目的:制备由卡波济肉瘤相关疱疹病毒(Kaposi's sarcoma-associated herpesvirus,KSHV)编码的病毒FLICE抑制蛋白(viral FLICE inhibitory protein,vFLIP)的多克隆抗体,通过vFLIP重组蛋白对该抗体特异性进行鉴定,并将此抗体初步应用于天然病毒vFLIP蛋白表达的检测.方法:对vFLIP蛋白抗原表位进行预测分析,设计并合成3条多肽,将合成肽与载体蛋白钥孔戚血蓝素(KLH)偶联作为抗原,免疫新西兰白兔,制备抗vFLIP的多抗;以pCDH-vFLIP质粒为模板扩增vFLIP片段,插入到真核表达载体pEF-MCS-Flag-IRES/Puro中,构建pEF-vFLIP表达载体,利用脂质体将其转染HEK293T细胞,并在其中表达vFLIP.采用ELISA、蛋白质印迹法鉴定vFLIP多克隆抗体的效价及特异性,将该抗体用于天然病毒vFLIP的检测.结果:经限制性内切酶鉴定和核酸序列测定证实成功构建了重组质粒pEF-vFLIP,Flag抗体可以特异性识别该质粒在HEK293T和EA.hy926细胞内表达的vFLIP-flag融合蛋白.进一步的ELISA结果显示,制备的兔抗vFLIP多克隆抗体效价为1:11 000以上.该抗体不但与HEK293T和EA.hy926细胞内表达的重组vFLIP-flag融合蛋白反应,而且能够特异性识别PEL细胞中天然的病毒vFLIP蛋白.结论:构建了含vFLIP基因的重组真核表达载体;采用人工合成肽作为半抗原制备的抗KSHV vFLIP多抗,可以成功地检测重组vFLIP蛋白和天然病毒蛋白.%Objective: To prepare rabbit polyclonal antibodies against the Kaposi's sarcoma-associated 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 an-ti-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 Lipofectamine 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 ex-pressed 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.
展开▼
