猪胸膜肺炎放线杆菌外膜蛋白基因(OMP-1)的克隆、表达及其ELISA方法的建立

Cloning and Expression of OMP-1 Gene of Actinobacillus Pleuropneumoniae and Establishment of Its ELISA Method

猪传染性胸膜肺炎由猪胸膜肺炎放线杆菌(APP)引起,是猪最重要的呼吸道传染病之一。通过筛选APP 3～8 kb随机基因组文库,获得1株阳性克隆。测序拯救质粒得到其DNA序列,通过BLAST分析确定此片段为APP外膜蛋白基因,预计成熟蛋白质的分子量为48.766 kD。根据序列设计特异性引物,用PCR扩增该基因,分子克隆表达该外膜蛋白。Western blot结果表明,该蛋白与APP康复期血清发生反应。纯化该重组蛋白,并将其作为抗原包被ELISA酶标板,对已知阳性血清、阴性血清和临床送检血清样品进行ELISA检测,为最终建立APP ELISA检测方法奠定了基础。

Actinobacillus pleuropneumoniae （APP） causes porcine contagious pleuropneumoniae, one of the most important infectious respiratory diseases. It is identified an out membrane protein （OMP- 1 ） by screening a phage library of 3 -8 kb random DNA fragments of APP. The inserted segment encoding the entire amino acid sequence of the OMP-1 was determined by sequencing the rescued plasmids and BLAST analysis. Specific primers were designed to amplify the OMP-1 gene. The OMP-1 was cloned, expressed and purified. Western blot results showed strong reactivity of the protein with convalescent sera （IgG） . The recombinant OPM-1 was coated into 96-weU plate as antigen to examine the known and unknown samples. The study is a preliminary step for ELISA of APP.