鸭疫里默氏杆菌pfs基因的克隆表达及单克隆抗体制备  

PREPARATION OF MONOCLONAL ANTIBODIES TO PFS OF RIEMERELLA ANATIPESTIFER

作　　者：范国博 韩先干[1] 张宇曦[1,2] 王少辉[1] 左佳坤 徐达[1,3] 于圣青[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]安徽农业大学动物科技学院,合肥230036 [3]扬州大学兽医学院,扬州225000

出　　处：《中国动物传染病学报》2015年第5期41-45,共5页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31370045;31572546;31272591)

摘　　要：将鸭疫里默氏杆菌的pfs基因进行原核表达,重组蛋白经纯化后作为抗原免疫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,利用间接ELISA方法筛选阳性杂交瘤细胞株。通过间接ELISA法测定腹水效价、Western blot法检测其免疫反应性,并鉴定抗体的亚型。重组蛋白SDS-PAGE检测结果验证得到26 k Da Pfs蛋白,免疫小鼠后最终获得1株阳性杂交瘤细胞株,命名为2E2E6,为Ig G1亚类,间接ELISA法检测腹水效价为1:64 000。Western blot结果表明制备的单克隆抗体具有良好的免疫反应性。本研究成功制备了Pfs的单克隆抗体,为进一步研究Pfs在鸭疫里默氏杆菌中的作用提供了参考。To prepare the monoclonal antibody against Pfs protein of Riemerella anatipestifer, the recombinant pfs（rpfs） was expressed and purified for immunization of BALB/c mice. After immunization for 3 times, mice were sacrificed for harvest of spleens. The splenocytes from the immunized mice were fused with murine myeloma SP2/0 cells. Positive clones were identified by screening with indirect ELISA and sub-cloned for 3 times. The hybridoma 2E2E6 producing anti-pfs monoclonal antibody（MAb） was obtained. The isotype of this MAb was determined to be Ig G1. Ascitic fluid was titrated to be 1：64 000 in ELISA. In Western blot, MAb 2E2E6 reacted with serotype 2 R.anatipestifer, but not with R.anatipestifer serotypes 1 and 10. The availability of MAb 2E2E6 would be useful for further study of pfs activity.

关 键 词：鸭疫里默氏杆菌 Pfs蛋白 单克隆抗体 

分 类 号：S852.612[农业科学—基础兽医学]