Abstract：Objective: To prepare rabbit polyclonal antibodies against the Kaposi′s sarcomaassociated herpesvirus(KSHV)encoded viral FLICE inhibitory protein (vFLIP) and appraise the specificity with the vFLIP recombinant protein, and initially applied to the detection of natural viral protein.Methods: With the analysis of the vFLIP conformational epitopes by means of bioinformatics, three sequences were chosen and synthesized. The synthesized peptides were conjugated to keyhole limpet hemocyanin(KLH) to increase anti-genicity. New zealand rabbits were immunized with KLH conjugated peptides to generate polyclonal antibodies against KSHV vFLIP; the fragment of vFLIP gene from pCDH-vFLIP was cloned into the eukaryotic expression vector pEF-MCS-Flag-IRES/Puro, then the recombinant vector pEF-vFLIP was transfected into the 293T cells by LipofectamineTM 2000 to obtain vFLIP-Flag fusion protein; the polyclonal antibodies induced were charactered by ELISA and Western blot, then applied to the detection of natural viral protein. Results: The recombinant expression vector carrying KSHV vFLIP was constructed successfully. By using the Flag antibody, vFLIP-Flag fusion protein was detected in 293T and EA.hy926 cells transfected with pEF-vFLIP. Further, ELISA results showed that the titer of induced polyclonal rabbit anti-vFLIP antibodies higher than 1∶11 000. The antibodies could specifically react with vFLIP-Flag fusion protein which expressed in 293T and EA.hy926 cells as well as natural viral protein. Conclusion: The recombinant expression vector pEF-vFLIP was constructed successfully; the synthesized vFLIP peptides was used to immunized rabbit to generate polyclonal antibodies against KSHV vFLIP; the antibodies could specifically react with vFLIP-Flag fusion protein as well as natural viral protein in PEL cell lines.
[1] Chang Y, Cesarman E, Pessin MS,et al. Identification of herpesvirus like DNA sequences in AIDS associated Kaposi′s sarcoma[J]. Science, 1994, 266(5192): 1865-1869.[2] Katano H, Sata T. Human herpesvirus 8 virology, epidemiology and related diseases[J]. Jpn J Infect Dis, 2000, 53(4): 137-155.[3] Dourmishev LA, Dourmishev AL, Palmeri D,et al. Molecular genetics of Kaposi′s sarcoma associated herpesvirus (human herpesvirus 8) epidemiology and pathogenesis[J]. Microbiol Mol Biol Rev, 2003, 67(2): 175-212. [4] Sarid R, Flore O, Bohenzky RA,et al. Transcription mapping of the Kaposi′s sarcoma associated herpesvirus (human herpesvirus 8) genome in a body cavity based lymphoma cell line (BC 1)[J]. J Virol, 1998, 72(2): 1005-1012.[5] Nicholas J. Human herpesvirus 8 encoded proteins with potential roles in virus associated neoplasia[J]. Front Biosci, 2007, 12: 265-281.[6] Schulz TF. Kaposi′s sarcoma associated herpesvirus (human herpesvirus 8): epidemiology and pathogenesis[J]. J Antimicrob Chemother, 2000, 45 (Suppl T3): 15-27.[7] Thome M, Schneider P, Hofmann K,et al. Viral FLICE inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors[J]. Nature, 1997, 386(6624): 517-521.[8] Grundhoff A, Ganem D. Mechanisms governing expression of the v-FLIP gene of Kaposi′s sarcoma associated herpesvirus[J]. J Virol, 2001, 75(4): 1857-1863.[9] Bieleski L, Talbot SJ. Kaposi′s sarcoma associated herpesvirus vCyclin open reading frame contains an internal ribosome entry site[J]. J Virol, 2001, 75(4): 1864-1869.[10] Low W, Harries M, Ye H,et al. Internal ribosome entry site regulates translation of Kaposi′s sarcoma associated herpesvirus FLICE inhibitory protein[J]. J Virol, 2001, 75(6): 2938-2945.[11] Chaudhary PM, Jasmin A, Eby MT,et al. Modulation of the NF kappa B pathway by virally encoded death effector domains containing proteins[J]. Oncogene, 1999, 18(42): 5738-5746.[12] Chugh P, Matta H, Schamus S,et al. Constitutive NF kappa B activation, normal Fas induced apoptosis, and increased incidence of lymphoma in human herpes virus 8 K13 transgenic mice[J]. Proc Natl Acad Sci U S A, 2005, 102(36): 12885-12890.