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

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(h UVECs)稳定表达。方法 (1)聚合酶链反应扩增目的基因,设计合成sh RNA,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(q RT-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h UVECs,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(h UVECs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q RT-PCR与Western blotting检测分别从m RNA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h UVECs中稳定表达。

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 2×10^8 TU/ml,2×10^8 TU/ml and 1×10^8 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.