1株抗猪流行性腹泻病毒N蛋白单克隆抗体的制备及其抗原表位的鉴定

Preparation of a monoclonal antibody against N protein of porcine epidemic diarrhea virus and identification of its antigenic epitopes

为鉴定猪流行性腹泻病毒(PEDV)核衣壳(N)蛋白的抗原表位,本研究通过原核表达N蛋白,纯化后免疫BALB/c小鼠,利用细胞融合和亚克隆技术,筛选出1株稳定分泌抗PEDV N蛋白抗体的杂交瘤细胞9B4。经鉴定单克隆抗体9B4的亚型为IgG1型,轻链为Kappa链。腹水抗体ELISA效价为10^(6)以上。ELISA、Western blot和免疫荧光鉴定结果显示该单克隆抗体反应原性和特异性良好。利用噬菌体展示技术对单克隆抗体9B4进行抗原表位鉴定,获得4个N蛋白的模拟抗原表位(C11、C12、C14和C26)。随后分别以噬菌体阳性克隆为模拟抗原,对临床PEDV血清样品进行检测,发现其均能与猪PEDV阳性血清发生特异性结合,而与猪传染性胃肠炎病毒(TGEV)阳性血清、猪德尔塔冠状病毒(PDCoV)阳性血清不反应,说明本研究获得的N蛋白模拟表位可用于PEDV抗体的检测,有助于PED流行病学调查和PEDV疫苗免疫效果的评价。

In order to identify the epitopes of the PEDV N protein,this protein was firstly obtained by prokaryotic expression.The purified recombinant protein was used to immunize BALB/c mice.A hybridoma cell with stable secretion of the anti-PEDV N protein antibody was screened by cell fusion and sub-cloning techniques,and the cell was named 9B4.The subtype identification results showed that the heavy chains of the monoclonal antibody(McAb)belonged to IgG1,and its light chains were of Kappa.The antibody titer of the McAb 9B4 mouse ascites exceeded 106.The results of ELISA,Western blot and immunofluorescence identification showed that the McAb had good reactivity and specificity.The epitopes of McAb 9B4 was identified by phage display,and four simulated epitopes(C11,C12,C14 and C26)were obtained.These four phage positive clones were used as antigens to detect PEDV antibodies in clinical serum samples,which showed that C11,C12,C14 and C26 could react specifically with PEDV positive serum,but did not react with TGEV positive serum or PDCoV positive serum.This finding was helpful for epidemiological investigation of PED and evaluation of the immune effect of PEDV vaccines.