波形蛋白真核表达质粒载体的构建及其在HepG2细胞中的表达

Construction of a Plasmid Containing Human Vimentin Intermediate Filaments and its Stable Expression in HepG2 Cells

目的构建重组人波形蛋白中间丝真核表达质粒载体pcDNA3.1-VIM并检测波形蛋白在肝癌HepG2细胞中的稳定表达。方法通过RT-PCR技术从人胎盘组织总RNA中扩增波形蛋白基因,经限制性核酸内切酶BamHⅠ-EcoRⅠ消化,然后插入pcDNA3.1(+)载体,脂质体介导重组质粒和空质粒分别转染HepG2细胞,G418筛选稳定表达细胞株,采用免疫细胞化学技术检测目的基因的表达。结果经DNA序列分析和限制性核酸内切酶消化,证实重组载体pcDNA3.1-VIM已正确克隆,建立的稳定细胞株高表达波形蛋白。结论成功地构建了重组质粒pcDNA3.1-VIM载体和建立了稳定表达波形蛋白的人肝细胞癌细胞株,为进一步研究波形蛋白与癌细胞生物学行为之间的相互关系奠定了基础。

Objective To construct the recombinant eukaryotic expression plasmid vector pcDNA3. 1-VIM and detect the stable expression of vimentin protein in HepG2 cells. Methods The eDNA encoding vimentin was amplified using RT-PCR from total RNA of human placenta tissue and inserted into the BamH I-EcoR I sites of pcDNA3.1 （ ＋ ） vector. Reconstructed plasmid and empty plasmid were respectively transfected into HepG2 ceils mediated by liposome. Two stable cell lines were selected using G418. The immunocytochemistry was used to evaluate the expression of vimentin. Results DNA sequencing and restriction cndonuelcases digestion analysis demonstrated that the recombinant vector pcDNA3. 1-VIM was correctly cloned. The stable cell line demonstrated a higher level of vimcntin expression. Conclusion Recombinant eukaryote plasmid pcDNA3.1-VIM is successfully constructed and a carcinoma cell line high ex- pressing vimentin is obtained. This work lays a good foundation for further study on the relationship between vimcntin and biological behavior of carcinoma cells.