人GLIPR-2基因慢病毒RNAi载体的构建及鉴定

目的 构建人GLIPR-2基因慢病毒RNA干扰（RNAi）载体,并鉴定其感染人肝癌细胞系HepG2后缺氧条件下的GLIPR-2基因表达变化.方法 根据GLIPR-2基因序列合成siRNA片段,克隆至慢病毒载体pMAGic7.1上,转染293T细胞进行包装,收获并浓缩重组慢病毒颗粒.感染HepG2细胞,流式细胞术检测GFP的表达率,并用Western blot检测目的 蛋白在缺氧条件下的表达变化.结果 重组RNAi质粒pMAGic7.1-shRNA-GLIPR-2经菌落PCR鉴定及测序证实构建正确;稳定感染HepG2 细胞后,GFP的表达率为分别为84.48%、69.97% 和39.82%;Western blot结果显示慢病毒转染组GLIPR-2蛋白的表达水平较对照组明显降低.结论成功构建了GLIPR-2基因慢病毒RNAi载体,该载体在缺氧条件下可明显抑制目的 蛋白在HepG2细胞中表达,为进一步研究GLIPR-2的生物学功能奠定了基础.

Construction and identification of RNAi lentiviral vectors targeting human GLIPR-2 gene

Objective To construct a lentiviral RNA interference（RNAi） vector targeting GLIPR-2 gene and identify the expres- sion of GLIPR-2 in infected hepatocellular carcinoma cell line HepG2 under hypoxia condition. Methods The siRNA fragment was synthesized according to GLIPR-2 gene sequence and cloned into lentiviral vector pMAGic7.1. The recombinant lentivirus plasmid was transfected to 293T cell line,and the packaged virus was determined for titer. Recombinant lentivirus particles were harvested and concentrated,then infected to HepG2 cells and determined the expression rate of GFP by flow cytometry and the expression of GLIPR-2 by Western blot under hypoxia condition. Results PCR amplification of bacterial colony and sequencing proved that re- combinant lentiviral plasmid were constructed correctly. The expression rate of GFP in HepG2 cells stably infected with the recom- binant lentiviral plasmid was 84. 48%,69.97% and 39. 82%, while the expression level of GLIPR-2 significantly decreased com- pared with the control. Conclusion The lentiviral RNA interference vector GLIPR-2 was constructed successfully and could sup- press GLIPR-2 expression in HepG2 cells,which laid a foundation of further study on biological function of GLIPR-2.