人EGFL7基因短发夹结构RNA慢病毒载体的构建及鉴定

Construction and identification of lentiviral vector-mediated short hairpin RNA of human EGFL7 gene

目的构建人类表皮生长因子域7(EGFL7)基因RNA干扰(RNAi)慢病毒载体。方法将靶向EGFL7基因的短发夹结构RNA(shRNA)表达序列连接到包含U6启动子及绿色荧光蛋白(GFP)报告基因的慢病毒载体pGCL-GFP中,获得重组质粒,命名为pGCL-GFP-vshEGFL7,经多聚酶链反应(PCR)和测序鉴定后,与慢病毒包装质粒pHelper 1.0及pHelper 2.0通过lipofectamine 2000共转染至包装细胞293T,包装产生病毒液,测定其滴度。结果PCR扩增和测序结果证实EGFL7shRNA核苷酸链序列插入正确,包装慢病毒产生病毒悬液的滴度为4.8×107TU/ml。结论成功构建人EGFL7基因shRNA慢病毒载体。

Objective To construct a lentiviral vector-mediated RNA interference （RNAi） of human epidermal growth factor-like domain 7 （ EGFL7 ） gene. Methods A EGFL7 gene targeting short hairpin RNA （shRNA） was cloned into lentiviral expression vector pC, CL-GFP, which contained U6 promoter and green fluorescent protein （GFP） reporter. The recombined vector named pGCL-GFP-vshEGFL7 was confirmed by polymerase chain reaction （PCR） and sequencing, and was eotransfeeted with pHelper 1.0 and pHelper 2. 0 packaging plasmids mixtured into 293T cells by lipofectamine 2000. Virus in the supernatant was collected and the virus titer was measured. Results PCR and DNA sequencing demonstrated that the inserted sequences were correct. The titer of virus was 4.8 × 107 TU/ml. Conclusion The lentivirus RNAi vector targeting EGFL7 has been constructed successfully.