Toll样受体4重组质粒及稳转HepG2细胞株的构建  

Establishment of HepG2 cell lines stably transfected with TLR4 gene

作　　者：黄萃园 张洪[1] 白瑞丹 HUANG Cuiyuan;ZHANG Hong;BAI Ruidan(Department of Pharmacy,Renmin Hospital of Whhan University,Whhan,Hubei 430060,China)

机构地区：[1]武汉大学人民医院药学部,湖北武汉430060

出　　处：《安徽医药》2018年第9期1714-1718,1856,共6页Anhui Medical and Pharmaceutical Journal

摘　　要：目的将Toll样受体(TLR4)野生型及突变型(包括单突变和双突变)基因分别转入HepG2细胞,建立稳定表达TLR4蛋白的细胞株。方法 PCR扩增用于合成TLR4的c DNA,设计引物应用PCR点突变的方法合成1196T及896G单突变质粒和TLR4-1196T,896G双突变质粒。经慢病毒包装后,将合成的p LVX-TLR4-Puro、p LVX-TLR4(A-G)-Puro、p LVX-TLR4(C-T)-Puro、p LVX-TLR4(A-G,C-T)-Puro质粒转染HepG2细胞。在细胞贴壁后加入嘌呤霉素进行筛选;Western blot检测各组细胞TLR4的表达量。结果嘌呤霉素筛选出含有抗性基因的细胞,说明TLR4重组质粒成功转入细胞;Western blot结果表明TLR4在HepG2细胞中稳定过表达。结论成功构建了4组TLR4过表达的细胞株,为研究TLR4基因多态性对HepG2细胞生物学功能的影响奠定基础。Objective To transfect TLR4 wild-type allele or variant（including single mutation and double mutation） into HepG2 cells and establish stably expressed cell lines for TLR4 protein. Methods Mutated plasmid designated TLR4 were produced by site-directed mutagenesis with model vector pc DNA 3. 1 with c DNA encoding TLR4 wild-type allele. After lentivirus packaging,the synthesized p LVX-TLR4-Puro,p LVX-TLR4（A-G）-Puro,p LVX-TLR4（C-T）-Puro,p LVX-TLR4（A-G,C-T）-Puro plasmids were transfected into HepG2 cells and were selected by puromycin. Western blot were applied to determine protein levels. Results The cells screened by puromycin contains resistance genes,indicating that TLR4 recombinant plasmids were successfully transferred into cells. Western blot results showed that TLR4 was over-expressed in HepG2 cells. Conclusion The recombinant cell lines were successfully constructed,which provide a great benefit for study the effect of TLR4 gene polymorphism on biological function of HepG2 cells.

关 键 词：Toll样受体4基因 肝癌细胞 基因多态性 过表达 

分 类 号：R735.7[医药卫生—肿瘤]