逆转录病毒载体介导的hLIF基因真核稳定表达体系的建立

Establishment of Eukaryotic Expression System of hLIF Gene via Retrovirus Vector

目的构建逆转录病毒载体介导的人白血病抑制因子(human leukemia inhibitory factor,hLIF)基因真核稳定表达细胞株。方法采用DNA重组技术,构建并鉴定重组逆转录病毒载体pEGZ/MCS-HA-hLIF;以脂质体转染法,将重组逆转录病毒载体与辅助质粒HIT456、HIT60共转染入包装细胞系293T包装逆转录病毒,感染人胚肾成纤维细胞系,经zeocin筛选获得真核稳定表达细胞株;采用RT-PCR法和ELISA法鉴定hLIF基因的转录和翻译,并检测表达上清中hLIF因子对人白血病细胞HL-60的生物学活性。结果PCR产物电泳后于609 bp处见特异性条带。双酶切出现两条带,其中位于609 bp处可见相应条带。核酸测序结果与GenBank中所报道的hLIF基因的CDS序列完全一致。RT-PCR结果显示表达细胞中存在hLIF基因转录,ELISA结果测定表达上清中hLIF的浓度为110.134 pg/ml。结论携带hLIF基因的重组逆转录病毒载体pEGZ/MCS-HA-hLIF构建成功,并且获得了稳定表达hLIF基因的人胚肾成纤维细胞株,这为此基因的临床应用和实验研究打下了基础。

Objective To establish the eukaryotic expressing cell strains of human leukemia inhibitory factor （hLIF） gene via retrovirus vector. Methods The recombined retrovirus vector pEGZ/MCS-HA-hLIF was contructed and identified by means of DNA recombination technology. The recombined vector and assisted plasmids HIT456 and HIT60 were co-transfected into 293T cell to get retrovirus.The human embryo kidney fibroblasts were tranfected and the eukaryotic expressing cell strains were established by zeocin; hLIF gene＇s transcription and translation were identified by RT-PCR and ELISA assay, meanwhile, the supernatant＇s biological activity on HL-60 cells--a kind of leukemia cell line was detected. Results Objective gene fragment appeared in PCR and endonucleases digestion products. The full cDNA sequence of hLIF were totally conformed with Genbank. The hLIF gene could be transcripted in the expressing cell strains. The hLIF concentration in the supernatant of 293T-hLIF was about 110.134 pg/ml. The supernatants could inhibit the growth of HL-60. The mechanism might induce the tumor cells＇ apoptosis and cell cycle arrest. Conclusion The recombined retrovirus vector pEGZ/MCS-HA-hLIF is successfully constructed, meanwhile human embryo fibroblasts rained, which may provide good basic for hLIF search. cell strains stably expressing gene ＇ s gene is obapplication and experiment research.