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.
出处
《中国生物制品学杂志》
CAS
CSCD
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)