EDAG-1基因真核表达载体的构建及其对细胞恶性生物学行为的影响  

Construction of eukaryotic expression vector of EDAG-1 and the functional research on malignant biological characteristics of cells

作　　者：郑贤芳[1] 杨帆[1] 储著朗[1] 汪思应[1] 

机构地区：[1]安徽医科大学病理生理学教研室,合肥230032

出　　处：《安徽医科大学学报》2011年第11期1133-1137,共5页Acta Universitatis Medicinalis Anhui

基　　金：安徽省十五攻关课题资助(编号:a01303006)

摘　　要：目的构建胚胎发育相关基因-1(EDAG-1)基因真核表达载体pcDNA3.1(+)-EDAG-1,并检测其对细胞的恶性生物学行为的影响。方法应用逆转录聚合酶链式反应(RT-PCR)法从白血病细胞株YAC-1中扩增到EDAG-1基因片段,构建重组真核表达载体pcDNA3.1(+)-EDAG-1。利用克隆PCR、限制性内切酶消化进行验证。将重组pcD-NA3.1(+)-EDAG-1真核表达载体和阴性对照的空载体pcDNA3.1分别转染到NIH3T3细胞和Ba/F3细胞中,应用软琼脂集落实验检测NIH3T3细胞转染前后的集落形成能力,流式细胞术检测Ba/F3细胞不依赖白介素-3(IL-3)抗凋亡能力以及其发生的机制。结果①扩增片段与预期片段大小相符,测序结果与GenBank公布一致,克隆成功,且鉴定证实pcDNA3.1(+)-EDAG-1真核表达载体构建成功。②转染真核表达载体pcDNA3.1(+)-EDAG-1后的NIH3T3细胞集落形成能力明显增高,而对照组不能形成集落。转染后Ba/F3细胞的不依赖IL-3抗凋亡能力明显提高。③在Ba/F3-EDAG细胞中,核因子κB的DNA结合活性明显高于对照细胞,而Stat3与Stat5的磷酸化未发生明显的变化。结论成功构建真核表达载体pcDNA3.1(+)-EDAG-1,为进一步研究EDAG-1基因在白血病和甲状腺癌细胞中的功能奠定了基础。Objective To construct the eukaryotic expression vector of full length EDAG-1 gene,and research the effects on malignant biological characteristics of cells.Methods The EDAG-1 gene was amplified by reverse transcriptase-polymerase chain reaction（RT-PCR）,then inserted it into pcDNA3.1（＋）.The recombinant plasmid pcDNA3.1（＋）-EDAG-1 was confirmed correctly through restriction enzyme digesting and PCR identification,which was transfected into NIH3T3 cell and Ba/F3 cells by lipid media transfection.The untransfected NIH3T3 and NIH3T3 transfected with pcDNA3.1（＋） were used as controls.EDAG-1 gene was detected by Western blot.The effects of EDAG-1 on cell proliferation and cell cycle progression were measured by soft agar colony formation and flow cytometry.Activity of NF-κB and Stat were used to study the effects of EDAG-1 gene.Results ① The amplified fragment by PCR conformed to the anticipated result,and its sequence was agreement with that published on GenBank.Therefore,the EDAG-1 gene was cloned successfully.Furthermore,the recombinant plasmid pcDNA3.1（＋）-EDAG-1 was constructed successfully through idenfication.② Overexpression of EDAG-1 in NIH3T3 cells resulted in anchorage-independent growth in soft agar,but the control group could not form colonies.Overexperssion of EDAG in IL-3-dependent Ba/F3 cells decreased the apoptosis in absence of IL-3.③In the Ba/F3-EDAG cells,NF-κB DNA binding activity was significantly higher than that in control,while Stat3 and Stat5 phosphorylation was not changed significantly.Conclusion The recombinant pcDNA3.1（＋）-EDAG-1 is constructed successfully,for further study of EDAG-1 gene function in leukemia and thyroid cancer cells.

关 键 词：EDAG-1 基因表达 克隆 基因转染 NF-ΚB 肿瘤 

分 类 号：R73[医药卫生—肿瘤] R341[医药卫生—临床医学]