慢病毒介导的GFP-Hsp90αE47A基冈在HepG2细胞表达及对细胞增殖性影响的研究

摘要

目的:建立真核细胞表达的GFP-Hsp90α FA7A基因重组慢病毒栽体三质粒包装细胞系统,并检测其对细胞增殖性的影响,为进一步研究HSP90分子伴侣功能奠定基础.方法:制备完整的重组慢病毒载体三质粒系统:转移质粒(Hsp90αE47A/psin-GFP),包装质粒(△NRF)及包膜蛋白质粒(VSV-G).磷酸钙法将三质粒共转染293T包装细胞,48 h后收集病毒上清.将制备好的慢病毒颗粒感染HepG2细胞,在荧光显微镜下观察报告基因GFP的表达情况,Western blot检测HepG2细胞GFP-Hsp90α表达.MTT法检测细胞增殖情况.结果:转染后的293T和感染后的HepG2细胞能观察到较强的绿色荧光,培养液上清病毒滴度约为3.0× 103ifu/μl,HepG2细胞中有GFP-Hsp90α蛋白的表达.内源性Hsp90α表达无明显上升(为对照组的1.05±0.15倍,P<0.05,t检验),有明显外源性GFP-HspgoaFA7A蛋白的表达,为对照组内源性Hspgoa的0.68±0.12倍.外源性GFP-Hsp90αE47A蛋白的表达HepG2细胞增殖活性于第4d有明显抑制.(1.051±0.03 vs 1.349± 0.05,P<0.05,t检验).结论:成功建立重组慢病毒载体的三质粒包装细胞系统,并将GFP-Hsp90αE47A基因在HepG2细胞中稳定表达,且并未引起细胞明显的热休克反应而导致的内源性Hsp90α增高:且能明显抑制细胞增殖,为后期Hsp90α分子伴侣功能进行研究奠定基础.%Objective: To construct the three-plasmid packaging cell line of the recombinant lentiviral vector encoding Hsp90α E47A/psin-GFP gene and investigate the molecular chaperon function of hsp90α. Methods: The three-plasmid recombinant lentiviral vector, which was made up of the vector plasmid (Hsp90αE47A/psin-GFP), the packaging plasmid (△ NRF) and the envelop plasmid encoding the vesicular stomatitis virus-glycoprotein (VSV-G), was isolated and purified. Human embryonic kidney 293T cells were co-transfected with the three plasmids by calcium phosphate method. 48 hours after the transfection, the viral supernatant was collected to infect HepG2cells. The expression of reporter gene GFP was detected by fluorescence microscope, and the GFP-Hsp90αE47A protein expressed in Hepg2 cells was detected by Western blot. Cell proliferation was tested by CCK-8 method. Results: There was strong expression of GFP s in 293T and Hepg2 cells after transfection. The viral titer was 3.0 × 103 ifu /μ1. The expression of GFP-Hsp90α in Hepg2 cells was confirmed by Western blot. Cell proliferation of GFP-Hsp90α F47A protein expressing Hepg2 cells was obviously inhibited at 4d. (1.051± 0.03 vs l.349± 0.05, P＞0. 05,t test). Conclusion: The three-plasmid packaging cell line system of recombinant lentiviral vector was successfully established, cell proliferation was obviously inbited by GFP-Hsp90αE47A gene transfection, which will provide a basis for exploring the molecular chaperon function ofHsp90α.