MCFP慢病毒干涉载体及HepG2稳定细胞系的构建

Construction of a lentiviral vector carrying an MCFP shRNA and generation of a HepG2 cell line stably transfected with this vector

目的:研究线粒体转运蛋白质超家族新成员SLC25A40(MCFP)RNA干扰(RNAi)慢病毒表达载体对人肝癌细胞系HepG2细胞中MCFP基因的沉默效果.方法:通过RNAi序列设计软件进行MCFP干涉片段设计,筛选出MCFP基因的RNAi有效靶序列,进一步合成靶序列的OligoDNA,构建pSiCoR-MCFP慢病毒载体并包装产生慢病毒干涉毒液.运用病毒感染HepG2细胞获得稳定干涉MCFP的细胞株,利用PCR和Westernblot方法检测稳定细胞株中MCFP的干涉效果.结果:成功构建具有MCFP干涉效果的慢病毒干涉载体并获得了稳定干涉MCFP及对照的HepG2细胞株.收集慢病毒上清,流式测定干涉病毒滴度为1.45×1010pfu/L,对照病毒滴度为1.78×1010pfu/L.PCR和Westernblot实验均证实干涉MCFP后,HepG2细胞株中MCFP蛋白表达水平明显降低(P<0.001).结论:成功构建具有MCFP干涉效果的慢病毒干涉载体及稳定干涉MCFP的HepG2细胞株.

AIM：To construct a lentiviral vector carrying a short hairpin RNA（shRNA）targeting the mitochondrial carrier functional protein SLC25A40（MCFP）gene and to detect the silencing effect of the vector in HepG2 cell line.METHODS：A doublestranded shRNA targeting the MCFP gene was designed,synthesized and cloned into the pSiCoR vector.The resulting lentiviral vector containing the MCFP shRNA was named pSiCoR-MCFP.HepG2 cells were transfected with the pSiCoR-MCFP lentivirus to obtain a cell line stably expressing the MCFP shRNA.After transfection,the mRNA and protein expression of MCFP in HepG2 cells was detected by RT-PCR and Western blot,respec-tively.RESULTS：A lentiviral vector carrying an shRNA targeting the MCFP gene was successfully constructed and a HepG2 cell line stably transfected with the vector was established.The recombinant lentivirus and control lentivirus harvested from 293 cells had a titer of 1.78×10 10 pfu/L and 1.45×10 10 pfu/L,respectively.RT-PCR and Western blot analyses confirmed that the expression of MCFP was down-regulated in HepG2 cell line stably transfected with the recombinant vector（both P0.001）.CONCLUSION：A lentiviral vector carrying an shRNA targeting the MCFP gene was successfully constructed and a HepG2 cell line stably transfected with the vector was established.