摘要
目的构建EGFR shRNA重组真核表达载体,并筛选抑制效果最好的EGFR shRNA序列。方法应用在线工具设计人EGFR siRNA序列,采用限制性内切酶BamHI和HindIII切割真核表达质粒pcDNA3.1(+),构建三种人EGFR的siRNA干扰序列载体(psilencer 4.1-CMV neo-EGFR siRNA)。将重组质粒载体转化大肠杆菌DH5α感受态并筛选阳性克隆,通过DNA测序鉴定重组质粒。应用脂质体lipo2000将三种人EGFR siRNA干扰序列载体转染到HepG2细胞,通过荧光显微镜观察转染效率,实时定量PCR检测EGFR mRNA表达水平,MTT法检测细胞活性。结果Psilencer 4.1-CMV neo-EGFR siRNA重组质粒被成功克隆。EGFR shRNA-1、EGFR shRNA-2和EGFR shRNA-3敲低EGFR mRNA的效率分别是80%、60%和70%以上。shRNA-2和shRNA-3使细胞活性分别下降50%(P<0.05),但shRNA-1对细胞活性无明显影响(P>0.05)。结论重组psilencer 4.1-CMV neo-EGFR siRNA质粒可下调肝癌细胞株EGFR表达水平和细胞活性。EGFR shRNA-3较EGFR shRNA-1和shRNA-2对HepG2细胞的抑制作用更显著。
Objective To construct the recombinant eukaryotic expression vector of EGFR shR⁃NA and screen the EGFR shRNA sequence with the best inhibitory effect.Methods An online tool was used to design the human EGFR siRNA sequence and synthesize the corresponding EGFR shRNA.The eukaryotic expression plasmid pcDNA3.1(+)was cleaved by the restriction endonucloenzymes BamHI and HindIII to construct the siRNA interference vector of three kinds of human EGFR(psilencer 4.1⁃CMV neo⁃EGFR siR⁃NA)and transform the recombinant plasmid vector into the Escherichia coli DH5αreceptor state.The positive clones were screened,and the recombinant plasmids were identified by DNA sequencing.HepG2 cells were transfected with the three siRNA interference sequence vectors of human EGFR using lipo2000.Fluorescence microscopy was used to observe the transfection efficiency,real⁃time quantitative PCR was used to detect the mRNA level of EGFR,and MTT assay was used to detect the cell activity.Results The psilencer 4.1⁃CMV neo⁃EGFR siRNA recombinant plasmid has been successfully cloned.The knockdown of EGFR shRNA⁃1 on EGFR mRNA was 80%,while EGFR shRNA⁃2 had a knockdown efficiency of 60%,and EGFR shRNA⁃3 had a knockdown efficiency of over 70%.Both shRNA⁃2 and shRNA⁃3 reduced cell activity by 50%(P<0.05),but shRNA⁃1 did not have a significant effect on cell activity(P>0.05).Conclusion The recombinant psilencer 4.1⁃CMV neo⁃EGFRsiRNA plasmid was able to downregulate the expression of EGFR and reduce the activity of hepatocellular carcinoma cell lines.Out of the three EGFR shRNA sequences tested,EGFR SHRNA⁃3 ex⁃hibited the most potent inhibitory effect on HepG2 cells.
作者
许楠
杨旭东
薛丽
宁启兰
王慧莲
耿燕
XU Nan;YANG Xudong;XUE Li;NING Qilan;WANG Huilian;GENG Yan(Department of Clinical Laboratory,the Second Affiliated Hospital of Xi'an Jiaotong University,Xi'an,Shanxi,China,710004;Department of Biochemistry and Molecular Biology,School of Basic Medicine Sciences,Xi'an Jiaotong University Health Sciences Center,Xi'an,Shaanxi,China,710061)
出处
《分子诊断与治疗杂志》
2024年第5期935-939,944,共6页
Journal of Molecular Diagnostics and Therapy
