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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 a recombinant lentiviral vector containing the viral FLICE inhibitory protein (FLFLP) gene encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV), and to obtain a human umbilical vein stably expressing vFLIP Human umbilical vein endothelial cells (HUVECs) were cultured and the ability of vFLIP to activate NF-κB signal pathway in HUVECs was detected. Methods: The vFLIP gene was amplified by eukaryotic expression plasmid pEF-vFLIP and inserted into lentiviral vector pHAGE-CMV-MCS-IZs-Green to construct recombinant plasmid pHAGE-vFLIP. 293T cells were co-transfected with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G by liposomes, and the filtered culture supernatant was collected to obtain a lentivirus suspension. After the virus titer was determined by gradient dilution method, HUVECs were infected with a certain number of infected lentiviruses and the expression of vFLIP was detected by Western blotting. HUVECs stably expressing vFLIP were obtained by green fluorescent protein (GFP) flow cytometry and Western blotting. Finally, the activity of NF-κB was detected by luciferase reporter assay, the localization of NF-κB subunit p65 was detected by immunofluorescence staining, and the expression of IκBα protein was detected by Western blotting to evaluate the function of HUVECs stably expressing vFLIP protein. Results: Nucleic acid sequence analysis confirmed that the lentiviral vector containing vFLIP gene was successfully constructed. The expression of vFLIP was detected after recombinant lentivirus infected HUVECs. HUVECs stably expressing vFLIP cells were obtained by flow cytometry, and NF-κB activation was activated by inhibiting the degradation of IκBα and hindering p65 entry into the nucleus. Conclusion: The recombinant lentivirus containing vFLIP gene of KSHV was successfully packaged and the HUVECs strain with the function of activating NF-κB signaling and expressing vFLIP stably was obtained.