人FBP17基因重组真核表达质粒的构建及其在LO2细胞中的表达

Construction of recombinant eukaryotic expression vector for human FBP17 gene and its expression in LO2 cells

目的构建人FBP17基因重组真核表达质粒,并检测其在人正常肝细胞LO2中的表达及对细胞存活率的影响。方法用EcoRⅠ和BamHⅠ双酶切pEGFP-C1-FBP17质粒,胶回收FBP17基因片段,与p3XFLAG-CMV-10载体连接,构建重组真核表达质粒p3XFLAG-CMV-10/FBP17,LipofectamineTM法转染人正常肝细胞LO2,RT-PCR和Western blot法检测转染细胞中FBP17基因mRNA的转录水平和蛋白的表达水平,XTT法检测其对转染细胞增殖的影响。结果重组表达质粒p3XFLAG-CMV-10/FBP17经双酶切及测序证实构建正确;转染该重组质粒的LO2细胞FBP17基因mRNA的转录水平和蛋白的表达水平均明显增高,且该质粒转染对细胞存活率无影响。结论成功构建了FBP17基因重组真核表达质粒,其可在LO2细胞中表达,且对细胞的存活率无影响,为进一步研究FBP17相互作用蛋白及其功能奠定了基础。

Objective To construct a recombinant eukaryotic expression vector for human FBP17 gene and investigate its ex- pression in human normal liver LO2 cells as well as its effect on eel1 survival. Methods Recombinant plasmid pEGFP-C1-FBP17 was digested with EeoR I and BamH I , and the FBP17 gene fragment was recovered and inserted into plasmid p3XFLAG-CMV-IO. Human normal liver LO2 cells were transfected with the constructed recombinant plasmid p3XFLAG-CMV-IO/FBP17 in mediation of LipofectamineTM, and determined for transcription level of FBP17 mRNA and expression level of FBP17 by RT-PCR and Western blot respectively. The effect of FBP17 overexpression on proliferation of LO2 cells was determined by XT＇I＂ method. Results Restriction analysis and sequencing proved that recombinant plasmid p3XFLAG-CMV-10/FBP17 was constructed correctly. Both the transcrip- tion level of FBP17 mRNA and expression level of FBP17 in LO2 calls transfected with the recombinant plasmid increased signifi- cantly. The transfection with recombinant plasmid p3XFLAG-CMV-10/FBP17 showed no significant effect on survival of LO2 cells. Conclusion The recombinant eukaryotic expression vector for human FBP17 gene was successfully constructed and expressed in LO2 cells, which showed no effect on cell survival and laid a foundation of further study on FBP17 interaction protein and its function.