靶向表皮生长因子受体shRNA表达载体的构建与鉴定.doc

靶向表皮生长因子受体shRNA表达载体的构建和鉴定 【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。 【关键词】 表皮生长因子受体;RNA干扰;短发卡状RNA;转染 　　Abstract： 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. 　　Key words: epidermal growth factor receptor; RNA interference; small hairpin RNA; transfection 　　表皮生长因子受体(epidermal growth factor receptor,EGFR)属于表皮生长因子家族[1] (erbB家族)，为原癌基因C-erbB(HER-1)的表达产物，是一种细胞膜表面的糖蛋白受体，它与表皮生长因子(epidermal growth factor,EGF)、转化生长因子(transferring growth factor ,TGF)等配体结合，能激活酪氨酸激酶受体，导致酪氨酸残基的磷酸化，促进细胞无节制地增殖分裂，导致恶性肿瘤的发生[2]。阻断EGFR可抑制肿瘤生长，其作用机制可能与抑制细胞周期进程、加速细胞凋亡、抑制血管生成、抑制浸润和转移、增强化、放疗敏感性等有关[3]。小干扰RNA(siRNA)[4]与同源性mRNA结合, 可靶向切断降解此mRNA, 在蛋白翻译前抑制基因表达[5]。我们构建编码EGFR的短发卡状RNA (shRNA) 质粒表达载体,并对其进行鉴定,为皮肤鳞癌的靶向 治疗 寻找新的途径。