摘要
构建pcDNA5/FRT-SlSCP-2和阳性对照pcDNA5/FRT-GFP真核表达载体,将斜纹夜蛾固醇转运蛋白SlSCP-2和绿色荧光蛋白GFP分别整合进入Flp-In^(TM)CHO细胞基因组中的FRT位点上,利用western blotting方法和荧光倒置显微镜观察鉴定SlSCP-2和GFP蛋白在CHO细胞的表达情况.结果表明,重组质粒瞬时转染CHO细胞24h后,pcDNA5/FRT-SlSCP-2和pcDNA5/FRT-GFP均能正确表达目的蛋白,SlSCP-2能够增加细胞对胆固醇的吸收.同时确定了筛选稳定转染细胞株合适的潮霉素浓度.
The pcDNA5/FRT-SlSCP-2 and positive control pcDNA5/FRT-GFP eukaryotic expression vector was constructed and transient transfected into CHO cells.The Spodoptera litura sterol carrier protein（SlSCP-2）protein expression in the cells was confirmed by western blotting and fluorescence inverted microscope respectively.After a 24 htransfection of CHO cells with the recombinant plasmids,the positive cell clones expressed SlSCP-2 and GFP correctly.SlSCP-2 can increase the uptake of cholesterol by cells.Meanwhile,the suitable concentration of hygromycin B was determined for the screening of stable transfected cell lines.The CHO cell lines with Eukaryotic expression of SlSCP-2 protein and determination of the screening concentration of Hygromycin B are the basis for the establishment of a stably cell line which was used for insecticide screening.
作者
张丽丽
万星
王娟
薛竞帆
武怡琼
靳爽
ZHANG Lili;WAN Xing;WANG juan;XUE Jingfan;WU Yiqiong;JIN Shuang(College of Life Sciences/Institute for Conservation and Utilization of Agro bioresources in Dabie Mountains,Xinyang Normal University,Xinyang 464000,China;Guangzhou Key I.aboratory of Insect Development Regulation and Application Research,Guangzhou 510631,China)
出处
《信阳师范学院学报（自然科学版）》
CAS
北大核心
2018年第3期396-401,共6页
Journal of Xinyang Normal University(Natural Science Edition)
基金
国家自然科学基金项目(31600837)
河南省科技公关计划项目(182102110034)
信阳师范学院"南湖学者奖励计划"青年项目(2016)
信阳师范学院博士科研启动基金项目(0201423)
信阳师范学院大学生科研基金项目(2017-DXS-141)
关键词
斜纹夜蛾
固醇转运蛋白
瞬时转染
高效表达
Spocloptera litura
sterol carrier protein
transient transfection
cukaryotic expression
