KSHV抗凋亡蛋白vFLIP编码基因的克隆表达及抗vFLIP多克隆抗体的制备与鉴定

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。

Recombinant expression of KSHV vFLIP and preparation of rabbit polyclonal antibodies against KSHV vFLIP

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 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.