iASPP真核表达载体构建及其功能鉴定

Construction of eukaryotic expression vector of iASPP and its identification of biological function

目的构建P53凋亡刺激蛋白(ASPP)家族的抑制成员iASPP的真核表达载体,并将其通过脂质体转染到结肠癌细胞株SW480及Lovo中,观察转染前后iASPP表达变化及其对细胞凋亡的影响。方法将解放军第十六医院检验科经测序鉴定的pMD19-T-iASPP质粒亚克隆至真核表达质粒pcDNA3.1(+),构建重组真核表达质粒pcDNA3.1(+)-iASPP,测序鉴定后用脂质体将重组质粒转染至结肠癌细胞株SW480及Lovo中,用逆转录聚合酶链反应(RT-PCR)检测iASPP的表达以及用流式细胞仪检测细胞凋亡的变化情况。结果重组表达质粒pcDNA3.1(+)-iASPP,经酶切测序与GenBank上记录的人iASPP cDNA序列(gi 60457962)完全一致。经pcDNA3.1(+)-iASPP质粒转染的结肠癌细胞株SW480及Lovo的iASPP mRNA表达增高,细胞凋亡率下降。结论成功构建了重组表达质粒pcDNA3.1(+)-iASPP,并成功在结肠癌细胞株SW480及Lovo中获得了表达,细胞株的凋亡率下降,提示抑制iASPP高表达有可能成为恢复P53抑癌功能的新策略。

Objective Construct the eukaryotic expression vector of inhibitory member of the ASPP family（iASPP）and transfect it into colon carcinoma cell lines SW480and Lovo by liposome.Then observe the expression of iASPP and detect the cell apoptosis by flow cytometry.Methods The amplified PCR product was digested and inserted into pMD19-T simple vector and subcloned into eukaryotic expression vector pcDNA3.1（＋）.The recombinant eukaryotic expression plasmid pcDNA3.1（＋）-iASPP was transfected into colon carcinoma cell lines SW480and Lovo by liposome,the iASPP expression was analyzed by RT-PCR.The cell apoptosis was detected by FCM.Results The eukaryotic expression plasmid pcDNA3.1（＋）-iASPP was constructed successfully,the gene squence of iASPP was consistent with that reported（gi 60457962）in GenBank.The mRNA expression levels of iASPP gene of SW480and Lovo cell lines which transfect the positive plasmid were increased,and the cell apoptosis rates were decreased.Conclusion We successfully constructed the recombinant expression plasmid pcDNA3.1（＋）-iASPP,and the plasmid were successfully expressed in colon carcinoma cell lines SW480and Lovo,the cell apoptosis rates of those cell lines were decreased.These facts indicated that reducing the high expression of iASPP may be a new strategy to renew the abilities of P53tumor suppressor.