EGFL7 基因RNAi慢病毒表达载体的构建及鉴定

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OligoDesigner 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× 108、2× 108和5× 108TU/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。

Construction of the Recombinant Lentivirus of RNAi Targeting Human EGFL7 Gene

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× 108, 2× 108 and 5× 108TU/mL, respectively. The interfering effect of each sequence was assessed and compared.Conclusion: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.