靶向表皮生长因子受体shRNA表达载体的构建和鉴定  

Establishment and identification of ShRNA expression vectors of recombinant targeting gene EGFR

作　　者：王慧[1] 魏志平[1] 刘彦群[1] 

机构地区：[1]徐州医学院附属医院皮肤科,江苏徐州221002

出　　处：《徐州医学院学报》2010年第5期321-324,共4页Acta Academiae Medicinae Xuzhou

摘　　要：目的构建靶向表皮生长因子受体(EGFR)的短发卡状RNA(shRNA)质粒表达载体并进行鉴定。方法①根据人类EGFR的mRNA序列,设计4条干扰片段,以PGPU6/GFP/Neo质粒为载体,构建shRNA重组体,转化入DH5a大肠杆菌,提取质粒,进行酶切和测序鉴定;②采用脂质体Lipofectamin 2000分别转染人皮肤鳞状细胞癌细胞株Colo-16细胞,用G418筛选阳性克隆。结果①经酶切及测序鉴定,成功构建了针对EGFR的4个重组干扰质粒;②成功转染Colo-16细胞,经筛选后得到稳定表达的细胞克隆。结论利用RNAi技术原理成功构建针对EGFR的shRNA重组质粒,并转染入Colo-16细胞。Objective To establish and identify the plasmid expression vector encoding short hairpin RNA（shRNA） targeting epidermal growth factor receptor（EGFR） of human gene.Methods ①Four short hairpin RNAs（shRNA） were designed according to the homo sapiens EGFR mRNA ID,and then recombinant shRNA was reconstructed,with PGPU6/GFP/Neo plasmids as vectors,respectively.The plasmids were transferred into E.coli DH5α for plasmid extraction and the subsequent identification by enzyme digesting and sequencing.②Four recombinant plasmids were transfected into Colo-16 cells with lipofectamine 2000 and were screened for positive clones by G418.Results ①The results of enzyme-digestion sequencing confirmed that four plasmids containing shRNA were successfully constructed.②Four recombinant plasmids were successfully transfected into Colo-16 cells and the cell clones with stable expression were obtained.Conclusion With RNAi technology,the recombinant shRNA plasmids targeting EGFR were successfully established and transfected into Colo-16 cells.

关 键 词：表皮生长因子受体 RNA干扰 短发卡状RNA 转染 

分 类 号：R739.5[医药卫生—肿瘤]