坏死梭杆菌120ku血凝素相关外膜蛋白的原核表达及免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of Hemagglutinin-related Outer Membrane Protein fromF.necrophorum

为了揭示坏死梭杆菌(F.necrophorum)120ku血凝素相关外膜蛋白的免疫原性,根据F.necrophorum120ku血凝素相关外膜蛋白基因的抗原表位分布情况设计特异性引物,扩增出3个彼此重叠、覆盖整个开放阅读框的基因片段p1、p2、p3,分别构建重组表达质粒pET-28a-p1、pET-32a-p2、pET-32a-p3,在大肠杆菌中进行诱导表达,通过SDS-PAGE电泳和Western blotting分析重组蛋白的表达情况和反应原性;以纯化的重组蛋白为抗原免疫试验兔,分析重组蛋白的免疫原性。结果显示,重组蛋白P1、P2、P3在大肠杆菌中均得到表达,分子质量分别约为48、59、62ku,能与F.necrophorum阳性的小鼠血清反应,可以刺激试验兔产生特异性抗体,说明F.necrophorum120ku血凝素相关外膜蛋白具有良好的免疫原性,为F.necrophorum基因工程亚单位疫苗的研制提供参考依据。

In order to analyze the immunogenicity of 120 ku hemagglutinin-related outer membrane protein fromF.necrophorum,specific primers were designed to amplify the 3truncated overlapping gene fragments covering the whole open reading frame（ORF）based on the result of antigenic epitope analysis.The gene fragments were ligated into the prokaryotic expression vector to construct pET-28a-p1,pET-32a-p2 and pET-32a-p3,respectively.Then recombinant plasmid was induced expression in E.coli.The SDS-PAGE results showed that the expression of recombinant proteins P1,P2 and P3,molecular mass of the expressed proteins were about 48,59 and 62ku,respectively.Western blotting indicated that the recombinant proteins could be recognized by antiserum against F.necrophorumfrom mouse.Humoral immunity response against recombinant proteins was detected in the immunized rabbits.Altogether,these findings suggested that 120 ku hemagglutinin-related outer membrane protein fromF.necrophorum had good immunogenicity,and this study provided a reference for further research on genetic engineering subunit vaccine of F.necrophorum.