vector pEGFP-N3.The recombinant vector was transfected into CHO cells by lipofectamine 2000.After screening with G418,stably transfected CHO cell line was established.The expression of HCV multi-epitopes was identified by RT-PCR and western-blot and immunofluorescence.Results The eukaryotic expression vector was constructed successfully.The stable transfected CHO cell line was established.Conclusion The establishment of stable transfected CHO cell line and the expression of the target gene provide solid foundation for further experimental studies.