EGFL7基因RNAi慢病毒表达载体的构建及鉴定（英文）  

Construction of the Recombinant Lentivirus of RNAi Targeting Human EGFL7 Gene

作　　者：王爱岳[1] 李强[2] 许琼冠[2] 刘达远 徐鹏翔[2] 余丹[1] 

机构地区：[1]海口市人民医院神经内科,海南海口570208 [2]海南省农垦总医院神经外科,海南海口570311

出　　处：《现代生物医学进展》2016年第26期5042-5046,共5页Progress in Modern Biomedicine

基　　金：Project of Natural Science Foundation of Hainan Province(814375);Project of Hainan Province Health Department(2010-31)~~

摘　　要：目的：构建人类表皮生长因子域7（EGFL7）基因RNA干扰（RNAi）重组慢病毒表达载体。方法：参照小分子干扰RNA的设计原则,应用Oligo Designer 3.0软件设计三条靶向人EGFL7基因的RNA干扰序列（hEGFL7-RNAi）,并将其分别插入含有绿色荧光蛋白（GFP）的慢病毒载体pLV3中,获得重组质粒,与包装质粒pRsv-REV、pMDlg-pRRE和pMD2G共同转染293T细胞,包装产生重组慢病毒,培养72 h后,应用qRT-PCR检测慢病毒感染人脐静脉内皮细胞（HUVEC）后靶基因mRNA的水平,以评价其基因沉默效果。结果：筛选出3条人EGFL7基因的RNAi序列,分别包装出重组慢病毒,其滴度分别为1×10^8、2×10^8和5×10^8TU/mL,将其转染入HUVEC后,EGFL7mRNA表达均受到明显抑制（P〈0.05）。结论：成功筛选出三条针对人EGFL7基因的RNAi有效靶序列,并成功构建重组慢病毒表达载体,证明该序列可沉默HUVECs中EGFL7基因的表达。Objective： To construct the recombinant lentivirus of RNAi targeting human Epidermal Growth Factor-like domain 7 （EGFL7） gene. Methods： The RNAi sequences targeting human EGFL7 gene were inserted into pLV3 lentivirus vectors containing green fluorescent protein. Recombinant plasmids were thus obtained and transfected to 293T cells along with packaging plasmids pRsv-REV, pMDlg-pRRE and pMD2G, so as to produce recombinant lentivirus, which later infected HUVECs. The gene silencing effect was then determined through examination of the resulting mRNA level of the targeted gene in the HUVECs. Results： Three RNAi sequences of human EGFL7 gene were selected out and packaged into recombinant lentivirus, with the titers of 1 × 10^8, 2 ×10^8 and 5 × 10^8TU/mL, respectively. The interfering effect of each sequence was assessed and compared. Conclusions： Three effective RNAi targeting sequences for human EGFL7 gene were selected out, and recombinant lentivirus was successfully constructed. It was demonstrated that the sequences can significantly silence the expression of the EGFL7 gene in HUVECs.

关 键 词：类表皮生长因子域7 RNA干扰 慢病毒 

分 类 号：R-33[医药卫生] Q78[生物学—分子生物学]