摘要
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 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.
出处
《江苏大学学报（医学版）》
CAS
2012年第1期1-8,共8页
Journal of Jiangsu University：Medicine Edition
基金
国家自然科学基金资助项目(30972619)
