胸膜肺炎放线杆菌3种Apx毒素及外膜蛋白Omp2的原核表达及免疫反应性鉴定  

Prokaryotic expression and immunological identification of ApxⅠ, ApxⅡ, ApxⅢ and omp2 of Actinobacillus pleuropneumoniae

作　　者：万玉萌 安家慧 纪玉洁 郑瑞程 李玉峰[1] WAN Yumeng;AN Jiahui;JI Yujie;ZHENG Ruicheng;LI Yufeng(Key Laboratory of Bacteriology,Ministiy of Agriculture ,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学/农业部动物细菌学重点开放实验室,江苏南京210095

出　　处：《畜牧与兽医》2019年第5期89-93,共5页Animal Husbandry & Veterinary Medicine

摘　　要：猪传染性胸膜肺炎是猪群一种常见的细菌性疾病,给养猪业造成了巨大的经济损失。为了对胸膜肺炎放线杆菌的感染情况进行监测,本研究设计了针对该菌毒素ApxⅠ、ApxⅡ、ApxⅢ和外膜蛋白Omp2的全基因PCR扩增引物,以实验室保存的血清2型和5型菌株基因组DNA作为模板成功扩增出特异性基因片段,分别将目的片段克隆入原核表达载体成功构建重组表达质粒pColdI-omp2、pColdI-ApxⅡ、pET-32am-ApxⅠ、pCold-ProS2-ApxⅢ。将重组质粒转化Rosetta感受态细胞,通过IPTG诱导表达,应用His单克隆抗体和临床阳性猪血清对表达的重组蛋白上清进行Western blot分析,结果表明表达蛋白的分子量大小与预期相符,通过诱导条件的优化可以稳定表达可溶性蛋白,可溶性蛋白能与His单克隆抗体和临床阳性猪血清发生特异性反应,证明重组蛋白具有良好的免疫反应性。该试验为后期建立胸膜肺炎放线杆菌抗体的ELISA检测方法奠定基础。Porcine pleuropneumonia is a common bacterial disease in swine industry and has caused great economic losses.In order to monitor the infection status of Actinobacillus pleuropneumoniae(APP),four pairs of primers against ApxⅠ,ApxⅡ,ApxⅢ,and omp2 were designed.The gene encoding proteins ApxⅠ,ApxⅡ,ApxⅢ and omp2 were amplified from the genomic DNA of APP serotypes 2 and 5 strains and were cloned into expression vectors to obtain recombinant expression plasmids pColdI-omp2,pColdI-ApxⅡ,pET-32 am-ApxⅠ and pCold-ProS2-ApxⅢ,respectively.The recombinant plasmids were transformed into Rosetta(DE3) cells and induced using different concentrations of IPTG.The SDS-PAGE results showed that the recombinant proteins were successfully expressed and their molecular sizes were 46 kDa,114 kDa,134 kDa,and 140 kDa,respectively.Western blot analysis showed that the recombinant proteins reacted with the 6×His monoclonal antibody and the polyclonal antibody in swine.Especially,the soluble proteins were stably expressed by optimization of the induction conditions.This study would provide a basis for developing a novel ELISA assay to monitor the infection of APP.

关 键 词：猪传染性胸膜肺炎放线杆菌 ApxI ApxⅡ ApxⅢ Omp2 原核表达 

分 类 号：S852.6[农业科学—基础兽医学]