CHO-DG44细胞瞬时转染条件的优化  

Optimization of condition for transient transfection of CHO-DG44 cells

作　　者：黎美香[1] 陈先金[1] 王晓慧[1] 王亚玉[1] 袁凤媚[1] 汪才坤[1] 谢秋玲[1] 

机构地区：[1]暨南大学生物医药研究院广东省生物工程药物重点实验室基因药物国家工程中心,广东广州510632

出　　处：《中国生物制品学杂志》2013年第4期558-562,566,共6页Chinese Journal of Biologicals

基　　金：国家"十二五"重大新药创制项目(2012ZX09202-301-001);广东省产学研项目(2010B090400500);暨南大学科研培育与创新基金(11612110);广州市重大科技专项计划项目(2010U1-E00541);"重大新药创制"国家科技重大专项(2011ZX09506-006)

摘　　要：目的优化CHO-DG44细胞瞬时转染的条件。方法采用TubeSpin一次性生物反应器,以绿色荧光蛋白(Green fluorescence protein,GFP)为报告基因,氯醚酰亚胺(Polyether imide,PEI)为转染试剂,将质粒pIRESneo3-eGFP瞬时转染CHO-DG44细胞,优化瞬时转染的基本条件[细胞密度、DNA浓度和DNA:PEI比例(w/w)]以及其他条件(换液、渗透压和温度),采用优化的条件转染质粒pIRESneo3-eGFP,流式细胞术检测细胞转染效率,生物发光仪检测相对荧光强度。结果 CHO-DG44细胞瞬时转染的最佳条件为:采用XLG-P8培养基进行转染,细胞密度为2×106个/ml,DNA浓度为6.25μg/5 ml,DNA∶PEI(w/w)比例为1∶5;转染后4 h更换新鲜培养基,添加30 mmol/L NaCl,并于31℃继续培养。在此条件下,CHO-DG44细胞的瞬时转染效率可达81.45%,相对荧光强度可达9×105RFU/106cells。结论优化了CHO-DG44细胞瞬时转染的条件,为下一步药物蛋白的研发奠定了基础。Objective To optimize the condition for transient transfection of CHO-DG44 cells.Methods CHO-DG44 cells were transiently transfected with plasmid pIRESneo3-eGFP in TubeSpin disposable bioreactor using green fluorescence protein（GFP） gene as a reporter and polyether imide（PEI） as a transfection reagent.The primary condition such as cell density,DNA concentration and ratio of DNA to PEI（w / w）,as well as other conditions such as osmotic pressure and temperature for changing medium,were optimized.The cells were transfected with pIRESneo3-eGFP under the optimized condition,and determined for transfection efficacy by flow cytometry,and for relative fluorescent intensity by bioluminescence analyzer.Results The optimal medium,cell density,DNA concentration,ratio of DNA to PEI（w / w） for transient transfection of CHO-DG44 cells were XLG-P8 medium,2 × 10^6 cells / ml,6.25 μg / 5 ml and 1： 5 respectively.The culture was transferred to fresh medium 4 h after transfection,added with 30 mmol / L sodium chloride and further incubated at 31℃.Under the optimized condition,the transient transfection efficiency of CHO-DG44 cells reached 81.45%,while the fluorescent intensity was about 9 × 10^5 RFU / 106 cells.Conclusion The condition for transient transfection of CHO-DG44 cells was optimized,which laid a foundation of further development of pharmaceutical proteins.

关 键 词：CHO-DG44细胞 转染 瞬时表达 优化 

分 类 号：Q819[生物学—生物工程]