人丙酮酸羧化酶异位表达提高中国仓鼠卵巢细胞补料分批培养后期活率  

Increase of Viability of Chinese Hamster Ovary Cells in Late Phase of Fed-Batch Culture by Ectopic Expression of Human Pyruvate Carboxylase

作　　者：付托[1,2] 张存超[2] 靖钰[1,2] 蒋成[2] 张学光[1] 寇庚[2] 

机构地区：[1]苏州大学医学生物技术研究所,江苏苏州215007 [2]第二军医大学肿瘤研究所,上海200433

出　　处：《化学与生物工程》2013年第6期39-42,共4页Chemistry & Bioengineering

基　　金：国家863计划资助项目(2009A02Z107)

摘　　要：构建了人丙酮酸羧化酶(hPC)异位表达的重组CHO细胞,并检测了重组细胞生长情况的改变。利用分子生物学实验技术构建hPC-pcDNA 3.0表达载体,将其转染CHO-K1细胞;转染细胞经过G418抗性筛选后,通过荧光定量PCR检测目的基因表达,并挑选表达量最高的克隆进行补料分批培养。电泳及测序结果显示,构建的hPC-pcDNA3.0表达载体序列与预期一致;在mRNA水平鉴定目的基因显示,4#克隆的mRNA表达水平最高;选其进行补料分批培养,生长曲线显示在培养后期,其活细胞密度和细胞活率均高于对照。成功构建了细胞生长和活率改善的重组CHO细胞,获得了生长改善的细胞株,为后续的重组蛋白表达研究与细胞培养工艺优化奠定了基础。Human pyruvate carboxylase （hPC） ectopic expression of recombinant CHO cells were construc- ted in this study, and the changes of the growth of the recombinant cells were detected. Molecular biology tech- niques were employed to build hPC-pcDNA 3. 0 expression vector, following by transfection into CHO-K1 cells. G418 resistant cell pools were selected from transfected cells, clones picked from which were then con- firmed by quantitative PCR in mRNA level. Qualified clones were verified by fed-batch culture,setting empty vector contained CHO-K1 cells as control. Electrophoresis and sequencing results indicated,sequence of hPC- pcDNA 3.0 expression vector built was consistent with expectations. The quantitative PCR results showed mR- NA expression level of CHO clone 4# was the highest. Choosing CHO clone 4# to do fed-batch culture,growth curves showed in the late culture, the viable cell density and cell viability were higher than those in control group. The experiment successfully constructed recombinant CHO cells with improved growth and viability, thus provided cell line for subsequent recombinant protein expression and cell culture process optimization.

关 键 词：CHO细胞 丙酮酸羧化酶 补料分批培养 活率 

分 类 号：Q813.110[生物学—生物工程]