猪胸膜肺炎放线杆菌结构蛋白基因ApxIVA特异性片段的表达和纯化

Expression and Bioactivity of ApxIVA Specific Fragment of Actinobacillus Pleuropneumoniae

以猪胸膜肺炎放线杆菌的血清8型分离株Hpn-405株基因组DNA为模板,PCR方法扩增ApxIVA特异性片段,PCR产物经纯化后与载体pMD18-T进行连接、转化,经酶切及序列分析鉴定后,亚克隆到原核表达载体pET-28a中,构建成重组表达质粒pET-ApxIVA3,转入到大肠埃希氏菌BL21(DE3)中,以IPTG进行诱导重组蛋白的表达,SDS-PAGE检测结果显示,表达的融合蛋白分子质量约为18 ku,与预期片段大小相符;将经过超声破碎的菌液离心取沉淀经尿素洗涤溶解后用镍金属螯合层析柱进行纯化。Western-blot分析证实,该融合蛋白具有免疫学活性。

The purpose of this study is to clone, identify, express and purify the secreted protein ApxlVA specific fragment of Actinobacillus pleuropneumoniae Hpn - 405 strain and to play a foundation for diagnosis and immunity. The gene encoding for protein ApxIVA specific fragment was amplified from APP Hpn -405 chromosomal DNA by using PCR technique. PCR product was cloned, and the strain containing the vectors was selected on LB - plus ampicillin （ 100 ug/ml） plates and digested with enzymes . Plasmids containing the right insertion were sequenced to confirm its identity and retransformed the combination into E. coll. BL21 （DE3） strain. Bacterial lyses prepared from 0.3mmoL/L IPTG induced cultures were loaded directly on to SDS -PAGE. Upon induction, the recombinant pET -ApxIVA3 produced indeed a new protein with an apparent MW of 18KDa. pET - ApxIVA 3was tagged with six histidine residuces at its C terminus and was purified by nicked chelation c phy. The fusion protein was immunogenic when detected by Westem- blot analysis.The purified protein was not detected in the inactivated APP bacteria vaccinated pigs, but was detected in A. pleuropneumoniae serotype 8 infected pigs. These results suggested that purified protein has Bioaetivity.