副猪嗜血杆菌ompP2基因的克隆、表达及间接ELISA抗体检测方法的建立

Cloning and expression of Haemoiphilus parasuis ompP2 gene and development of an indirect enzyme-linked immunosorbent assay for detecting antibody against Haemophilus parasuis

用PCR方法扩增副猪嗜血杆菌的外膜蛋白ompP2基因,序列测定结果表明扩增片段全长1 188bp。将扩增片段插入表达载体pET-32a,转化BL21进行诱导表达,SDS-PAGE电泳结果证实重组融合蛋白约为60 000,West-ern-blot结果表明重组表达蛋白具有免疫反应性。以纯化的重组蛋白ompP2作为抗原,建立副猪嗜血杆菌的抗体间接ELISA检测方法。通过试验确定最佳反应条件为抗原包被质量浓度4.75mg/L,4℃包被过夜,待检血清的稀释浓度为1∶40,阳性判断标准为D630大于0.383。特异性和重复性试验表明,建立的间接ELISA方法具有良好的特异性和敏感性,可用于副猪嗜血杆菌的检测。

The ompP2 gene which encode Haemophilus parasuis outer membrane protein was amplified by PCR.Sequencing results showed that the amplified ompP2 gene was 1 188 bp fragment,the gene was inserted into the expression vector pET-32a.A fusion protein was expressed in BL21 that transfected by pET-32a-ompP2 and induced by IPTG.The molecular weight of the recombinant was about 60 000 by SDS-PAGE,and the immunoreaction activity of the recombinant protein was confirmed by Western-blot.An indirect enzyme-linked immunosorbent assay for detecting antibody against HPS was developed by using expressed protein ompP2 as coating antigen.The results of tests showed that coating antigen concentration for ompP2 is 4.75 mg/L incubated optimally overnight at 4℃,and the serum dilution fold is 1∶40.The positive criterion for this ELISA assay is D6300.383.Specificity and sensitivity tests conformed that the developed ELISA had good stability and specificity.So this assay can be used as a tool of diagnosis and quarantine of Haemophilus parasuis