埃博拉病毒NP蛋白的单克隆抗体制备及抗原肽的定位

Generation and epitope mapping of a monoclonal antibody against nucleoprotein of Ebola virus

摘要:

埃博拉病毒 (Ebola virus，EBOV) 是一种能导致人类及脊椎动物出血热的致死性病毒，对公共卫生具有较严重的危害。EBOV的NP蛋白在病毒复制中具有重要作用，也是诊断该病重要的靶蛋白。文中原核表达重组扎伊尔型EBOV的NP蛋白，重组蛋白免疫bal/c小鼠，制备了一株小鼠抗EBOV-NP的单克隆抗体。利用Western blotting方法，该抗体能特异识别真核表达和原核表达的重组EBOV-NP，并能同莱斯顿型 (Reston Ebola virus, REBOV)、科特迪瓦型 (Cote-d’Ivoire Ebola virus, CIEBOV) 和本迪布焦型 (Bundibugyo Ebola virus, BEBOV) 埃博拉病毒产生交叉反应，而不与苏丹型 (the Sudan Ebola virus, SEBOV) 和马堡型 (the Marburg virus, MARV) 埃博拉病毒产生反应。利用突变PCR和Western blotting方法，定位了该抗体识别的抗原决定簇序列，该序列 (PPLESD) 位于EBOV-NP蛋白的C端583-588aa。生物信息学研究表明，该序列在已经公布的ZEBOV、CIEBOV、BEBOV共16个型和REBOV的4个型中高度保守。研究结果为建立以上各型埃博拉病毒的检测方法提供了工具，也为研究埃博拉病毒复制及致病机理提供了基础。

Abstract:

Ebola virus (EBOV) causes highly lethal hemorrhagic fever in humans and nonhuman primates and has a significant impact on public health. The nucleoprotein (NP) of EBOV (EBOV-NP) plays a central role in virus replication and has been used as a target molecule for disease diagnosis. In this study, we generated a monoclonal antibody (MAb) against EBOV-NP and mapped the epitope motif required for recognition by the MAb. The MAb generated via immunization of mice with prokaryotically expressed recombinant NP of the Zaire Ebola virus (ZEBOV-NP) was specific to ZEBOV-NP and able to recognize ZEBOV-NP expressed in prokaryotic and eukaryotic cells. The MAb cross-reacted with the NP of the Reston Ebola virus (REBOV), the Cote-d’Ivoire Ebola virus (CIEBOV) and the Bundibugyo Ebola virus (BEBOV) but not with the NP of the Sudan Ebola virus (SEBOV) or the Marburg virus (MARV). The minimal epitope sequence required for recognition by the MAb was the motif PPLESD, which is located between amino acid residues 583 and 588 at the C-terminus of ZEBOV-NP and well conserved among all 16 strains of ZEBOV, CIEBOV and BEBOV deposited in GenBank. The epitope motif is conserved in four out of five strains of REBOV.