猪胸膜肺炎放线杆菌毒素I基因的克隆、表达及其ELISA检测方法的建立  

Cloning, Expression and Establishment of the ELISA Detection Method of theapxⅠCA Gene of Actinobacillus pleuropneumoniae

作　　者：刘建杰[1] 何启盖[1] 陈焕春[1] 吴斌[1] 徐晓娟[1] 刘军发[1] 唐先春[1] 贝为成[1] 

机构地区：[1]华中农业大学动物医学院动物病毒室,武汉430070

出　　处：《中国农业科学》2004年第1期148-151,共4页Scientia Agricultura Sinica

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

摘　　要：根据胸膜肺炎放线杆菌S4 0 74菌株毒素I的序列 ,设计了 1对引物 ,从本室自己分离的菌株中扩增了猪胸膜肺炎放线杆菌毒素I的结构基因 (apxICA) ,扩增的DNA片段大小为 36 4 0bp(4 6 87～ 832 6bp) ,将其克隆到pMD18 T载体中 ,酶切鉴定和序列测定后 ,进一步将其插入pET 2 8a中构建了原核表达载体 ,SDS PAGE和Westernblotting分析表明 ,该基因在大肠杆菌BL2 1(DE3 )中获得了表达 ,而且表达的蛋白质具有免疫学活性。利用表达的蛋白作为抗原包被酶标板 ,建立了检测毒素I血清抗体的ELISA方法。临床应用表明 。A pair of primers were designed according to the sequence of the apxICA of Actinobacillus pleuropneumoniae (S4074) and a 3 640 bp(4 687-8 326 bp)gene segment was amplified by PCR with the template of Actinobacillus pleuropneumoniae we isolated. The amplified DNA fragment was cloned into pMD18-T and identified by R.E. analysis and sequencing, then inserted into an expression vector( pET-28a) to yield the expression plasmid. SDS-PAGE and Western blotting analysis showed that the apxICA gene was expressed in BL 21(DE 3) and the expression products had immunogenicity. The ELISA method was established using the expressed protein to detect ApxI serum antibody, the clinical application showed that it could be used to detect highly virulent APP strains.

关 键 词：猪 胸膜肺炎 放线杆菌毒素 I基因克隆 基因表达 ELISA检测 

分 类 号：S852.61[农业科学—基础兽医学]