猪源产毒素多杀性巴氏杆菌HN-13株外膜蛋白H的基因克隆与特征

摘要

Progressive atrophic rhinitis is an important respiratory disease in swine industry, caused by toxigenic Pasteurella multocida. The outer membrane protein H (OmpH) of P. mumtocida is a major outer membrane protein involved in the pathogenesis and immunity. In this study the 1 020 bp coding sequence was amplified from P. multocida serotype D strain HN-13. DNA sequencing and bioinformatics analysis revealed that OmpH has the typical characteristics of membrane channel protein (porin). Then the ompH gene was cloned into the prokaryotic expression vector pET-28a producing recombinant plasmid pET-ompH. SDS-PAGE analysis showed that the gene was highly expressed in Escherichia coli BL21 (DE3), and the recombinant protein (rOmpH) existed mostly in form of inclusion bodies. Polyclonal antisera were generated by immunizing rabbits with the purified rOmpH. Western blotting analysis showed that the antisera could specifically react with the proteins of HN-13 cells. An indirect enzyme-linked immunosorbent assay(ELISA) was established using the rOmpH. The optimal antigen coating concentraation was set as 2.89 μg/mL, and the cut-off value(OD630) was 0.36. A hundred and ninety-two clinical swine serum samples were screened using the ELISA, showing a positive rate of 51.6% (99/192), and negative rate of 48.4% (93/192). This study provideds useful data for further use of the OmpH.%传染性萎缩性鼻炎是养猪业中的一种重要的呼吸道传染病,产毒素多杀性巴氏杆菌(Pasteurella multocida)是其重要的病原之一,外膜蛋白H(OmpH)在该菌的致病和免疫中发挥重要作用.以产毒素多杀性巴氏杆菌血清D型菌株HN-13基因组为模板,扩增了ompH基因1 020bp编码区,经过序列测定和生物信息学分析,发现OmpH蛋白具有典型的膜通道蛋白的特征.将ompH基因克隆到原核表达载体pET-28a中构建了重组表达质粒pET-ompH,转化大肠杆菌(Escherichia coli)BL21(DE3),经IPTG诱导,实现了高效表达,表达产物主要以包涵体的形式存在.用纯化的重组蛋白免疫家兔制备OmpH的抗血清,Western杂交显示抗血清能够与菌体蛋白发生特异性反应.以重组OmpH为包被抗原建立了间接ELISA方法,方阵滴定确定最佳抗原包被浓度为2.89μg/mL,用56份参考阴性猪血清确定阴阳界限值为0.36(OD630).用建立的ELISA方法对192份临床猪血清样品进行检测,结果显示OmpH抗体阳性率为51.6%(99/192),阴性率为48.4%(93/192).研究为OmpH的进一步应用奠定了基础.