【摘要】 目的构建干扰表皮生长因子受体(EGFR)的pSilencerTM-EGFR表达质粒,转染入非小细胞肺癌(NSCLC)A549细胞株,为观察对EGFR的沉默效应奠定基础。方法①根据人类EGFR的mRNA序列,设计4条干扰片段,以pSilencerTM2.1-U6-hygro质粒为载体,构建shRNA重组体,转化入DH5α大肠杆菌,提取质粒,进行酶切和测序鉴定;②采用脂质体2000分别转染人类非小细胞肺癌细胞株A549,用潮霉素(Hygromycin B)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种重组干扰质粒,分别命名为pSilencerTM-EGFRⅠ、pSi-lencerTM-EGFRⅡ、pSilencerTM-EGFRⅢ和pSilencerTM-EGFRⅣ;②成功转染A549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A549细胞。更多还原

【Abstract】 Objective To observe inhibition ratio of EGFR expression,we constructed four plasmids which can inhibit expression of EGFR,and transfect them into cell line A549.Methods①Four short hairpin RNAs (shRNA) were designed according to the homo sapiens EGFR mRNA ID,and then reconstructed to pSilencerTM 2.1-U6 hygro plasmids respectively.The recombinant plasmids were transferred into DH5α E.coil,the plasmids were extracted,identified by enzyme digesting and sequencing.②Four recombinant plasmids were transfect to A549 cells with lipofectamine 2000 and selected positive clone by Hygromycin B,respectively.Results ①The sequencing results showd four plasmids containing shRNA were constructed successfully.②Four recombinant plasmids were successfully transfected into A549 cells and the cell clones that expressed stably were obtained.Conclusion The recombinant shRNA plasmids were established successfully and transfect efficiently into A549 cell.更多还原