KSHV vFLIP在血管内皮细胞中的表达及其功能验证

Construction of the recombinant expression lentivirus vector carrying KSHV vFLIP gene and its protein expression in human umbilical vein endothelial cells

目的:构建含卡波氏肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus,KSHV)编码的病毒FLICE抑制蛋白(viral FLICE inhibitory protein,vFLIP)基因的重组慢病毒表达载体,获得稳定表达vFLIP的人脐静脉内皮细胞株(human umbilical vein endothelial cells,HUVECs),并检测vFLIP在HUVECs中活化NF-κB信号通路的能力。方法:以真核表达质粒pEF-vFLIP为模板扩增vFLIP基因,插入至慢病毒载体pHAGE-CMV-MCS-IZs-Green中构建重组质粒pHAGE-vFLIP。利用脂质体将其与包装质粒psPAX2和包膜质粒pMD2.G共转染293 T细胞,收集过滤后的培养上清获得慢病毒悬液。利用梯度稀释法测定病毒滴度后,以一定感染复数的慢病毒感染HUVECs,通过蛋白质印迹法检测vFLIP的表达。经绿色荧光蛋白(GFP)流式分选以及蛋白质印迹法验证获得稳定表达vFLIP的HUVECs。最后,通过虫荧光素酶报告实验检测NF-κB的活性,免疫荧光染色检测NF-κB亚基p65的细胞定位,蛋白质印迹法检测IκBα蛋白的表达来评价稳定表达vFLIP蛋白的HUVECs功能。结果:核酸序列测定证实含vFLIP基因的慢病毒表达载体已构建成功。重组慢病毒感染HUVECs后可检测到vFLIP的表达。通过流式分选获得了稳定表达vFLIP的HUVECs细胞,并且能通过抑制IκBα的降解,阻碍p65入核来活化NF-κB,从而激活经典NF-κB信号通路。结论:成功包装了含KSHV vFLIP基因的重组慢病毒,并获得具有激活NF-κB信号通路功能、稳定表达vFLIP的HUVECs株。

Objective： To construct the recombinant lentivirus carrying Kaposi＇s sarcoma-associated herpesvirus（KSHV）-encoded viral FLICE inhibitory protein（vFLIP） gene and to obtain human umbilical vein endothelial cells （HUVECs） stably expressing vFLIP protein. Methods： The fragment of vFLIP gene from expression plasmid pEF-vFLIP was cloned into the lentivirus vector pHAGE-CMV-MCS-IZs-Green. Then, the recombinant plasmid pHAGE-vFLIP, packaging plasmid psPAX2 and envelope plasmid pMD2. G were co-transfeeted into 293 T cells. The filtered culture media were harvested and the recombinant lentivirus was obtained. After detecting the titer of recombinant lentivirus, the expression of vFLIP protein in ]entivirus-infected HUVECs was detected by Western blot. Next, HUVECs stably expressing vFLIP were obtained by flow cytometry assay（FCM） and verified the expression of vFLIP by Western blot. Finally, the functional verifications were performed in HUVECs stably expressing vFLIP, including detecting the activity of NF-κB by luciferase assay,the location of p65 by immunofluorescence assay（IFA）, and the expression of IkBα protein by Western blot. Results： The recombinant lentivirus vector containing vFLIP was constructed successfully, and the expression of vFLIP in HUVECs infected with lentivirus-vFLIP was detectable. The HUVECs stably expressing vFLIP were gained successfully by FCM, which could inhibit the degradation of IκBα and the transloeation of p65 to activate NF-κB pathway. Conclusion： The recombinant lentivirus carrying KSHV vFLIP gene was successfully constructed, and the HUVECs stably expressing vFLIP with the ability of activating NF-κB pathway were obtained.