蓬乱蛋白2 干扰和过表达慢病毒载体的构建 及其在hUVECs 中的表达

Construction of lentivirus vector of DVL2 in hUVECs

摘要:

目的 获得蓬乱蛋白2（DVL2）基因干扰和过表达慢病毒表达系统，并在人脐静脉内皮细胞中（hUVECs） 稳定表达。方法 ①聚合酶链反应扩增目的基因，设计合成shRNA，以慢病毒表达质粒为基础构建DVL2 干扰 和过表达载体，酶切电泳、实时荧光定量聚合酶链反应（qRT-PCR）和测序技术鉴定载体构建是否成功；② 以3 质粒系统在HEK293T 细胞中包装病毒，通过荧光显微镜观察计数并计算病毒滴度；以嘌呤霉素筛选稳定转 染的hUVECs，通过荧光显微镜观察计数获得转染效率。将感染好的细胞分为BC 组（hUVECs 空白对照）、NC 组（HBLV-GFP-PURO 阴性对照）、干扰组（pHB-shRNA-HDVL2）及过表达组（pHBLV-HDVL2）。③通过 qRT-PCR 与Western blotting 检测分别从mRNA 和蛋白表达水平验证目的基因的干扰和过表达水平。结果 ①插 入慢病毒表达载体的基因片段与目的基因的碱基序列完全一致。干扰序列峰形图为单峰，无突变。②病毒包装 后，NC 组、干扰组、过表达组滴度分别为2×108、2×108 和1×108 TU/ml，感染人脐静脉内皮细胞的感染效率 达98%。③干扰组DVL2 的表达较BC 组降低，差异有统计学意义（P <0.05），过表达组较BC 组升高，差异有统 计学意义（P <0.05），其中干扰效率为61%，蛋白质过表达水平为BC 组的2.7 倍。结论 DVL2 基因干扰和过表 达慢病毒载体构建成功，并能够在原代hUVECs 中稳定表达。

Abstract:

Objective To obtain interference and overexpression of DVL2 through lentivirus, and its expression in hUVECs. Methods ShRNAs were designed and synthesized, and DVL2 interference and overexpression vectors were constructed based on lentivirus expression plasmids. PCR and sequencing techniques were utilized to confirm successful construction of vector. Three plasmids system were performed to package the virus in HEK293T cells. Virus titer was measured by fluorescence microscope. HUVECs transfected with virus were identified by Purinemycin. Transfection efficiency was measured by fluorescence microscope. Further, the interference and overexpression of the target gene was verified by qRT-PCR and WB. Results Electrophoretic and sequencing results showed that the gene fragment was successfully inserted into the lentivirus vector. The titer of NC, pHB-shRNAHDVL2 and pHBLV-HDVL2 was 2×108 TU/ml, 2×108 TU/ml and 1×108 TU/ml, respectively. Infection efficiency was 98%. qRT-PCR and Western blotting confirmed stable expression of DVL2. Expression of DVL2 was decreased in interference group and increased significantly when compared with that in NC group (P < 0.05). Interference efficiency was 60%, and overexpression efficacy was 270% compared with control group. Conclusions Interfering and overexpression of DVL2 through lentivirus vector is constructed successfully in HUVECs.