质粒构象对转染后蛋白表达时间曲线的影响  

The effect of plasmid conformations on protein time-course dependent expression after transfection

作　　者：王志平[1] 霍玉奇[2] 王明臣[2] 

机构地区：[1]郑州人民医院药务科,河南郑州450003 [2]郑州大学基础医学院生物化学与分子生物学教研室,河南郑州450052

出　　处：《中国医学工程》2012年第1期4-5,10,共3页China Medical Engineering

基　　金：河南省科技厅自然科学基金资助项目(0611043400)

摘　　要：目的探讨质粒的不同构象下磷酸钙转染后对蛋白表达量时间曲线的影响。方法经过处理的pcDNA3-MADCAM-1-Fc,pcDNA3.1/Hygro(+)-Alpha4,pcDNA3.1/Hygro(+)-Beta7三种质粒磷酸钙转染293T细胞,第一种表达分泌到胞外的蛋白通过收集上清,ProteinA纯化,另外两种表达于细胞外膜,通过流式细胞仪检测荧光强度。结果 pcDNA3-MADCAM-1-Fc磷酸钙转染后处理组(缺口开环)比对照组表达蛋白量多(t=5.009,p=0.0376)。pcDNA3.1/Hygro(+)-Alpha4,pcDNA3.1/Hygro(+)-Beta7两种质粒共转后处理组荧光强度36h后略高于对照组,48h后对照组明显升高。结论质粒的不同构象下磷酸钙转染后蛋白表达量有时间依赖性。【Objective】 To explore the effect of plasmid conformations on its coded protein expression after calcium phosphate transfection.【Methods】 pcDNA3-MADCAM-1-Fc,pcDNA3.1/Hygro（＋）-Alpha4 and pcDNA3.1/Hygro（＋）-Beta7 were pretreated before transfection into 293T cells with calcium phosphate precipitation method.The first plasmid coding a protein secreting into extracellular matrix was purified by Protein A agarose beads column while the latter two plasmids coding two subunits of integrin alpha4beta7 which were located on the extramembrane were assayed by flow cytometry after transfection.【Results】 pcDNA3-MADCAM-1-Fc pretreated showed a higher expression level versus control（t=5.009,P=0.0376）.pcDNA3.1/Hygro（＋）-Alpha4 and pcDNA3.1/Hygro（＋）-Beta7 cotransfected cells showed a minor higher expression in pretreated group at 36hr after transfection while at 48hr the control group showed a significant higher expression level.【Conclusion】 plasmid conformations can affect its coded protein expression in a time-course dependent manner.

关 键 词：质粒构象 碱裂解法 转染 缺口开环 荧光强度 

分 类 号：R394[医药卫生—医学遗传学]