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| Construction of the recombinant expression lentivirus vector carrying KSHV vFLIP gene and its protein expression in human umbilical vein endothelial cells |
| JIN Liuyang, YAN Qin, LU Chun |
| Department of Microbiology and Immunology, Nanjing Medical University, Nanjing Jiangsu 210029, China |
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Abstract Objective: To construct the recombinant lentivirus carrying Kaposi′s sarcomaassociated 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 pEFvFLIP was cloned into the lentivirus vector pHAGECMVMCSIZsGreen. Then, the recombinant plasmid pHAGEvFLIP, packaging plasmid psPAX2 and envelope plasmid pMD2.G were cotransfected 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 lentivirusinfected 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 NFκB by luciferase assay,the location of p65 by immunofluorescence assay(IFA),and the expression of IκBα protein by Western blot. Results: The recombinant lentivirus vector containing vFLIP was constructed successfully,and the expression of vFLIP in HUVECs infected with lentivirusvFLIP was detectable.The HUVECs stably expressing vFLIP were gained successfully by FCM, which could inhibit the degradation of IκBα and the translocation of p65 to activate NFκB pathway. Conclusion: The recombinant lentivirus carrying KSHV vFLIP gene was successfully constructed, and the HUVECs stably expressing vFLIP with the ability of activating NFκB pathway were obtained.
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Received: 17 October 2013
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2014, Vol. 24 
