猪瘟病毒糖蛋白E^rns中和表位的鉴定和比较

猪瘟病毒 (CSFV)囊膜结构糖蛋白Erns(gp4 8)是诱导机体产生中和抗体及激发保护性免疫应答的第二抗原蛋白。E2和Erns与细胞表面受体的相互作用介导CSFV感染细胞的过程。Erns具有RNA酶活性 ,影响病毒自身复制并涉及对病毒的中和效应。采用抗CSFValfortT櫣ｂｉｎｇｅｎ毒株Ｅｒｎｓ糖蛋白的 1B5 ,b4_2 2和 2 4 16单克隆中和抗体 ,筛选噬菌体展示的 12肽随机肽库 ,进行Erns中和表位的鉴定和比较 ,获得分别针对 1B5、b4_2 2和 2 4 16单克隆抗体的 3个主要中和表 (拟 )位基序WxNxxP、DKNR (Q)G和A(T)CxYxKN ,分别定位于Erns的 35 1位～ 35 6位或 348位～ 35 0位、384位～ 386及 32 2位～ 32 3位、380位～ 386位氨基酸区域。分析表 (拟 )位基序与单克隆抗体的免疫反应性差异。b4_2 2和 2 4 16单克隆抗体识别基序存在共有序列KN ,识别Erns中的相似抗原区 ,但其侧翼序列及免疫印迹、免疫荧光抗体抑制试验结果均存在显著差异

Identification and comparison of neutralizing epitopes of glycoprotein Erns of Classical swine fever virus

Structural and envelope glycoprotein E(rns) (gp48) of classical swine fever virus (CSFV) is the second antigenic protein being responsible for eliciting neutralizing antibodies and conferring protective immunity. Infection of cells with CSFV is mediated by the interaction of glycoprotein E(rns) and E2 with the cell surface receptors. The glycoprotein E(rns) has been shown to contain RNase activity, which plays a role in the viral life cycle and is also involved in virus neutralization. Neutralizing epitopes of glycoprotein E(rns) had been mapped by screening a 12-mer random peptide phage display library using the neutralizing monoclonal antibodies (MAbs) 1B5, b4-22 and 24/16, raised against CSFV strain alfort T bingen and reacted with glycoprotein E(rns). Three major epitope (mimotope) motifs, WxNxxP, DKNR (Q) G and A (T) CxYxKN (around amino acid position aa351-aa356 or aa348-aa350, aa384-aa386 and aa322-aa323, aa380-aa386 of glycoprotein E(rns) of CSFV) were identified respectively and characterized immunologically by the MAbs, 1b5, b4-22 and 24/16. MAbs b4-22 and 24/16 shared a part of binding motif sequence KN, and recognized the similar antigenic domain on the glycoprotein E(rns) but showed a distinct pattern of flank sequence and reactivities with the mimotopes by Western blot and inhibition of immunofluorescent antibody analysis.