胸膜肺炎放线杆菌apxⅡCA基因的克隆、表达及ApxⅡ-ELISA检测方法的初步建立  

Cloning and Expression of the Gene apxⅡCA of Actinobacillus pleuropneumoniae and Primary Development of Enzyme-linked Immunosorbent Assay

作　　者：徐晓娟[1] 何启盖[1] 徐高原[1] 陈焕春[1] 

机构地区：[1]华中农业大学动物医学院,湖北武汉430070

出　　处：《中国兽医学报》2004年第4期335-337,共3页Chinese Journal of Veterinary Science

基　　金：湖北省重点科技攻关项目 ( 2 0 0 1AA2 0 1B0 2 ) ;国家自然科学基金资助项目 ( 3 0 2 0 0 0 11)

摘　　要：根据国外已发表的胸膜肺炎放线杆菌 (APP) Apx 的基因序列设计了 1对引物 ,从我国 APP武汉株扩增出约3.5 kb的 apx CA基因。测序结果表明 ,该基因的大小与国外已报道的 1型和 9型 APP的 apx CA基因大小一致 ,同源性达 99%。将该基因连接到原核表达载体 p ET- 17b,构建了原核表达质粒 p ET- apx CA,并在大肠杆菌 BL 2 1(DE3)中表达出了大小约 10 5 0 0 0的 Apx 蛋白。提取包涵体蛋白作为抗原包被 EL ISA酶标板 ,对已知阳性、阴性血清和临床送检血清样品进行了 EL ISA检测 ,从而为最终建立 Apx - ELThe 3.5 kb toxic gene apxⅡCA of Actinobacillus pleuropneumoniae was got and amplified by PCR after a pair of particular primer was deviced according to the published sequence abroad.The sequencing result showed that the size of this gene was the same as that published in serotype 1 and 9,and it reached 99% homogeneity of the corresponding gene in serotype 1 and 9.The gene apxⅡCA was cloned into prokaryotic expression vector pET-17b to develop the recombinant plasmid pET-apxⅡCA,which was transformed into BL(DE3),as a result,a particular (105 000) protein was produced.The inclusion protein was exacted and used as immunogent to coat the polystyrene U-bottom microtiter plates to detect clinical sera.This study is a preliminary step for enzyme-linked immunosorbent assay for Actinobacillus pleuropneumoniae.

关 键 词：胸膜肺炎放线杆菌 apxⅡCA基因 克隆 表达 ELISA 

分 类 号：S852.619[农业科学—基础兽医学]