E2F-1真核表达载体的构建和Genectin抗性细胞的筛选  

Construction of E2F-1 gene eukaryotic expression vector and selection of Genectin-resistant gastric carcinoma cells

作　　者：王长青[1] 尹永硕[2] 李雷[3] 谢玉波[3] 肖强[3] 

机构地区：[1]深圳市宝安人民医院,广东深圳518101 [2]东莞市人民医院 [3]广西医科大学附属第一医院

出　　处：《山东医药》2010年第48期19-21,共3页Shandong Medical Journal

基　　金：广西省自然科学基金资助项目(0640085)

摘　　要：目的通过构建含E2F-1基因的真核表达载体,转染胃癌细胞MGC-803,获得Genectin抗性的稳定过表达E2F-1的胃癌细胞株。方法 PCR扩增E2F-1基因片段,经限制性内切酶EcoRⅠ和HindⅢ双酶切后将E2F-1定向克隆到真核表达载体pCMV-HA2中转化筛选。用脂质体Lipofectamine 2000将该载体转染MGC-803胃癌细胞,然后用含G418的培养基筛选获得具Genectin抗性的过表达E2F-1的胃癌细胞株。RT-PCR和Western blot技术检测MGC-803/E2F-1细胞内E2F-1表达情况。结果重组载体pCMV-E2F-1-HA2经EcoRⅠ和HindⅢ双酶切后得到预期片段,测序结果与报道的E2F-1基因完全一致。获取具Genectin抗性的细胞克隆,并进行了扩增。RT-PCR和Western blot证实重组载体pCMV-E2F-1-HA2成功转入胃癌细胞内,并稳定过表达E2F-1。结论成功获取具Genectin抗性的稳定过表达E2F-1的胃癌细胞株,为下一步的功能实验奠定了基础。Objective To construct an eukaryotic expression vector containing E2F-1 gene and transfect the vector into gastric cancer cell line MGC-803.Methods Total RNA was extracted from normal human liver.E2F-1 cDNA was cloned by using RT-PCR method and transferred into eukaryotic expression vector pCMV-HA2 after digested by restrictive endonuclease enzyme Hind Ⅲ and EcoRⅠ.Then pCMV-E2F-1-HA2 was transfected into MGC-803 cells with Lipofectamine 2000.The genectin-resistant cells were selected and harvested in medium containing G418.RT-PCR and Western blot were used to identify the expression of E2F-1.Results pCMV-E2F-1-HA2 prospective fragments was obtained and the result of sequencing was completely consistent with the reported E2F-1 gene sequences.Cell clones with resistance to Genetecin were obtained and amplified.Experimental results of RT-PCR and Western blot confirmed that the recombinant vector pCMV-E2F-1-HA2 was successfully transfected into gastric cancer cell line MGC-803 and stably overexpressed the E2F-1 gene.Conclusion Eukaryotic expression vector pCMV-E2F-1-HA2 had been successfully constructed.Gastric cancer cell line MGC-803/E2F-1 with stable overexpression of the E2F-1 gene was established.

关 键 词：E2F-1 胃肿瘤 MGC-803细胞 转染 

分 类 号：R735.2[医药卫生—肿瘤] R73-35[医药卫生—临床医学]