鸡SIgA重链单克隆抗体的制备及新城疫病毒和禽流感病毒特异黏膜SIgA检测方法的建立  

Preparation of the monoclonal antibody against chicken SIgA heavy chain and establishment of ELISA for detection of Newcastle disease virus and avian influenza virus specific mucosal SIgA

作　　者：肇慧君[1] 陈伟业[2] 葛金英[2] 王喜军[2] 步志高[2] 

机构地区：[1]辽宁出入境检验检疫局,辽宁大连116001 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2012年第2期138-142,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家攻关项目(2004BA519A19;2005BA711A10);国家973项目(2005CB523200)

摘　　要：为建立快速检测禽类黏膜组织中SIgA含量的方法,本研究原核重组表达鸡SIgA重链片段蛋白,纯化后免疫BALB/c鼠。以鸡胆汁中分离纯化的SIgA作为检测抗原,常规单克隆技术筛选出1株能够稳定分泌抗鸡SIgA单克隆抗体(MAb)的IgG2a、κ型MAb。经ELISA和western blot分析,所获得的1株MAb亲和力高、特异性强;采用生物素标记纯化MAb腹水IgG为检测二抗,以禽流感-新城疫重组二联活载体疫苗免疫SPF鸡为模型,初步建立了新城疫病毒和H5亚型禽流感病毒特异的黏膜SIgA间接ELISA检测方法。本研究为开展特异性家禽黏膜免疫机制的研究提供了重要技术手段。To establish the method for detection of specific mucosal SlgA, BALB/c mice was immunized with puriticdrecombinant chicken partial SIgA heavy protein expressed in E. coli. A hybridoma cell line which steadily secreted monoclonalantibody （MAb） against chicken SIgA was prepared by fusion the mouse spleen cells with SP2/0 cells and screened by H.ISA coating with SIgA from chicken bile. Isotope of the MAb was lgG2a, K type. ELISA and western blot showed the MAb was highaffinity and specificity. An indirect ELISA method based on biotin-labeled MAb for the detection of Newcastle disease virus （NDV） and H5 avian influenza virus （H5 AIV） specific mueosal SIgA was established, and the method was successfully used forthe detection of NDV and H5 AIV specific SIgA in salivary of chickens immunized with recombinant NDV live vaccine. Our study provided technological evidence for mucosal immunity mechanism in poultry.

关 键 词：SIGA 单克隆抗体 新城疫病毒 H5亚型禽流感病毒 ELISA 

分 类 号：S852.65[农业科学—基础兽医学]