鸭疫里默氏杆菌GroEL蛋白的原核表达和单克隆抗体制备

GENERATION OF MONOCLONAL ANTIBODIES TO GROEL OF RIEMERELLA ANATIPESTIFER

本试验旨在研制鸭疫里默氏杆菌GroEL蛋白的单克隆抗体。将鸭疫里默氏杆菌WJ4菌株的groEL基因进行原核表达,重组蛋白经过纯化后作为免疫原免疫BALB/c小鼠。经过3次免疫后,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行细胞融合,用间接ELISA筛选阳性细胞株并进行3次亚克隆后制备小鼠腹水单克隆抗体。共获得2株稳定分泌鸭疫里默氏杆菌GroEL单克隆抗体的杂交瘤细胞株,分别命名为1G2B6和1G2F10。2株单克隆抗体均为IgG1亚类。腹水单克隆抗体ELISA效价达到1:102 400。Western blot检测结果表明2株单克隆抗体均能与鸭疫里默氏杆菌血清1、2和10型菌株发生特异性结合,而与禽致病性大肠杆菌、沙门氏菌和禽巴氏杆菌菌株无反应性。本研究成功制备了稳定性好、特异性强的鸭疫里默氏杆菌GroEL单克隆抗体,为进一步研制基于抗体检测鸭疫里默氏杆菌抗原的试剂盒奠定了基础。

The objective of this study was to generate monoclonal antibodies (MAbs) against GroEL protein of Riemerellaanatipestifer. The GroEL gene was cloned from R. anatipestifer strain WJ4 and the recombinant GroEL was expressed in E. coli. After purification, the recombinant GroEL was used to immunize BALB/c mice 3 times. Spleen cells of immunized mice were fused with murine myeloma SP2/O cells. Specific MAb secreting hybridomas were screened using indirect ELISA and positive hybridomas were cloned 3 times. As a result, two hybridoma cell lines (1G2B6, 1G2F10) stably producing anti-GroEL MAbs were identified and established. The isotype of both MAbs was IgG1. The ELISA titer of ascetic fluids was determined to be 1:102 400. In Western blot, both MAbs reacted with R. anatipestifer serotypes 1, 2 and 10 strains but not with Escherichia coli, Salmonella enterica and Pasteurella multocida strains. Besides, both MAbs were specific for R. anatipestifer serotypes 1, 2 and 10 strains tested. The availability of MAbs specific for GroEL paved a path for further development of diagnostic kit for R.anatipestifer.