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

马新廷， 郝婷婷， 朱小飞， 卢春

江苏大学学报（医学版） ›› 2012, Vol. 22 ›› Issue (1) : 1.

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江苏大学学报（医学版） ›› 2012, Vol. 22 ›› Issue (1) : 1.
基础医学

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

  • 马新廷， 郝婷婷， 朱小飞， 卢春
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Recombinant expression of KSHV vFLIP and preparation of rabbit polyclonal antibodies against KSHV vFLIP

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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。

Abstract

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.

关键词

病毒FLICE抑制蛋白 / 合成肽 / 多克隆抗体 / 卡波济肉瘤相关疱疹病毒

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马新廷, 郝婷婷, 朱小飞, 等. 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[J]. 江苏大学学报（医学版）, 2012, 22(1): 1
MA Xin-Ting, Hao-Ting-Ting, Zhu-Xiao-Fei, et al. Recombinant expression of KSHV vFLIP and preparation of rabbit polyclonal antibodies against KSHV vFLIP[J]. Journal of Jiangsu University(Medicine Edition), 2012, 22(1): 1

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基金

国家自然科学基金资助项目（30972619）


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