猪脑心肌炎病毒VP2蛋白单克隆抗体的制备及其识别表位分析

PREPARATION OF MONOCLONAL ANTIBODIES FOR VP2 PROTEIN OF PORCINE ENCEPHALOMYOCARDITIS VIRUS AND IDENTIFICATION OF RECOGNIZED EPITOPES

为获得针对猪源EMCV结构蛋白VP2的特异性单克隆抗体,本试验以EMCV结构蛋白VP2为对象,将EMCV BJC3株VP2基因定向插入穿梭质粒中,构建重组腺病毒AdV-VP2,免疫BALB/c小鼠制备单克隆抗体,利用Pepscan技术对单抗识别的抗原位点进行分析。结果显示,间接ELISA及间接免疫荧光法筛选获得了2个阳性细胞克隆株,命名为C11和G8。经检测杂交瘤细胞培养上清抗体效价为1:1 600,腹水效价分别为1:2.56×106和1:1.28×106,中和试验鉴定均无中和活性。Western-blot鉴定表明获得的2株单抗均能识别原核表达的重组VP2蛋白。亚类鉴定结果显示2株单抗均为IgG2b亚类,轻链均为κ型。Pepscan分析结果显示,这2株单抗可分别识别EMCV VP2蛋白上的2个B细胞线性表位。本研究获得的蛋白十分接近于其天然构象,很大程度上保证了病毒表面抗原位点的结构和功能。

To prepare specific monoclonal antibodies against structural protein VP2 of porcine encephalomyocarditis virus, the recombinant adenovirus was chosen as immunogen in this work. Using hybridoma technique, two positive cell clones named as C11 and G8 were obtained after screening by indirect ELISA and IFA. Both of the titres of culture supernatants were 1：1600 and ascites titres were 1： 2.56× 106 and 1：1.28× 106,respectively. The two MAbs had no neutralization activity as determined by using NT. The analysis of Western - blotting indicated that the two MAbs could recognize prokaryotic expressed VP2 protein. The subtype of them was IgG2b with （ light chain. The results of pepscan showed that two MAbs were able to recognize two linear B cell epitopes in EMCV VP2.