[13] Matta H, Mazzacurati L, Schamus S,et al. Kaposi′s sarcoma associated herpesvirus (KSHV) oncoprotein K13 bypasses TRAFs and directly interacts with the Ikappa B kinase complex to selectively activate NF kappa B without JNK activation[J]. J Biol Chem, 2007, 282(34): 24858-24865.[14] Ye FC, Zhou FC, Xie JP,et al. Kaposi′s sarcoma associated herpesvirus latent gene vFLIP inhibits viral lytic replication through NF kappa B mediated suppression of the AP1 pathway: a novel mechanism of virus control of latency[J]. J Virol, 2008, 82(9): 4235-4249.[15] Zhao J, Punj V, Matta H,et al. K13 blocks KSHV lytic replication and deregulates vIL6 and hIL6 expression: a model of lytic replication induced clonal selection in viral oncogenesis[J]. PLoS One, 2007, 2(10): e1067.[16] Xu Y, Ganem D. Induction of chemokine production by latent Kaposi′s sarcoma associated herpesvirus infection of endothelial cells[J]. J Gen Virol, 2007, 88(Pt 1): 46-50.[17] An J, Sun Y, Sun R,et al. Kaposi′s sarcomaassociated herpesvirus encoded vFLIP induces cellular IL-6 expression: the role of the NFkappa B and JNK/AP1 pathways[J]. Oncogene, 2003, 22(22): 3371-3385.[18] Godfrey A, Anderson J, Papanastasiou A,et al. Inhibiting primary effusion lymphoma by lentiviral vectors encoding short hairpin RNA[J]. Blood, 2005, 105(6): 2510-2518.[19] Guasparri I, Keller SA, Cesarman E. KSHV vFLIP is essential for the survival of infected lymphoma cells[J]. J Exp Med, 2004, 199(7): 993-1003.[20] Alkharsah KR, Singh VV, Bosco R,et al. Deletion of Kaposi′s sarcoma associated herpesvirus FLICE inhibitory protein, vFLIP, from the viral genome compromises the activation of STAT1 responsive cellular genes and spindle cell formation in endothelial cells[J]. J Virol, 2011, 85(19): 10375-10388.[21] Sturzl M, Hohenadl C, Zietz C,et al. Expression of K13/v-FLIP gene of human herpesvirus 8 and apoptosis in Kaposi′s sarcoma spindle cells[J]. J Natl Cancer Inst, 1999, 91(20): 1725-1733.[22] Thurau M, Marquardt G, Gonin Laurent N,et al. Viral inhibitor of apoptosis vFLIP/K13 protects endothelial cells against superoxide induced cell death[J]. J Virol, 2009, 83(2): 598-611.[23] Matta H, Chaudhary PM. Activation of alternative NF kappa B pathway by human herpes virus 8 encoded Fas associated death domain like IL-1 beta converting enzyme inhibitory protein (vFLIP)[J]. Proc Natl Acad Sci U S A, 2004, 101(25): 9399-9404.[24] Bagneris C, Ageichik AV, Cronin N,et al. Crystal structure of a vFlip IKKgamma complex: insights into viral activation of the IKK signalosome[J]. Mol Cell, 2008, 30(5): 620-631.[25] Field N, Low W, Daniels M,et al. KSHV vFLIP binds to IKK gamma to activate IKK[J]. J Cell Sci, 2003, 116(Pt 18): 3721-3728.[26] Sun Q, Matta H, Lu G,et al. Induction of IL-8 expression by human herpesvirus 8 encoded vFLIP K13 via NF kappaB activation[J]. Oncogene, 2006, 25(19): 2717-2726.[27] Emuss V, Lagos D, Pizzey A,et al. KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia[J]. PLoS Pathog, 2009, 5(10): e1000616.[28] Lee JS, Li Q, Lee JY,et al. FLIP mediated autophagy regulation in cell death control[J]. Nat Cell Biol, 2009, 11(11): 1355-1362.[29] Kessenbrock K, Raijmakers R, Fritzler MJ,et al. Synthetic peptides: the future of patient management in systemic rheumatic diseases?[J]. Curr Med Chem, 2007, 14(26): 2831-2838.[30] Chen SW, Van Regenmortel MH, Pellequer JL. Structure activity relationships in peptide antibody complexes: implications for epitope prediction and development of synthetic peptide vaccines[J]. Curr Med Chem, 2009, 16(8): 953-964.[31] Bucher MH, Evdokimov AG, Waugh DS. Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin binding protein[J]. Acta Crystallogr D Biol Crystallogr, 2002, 58(Pt3): 392-397.
2012, Vol. 22 
