基因C型鸭甲肝病毒单克隆抗体制备及其抗原表位鉴定

Monoclonal antibody preparation and the epitope identification of genotype C duck hepatitis A virus

为制备基因C型鸭甲肝病毒(DHAV-C)单克隆抗体(MAb),本研究根据DHAV-C ORF编码氨基酸序列与基因A型DHAV(DHAV-A)相应序列比对结果,利用生物信息学软件筛选位于DHAV-C ORF编码蛋白序列上的不同抗原表位,合成5条十肽表位序列,分别与KLH载体蛋白偶联后免疫BALB/c小鼠,经细胞融合筛选获得5株稳定分泌抗DHAV-C MAb杂交瘤细胞株。Western blot显示,5株MAb均能够与DHAV-C发生特异性反应,其中5H24-9、3E18-4和5E3-9 3株MAb不与DHAV-A、鸭瘟病毒、新城疫病毒和禽流感病毒反应,为DHAV-C的特异性MAb,同时也证明与这3株MAb结合的十肽序列为DHAV-C的抗原表位。这3株MAb亚类分别为IgG2b、IgG1和IgG2b。MAb的制备为建立DHAV-C的检测方法和致病机理奠定了基础。

To identify antigen epitopes of genotype C duck hepatitis A virus （DHAV-C） and prepare corresponding monoclonal antibodies （MAbs）, the potential antigen epitopes of DHAV-C were predicted by bioinforrnatic soflwares according to its complete deduced amino acid sequences and artificially synthetized as different 10-amino-acid polypeptides to immune BALB/c mice for preparation of MAbs. In addition, five MAbs were prepared by cell fusion technique. Western blot results showed that three of the five MAbs reacted specifically with DHAV-C, but not with genotype A duck hepatitis A virus （DHAV-A）, duck plaque virus, Newcasstle disease virus （NDV） and avian influenza virus （AIV）. The MAb subclasses of the three specific MAbs were IgG2b, IgG1 and IgG2b, respectively. The preparation of MAbs and the verification of DHAV-C antigen epitopes provided the basis for the further development of detection method for DHAV-C.