摘要： 为获得副猪嗜血杆菌外膜蛋白P5的分段表达产物,并对各片段的免疫学性质进行鉴定。采用生物信息学软件分析Omp P5全长基因,设计3对分段引物,PCR分别从HPS汕头分离株(H0801)Omp P5中扩增出P5F1、P5F2和P5F3基因片段,构建于pET-32a(+)原核表达载体上,并将重组质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达,利用SDS-PAGE分析蛋白表达情况。将蛋白纯化后,利用ELISA及Western Blot分别鉴定其免疫原性及抗原性。结果表明,扩增得到的Omp P5的3个基因片段与预期大小相符合,基因测序结果与GenBank公布的序列一致,经SDS-PAGE分析,3个蛋白表达相对分子质量大小都与预期相符。ELISA和Western Blot分析显示,表达的3个蛋白表现出良好的抗原性和免疫原性。HPS Omp P5的分段表达为进一步研究HPS保护性表位及疫苗鉴定奠定了新的基础。

关键词: 副猪嗜血杆菌, 外膜蛋白P5, 抗原性分析

Abstract: This experiment was conducted to express outer membrane protein P 5 of Haemophilus parasuis (HPS) and analysis immunological claracteristics. Three overlapping fragments P5F1,P5F2 and P5F3 of P5 gene were amplified by PCR and cloned into the expression vector pET 32a( +),respectively.It was confirmed that the recombinant plasmids pET32a-P5F1,pET32a-P5F2 and pET32a-P5F3 were constructed correctly by using restric-tion endonuclease digestion and sequencing. The plasmid were transformed into E.coli BL21 ( DE3 ) and the target proteins of 38,32,27 kDa were expressed by induction using IPTG. The immune activity of the proteins was ana-lyzed by Western Blot and ELISA. The results showed that three proteins could react with HPS positive serum. The above results indicated that these recombinant fusion proteins might be useful as an antigen for vaccine and the spe -cific diagnosis antigen for ELISA assay.

Key words: Haemophilus parasuis, Outer membrane protein P5, Analysis of antigenicity

中图分类号: 

• S852.61