猪胸膜肺炎放线杆菌毒素ⅢA基因的克隆、序列分析及原核表达  

Cloning and Expression of the apxⅢA Gene of Actinobacillus pleuropneumonie

作　　者：陈汉阳[1] 刘军发[1] 何启盖[1] 肖少波[1] 陈焕春[1] 

机构地区：[1]华中农业大学畜牧兽医学院动物病毒室,武汉430070

出　　处：《微生物学报》2003年第3期324-329,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金 (30 2 0 0 0 11);湖北省"十五"重点科技攻关项目 (2 0 0 1AA2 0 1B0 2 )~~

摘　　要：因胸膜肺炎放线杆菌的致病性主要是由毒素决定的 ,故参照猪胸膜肺炎放线杆血清2型菌株的序列 (GenBankL1 2 1 4 5)设计了一对特异性引物 ,用PCR的方法扩增apxⅢA基因并得到了长 3 466bp的片段 ,然后将其克隆到pMD 1 8T中 ,经酶切鉴定和序列分析表明克隆是成功的 ;再将apxⅢA插入到原核表达载体pET 2 8b后 ,转化BL2 1 (DE3) ,在IPTG诱导下获得高效表达 ,经Westernblotting检测证实表达产物有活性。以表达产物包被ELISA板 ,建立了特异、敏感的ELISA诊断方法。The apxⅢA gene of Actinobacillus pleuropneumonie (App) was amplified by PCR. The amplified DNA fragment 3 466bp was cloned into pMD18-T. After R.E. analysis and sequencing, the apxⅢA gene in pMD18-T was ligated into pBluescripⅡSK(+), the recombinant expression plasmid pET-28b/apxⅢA was constructed and analysed with R.E., the protein of apxⅢA gene expressed in E.coli BL21 was detected by Western blotting.Based on expressed apxⅢA protein as antigen,empty expression vector as control,the ELISA to detect antibody against apxⅢA was developed and was primarily used to detect serum samples.

关 键 词：猪 胸膜肺炎放线杆菌 毒素ⅢA基因 克隆 序列分析 原核表达 

分 类 号：S852.6[农业科学—基础兽医学]