外源人gp96基因在肿瘤细胞中的高效表达

背景与目的：从肿瘤细胞中分离出的热休克蛋白gp96制备物免疫小鼠可产生抗相应肿瘤的特异性保护性细胞毒性T细胞免疫应答,为肿瘤免疫治疗开辟了一条新的可行性途径,但gp96害细胞中的表达水平较低,从有限的细胞或组织中获得的gp96制备物难以满足研究的需要。因此,本研究拟为制备高质量和充足的gp96而建立高表达gp96的单克隆细胞株。方法：构建人gp96cDNA的重组表达质粒pcDNA-hgp96并将其转染HeLa细胞,G418筛选稳定转染的阳性细胞克隆,然后用Western印迹分析单克隆细胞株的gp96表达时。结果：成功构建了人gp96的重组表达质粒,建立了稳定转染的单克隆细胞株,并筛选出一株高表达gp96的单克隆细胞。结论：成功建立了高表达gp96的单克隆细胞株,为gp96抗肿瘤免疫的研究奠定了基础。

Efficient expression of exogenous human gp96 gene in tumor cells

Background &Objective:Immunization of mice with preparati ons of heat shock protein(HSP)gp96isolated from cancer cells has been s hown to elicit specific protective c ytotoxic T lymphocyte response against cells from which gp96originate.This phenomenon exp loits a new practicable pathway for c ancer immunotherapy.But gp96is gen erally expressed at low level in cells.Gp96preparations from limited cells or t issue are difficult to meet the needs of study.So the current study aims to acquire monoclonal cell lines expressing gp96at hi gh level in order to prepare enough gp96with high quality.Methods:The recombinant plasmid pcDNA-hgp96of human gp96cDNA was constructed b y ligating the fragment of gp96cDNA into the pcDNA3 plasmid,a eucaryotic expressing vector.Then the recombinant plasmid w as transfected into Hela cells and the stable transfecta nts were selected with G418.The expr ession level of gp96of the positive monoclonal cells was detected by Western blot analysis.Results:The recombinant expression plasmid of human gp96cDNA was successfully constructed.The monoclonal cell lines with stable transfection were ob tained.A monoclonal cell line expre ssing gp96on high level was selected out.Conclusions:The monoclonal cell line expressing gp96at high level has been successfu lly established,which lays the groundw ork for the study of its antitumor imm unity.