禽传染性支气管炎病毒S1蛋白单克隆抗体制备及其中和抗原表位的鉴定

Preparation of monoclonal antibody against S1 protein of avian infectious bronchitis virus and identification of its epitopes

为鉴定禽传染性支气管炎病毒(IBV)刺突(S1)蛋白的中和抗原表位,本研究通过IBV全病毒免疫BALB/c小鼠,经融合、亚克隆筛选获得了4株稳定分泌抗IBV S1蛋白抗体的杂交瘤细胞株4A11、1B11、5E5和7C9。经鉴定制备的单克隆抗体腹水抗体ELISA效价为106以上,Western blot和间接免疫荧光试验分析显示该单克隆抗体反应性和特异性良好。随后用8种基于S1分型的IBV毒株为试验毒株,同实验室已有的8株IBV S1单抗(1E9、1H1、1E4、3C6、3C7、2F3、2E5、4F9)共12株单抗进行气管环中和活性检测,发现S1蛋白单抗与8种S1亚型的毒株均有不同程度的中和作用。随后,在抗原表位和Western blot分析的基础上,鉴定出单克隆抗体识别的抗原表位区域,获得了6个中和抗原表位,其中除^(416)IQTRTEP^(422)表位,其他5个表位仍未有相关报道。本研究成功制备出4株特异性识别S1蛋白且具有中和活性的单克隆抗体,不仅丰富了IBV的单克隆抗体库,为今后研究IBV S1蛋白分子结构提供了关键生物材料;获得的6个S1蛋白中和抗原表位也为后续建立IBV疫苗免疫效果评价方法和亚单位疫苗开发提供了工具。

In order to identify the neutralizing epitope of the spike(S1)protein of avian infectious bronchitis virus(IBV),four monoclonal antibody(mAb)hybridoma cell lines 4A11,1B11,5E5and 7C9 stably secreting anti-IBV S1 protein antibodies were prepared from BALB/c mice immunized with IBV whole virus through cell fusion and subcloning.The mAb ascites were further prepared with ELISA titer more than 10~6.Western blot and indirect immunofluorescence analysis showed that the mAbs had good reactivity and specificity.Subsequently,8 strains of different IBV genotypes based on S1 gene and total of 12 mAbs including 8 mAbs(1E9,1H1,1E4,3C6,3C7,2F3,2E5,4F9)previously prepared in our lab were selected to perform a tracheal ring neutralization test.The mAbs all had certain degree neutralizing activity to the remaining 8 strains with different genotypes.Based on the epitope and Western blot analysis,a series of eukaryotic vectors carrying truncated S1 gene fragments were constructed,and total of six neutralizing epitopes recognized by the mAbs were obtained.Except for^(416)IQTRTEP^(422),the other five epitopes have not been reported so far.In this study,four mAbs to IBV S1 protein with neutralizing activity were successfully prepared,which not only enriched the mAb library of IBV S1,but also provided key biomaterials for the future study of the molecular structure and function of IBV S1 protein.The six neutralizing epitopes of S1 protein obtained here also provide a tool for the further development of a detection method for evaluation of IBV vaccine efficacy and novel subunit vaccine for IBV control.