不同核心启动子对中国仓鼠卵巢细胞重组蛋白表达的影响  

Effect of different core promoters on expression of recombinant protein in Chinese hamster ovary cells

作　　者：肖梦珂 华宇 冯莹莹 路江涛 翟海晖 王天云[3] 贾岩龙[2] XIAO Mengke;HUA Yu;FENG Yingying;LU Jiangtao;ZHAI Haihui;WANG Tianyun;JIA Yanlong(School of Public Health,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;School of Pharmacy,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;School of Basic Medical Sciences,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)

机构地区：[1]新乡医学院公共卫生学院,河南新乡453003 [2]新乡医学院药学院,河南新乡453003 [3]新乡医学院基础医学院,河南新乡453003

出　　处：《新乡医学院学报》2021年第6期501-504,共4页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:32071468);河南省科技攻关项目(编号:192102310149);河南省高校国家级大学生创新创业训练计划项目(编号:202010472008)。

摘　　要：目的探讨不同核心启动子对中国仓鼠卵巢(CHO)细胞重组蛋白表达的影响。方法以增强型绿色荧光蛋白(EGFP)基因为报告基因,分别构建以巨细胞病毒(CMV)启动子、Natural CMV启动子、超级核心启动子(SCP)2和SCP3驱动的表达载体并转染CHO细胞,根据转染表达载体的不同将细胞分为对照组、Natural CMV组、SCP2组和SCP3组。应用荧光倒置显微镜观察报告基因EGFP的瞬时转染效率,流式细胞术检测各组第1代和第30代细胞中EGFP表达水平,实时荧光定量聚合酶链反应法检测各组第30代细胞中EGFP mRNA相对表达量。结果对照组、Natural CMV组、SCP2组细胞的瞬时转染效率比较差异无统计学意义(P>0.05);SCP3组细胞的瞬时转染效率显著高于对照组(P<0.05)。对照组、Natural CMV组、SCP2组、SCP3组第1代多克隆细胞中EGFP表达水平两两比较差异均无统计学意义(P>0.05),对照组、Natural CMV组、SCP2组第30代多克隆细胞中EGFP表达水平两两比较差异无统计学意义(P>0.05),SCP3组第30代多克隆细胞中EGFP表达水平显著高于对照组、Natural CMV组和SCP2(P<0.05)。对照组与Natural CMV组第30代多克隆细胞中EGFP mRNA相对表达量比较差异无统计学意义(P>0.05),SCP2组、SCP3组第30代多克隆细胞中EGFP mRNA相对表达量显著高于对照组、Natural CMV组(P<0.05),SCP3组第30代多克隆细胞中EGFP mRNA相对表达量显著高于SCP2组(P<0.05)。结论SCP3能够显著提高CHO细胞中重组蛋白的表达及稳定性。Objective To investigate the effect of different core promoters on the expression of recombinant protein in Chinese hamster ovary(CHO)cells.Methods The enhanced green fluorescent protein(EGFP)was used as the reporter gene,and the expression vectors which were driven by the promoters of cytomegalovirus(CMV),the promoters of Natural CMV,super core promoter(SCP)2 or SCP3 were constructed.The four expression vectors were transfected into CHO cells,respectively.The CHO cells were divided into the control group,the Natural CMV group,the SCP2 group and the SCP3 group according to the transfected expression vectors.The transient transfection efficiency of EGFP was observed by fluorescence inversion microscopy.The expression of EGFP of the 1 st and the 30 th generations of CHO cells in each groups was detected by flow cytometry.The relative expression level of EGFP mRNA of the 30 th CHO cells was detected by real-time fluorescence quantitative polymerase chain reaction.Results There was no statistic difference in the transient transfection efficiency of cells among the control group,the Natural CMV group and the SCP2 group(P>0.05).The transient transfection efficiency of cells in the SCP3 group was significantly higher than that in the control group(P<0.05).There was no statistic difference in the expression of EGFP in the 1 st generation of the polyclonal CHO cells among the control group,the Natural CMV group,the SCP2 group and the SCP3 group(P>0.05).There was no statistic difference in the expression of EGFP in the 30 th generation of the polyclonal CHO cells among the control group,the Natural CMV group and the SCP2 group(P>0.05).The expression of EGFP of the 30 th generation of the polyclonal CHO cells in the SCP3 group was significantly higher than that in the control group,the Natural CMV group and the SCP2 group(P<0.05).There was no statistic difference in the relative expression le-vel of EGFP mRNA in the 30 th generation of the polyclonal CHO cells between the control group and the Natural CMV group(P>0.05).The

关 键 词：核心启动子 中国仓鼠卵巢细胞 重组蛋白 巨细胞病毒 超级核心启动子 

分 类 号：Q78[生物学—分子生物学]