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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 an eukaryotic expression vector containing hepatitis C virus (HCV) multi-epitope gene and transfect it into CHO cells to establish a stable transfected cell line. Methods A number of cytotoxic T lymphocyte (CTL) epitopes covering different genotypes of HCV and mouse H2 complex were obtained by bioinformatics prediction and analysis. Synthetic gene sequences were tandemly linked and cloned into eukaryotic expression vector pEGFP-N3. Then, CHO cells were transfected by lipofectamine and stable transfected cell lines were selected by continuous G418 screening. Finally, the expression of multi-epitope genes was confirmed by fluorescence microscopy, RT-PCR and Western Blot. Results The eukaryotic expression vector pEGFP-mEpi containing HCV multi-epitope gene was stably expressed in CHO cells. Conclusion The successful construction of eukaryotic expression vector and the establishment of stable transfected CHO cell line laid the foundation for further study on the gene immunization of multi-epitope vaccine.