副猪嗜血杆菌oppA基因的克隆、表达及间接ELISA检测方法的建立

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

采用PCR方法扩增副猪嗜血杆菌(Hps)的寡肽结合蛋白(Opp A)基因,序列测定结果表明,扩增片段全长1654 bp.将扩增片段插入表达载体p GEX-6P-1中,转化BL21进行诱导表达,SDS-PAGE电泳结果表明,重组融合蛋白大小约为87 ku,以纯化的重组蛋白Opp A作为抗原,建立Hps抗体的间接ELISA检测方法.通过试验确定最佳的反应条件为:抗原包被浓度为1.0μg·孔-1,于37℃包被1 h,待检血清的稀释度为1∶50,阳性判断标准(D450 nm)大于0.643.特异性和重复性试验结果表明,建立的间接ELISA方法具有良好的特异性和稳定性,可用于Hps抗体的检测.

The opp A gene which encodes Haemophilus parasuis oligopeptide binding protein A（ Opp A） was amplified by PCR. Sequencing results showed that amplified opp A gene was 1654 bp fragment,the gene was inserted into the expression vector p GEX-6p-1. A fusion protein was expressed in BL21 that transfected by p GEX-6p-1-opp A and induced by IPTG. The molecular weight of the recombinant was about 87 ku by SDS-PAGE. An indirect enzyme-linked immunosorbent assay for detecting antibody against H. parasuis was developed by using Opp A protein expressed as coating antigen. The results of tests showed that coating antigen concentration for Opp A is 1 μg·well- 1incubated optimally 1 h at 37 ℃,and the serum dilution fold is 1 ∶ 50. The positive criterion for this ELISA assay is D450 nm 0. 643. Specificity and repeatability tests conformed that developed ELISA had good specificity and stability. So this assay can be used as a tool of diagnosis and quarantine of H. parasuis.