副猪嗜血杆菌转铁结合蛋白A基因的原核表达与鉴定

Prokaryotic expression of transferrin-binding protein A gene from Haemophilus parasuis and immunological analysis of the recombinant protein

为获得副猪嗜血杆菌(HPS)转铁结合蛋白A基因(tbpA)的表达产物,根据GenBank发表的HPS(SH0165株)tbpA的核苷酸序列设计合成1对特异性引物,以HPS血清13型安徽分离株(LJ3)的基因组DNA为模板,利用PCR扩增tbpA基因,然后将其克隆至pET-SUMO载体,构建重组原核表达质粒pET-SUMO-tbpA,通过PCR鉴定和测序确认,将重组质粒转化大肠杆菌Rosetta(DE3)中,并进行IPTG诱导表达和His-Ni-resin纯化目的蛋白。经SDS-PAGE检测,获得了110ku的表达产物,与预期大小的转铁结合蛋白A(TbpA)的分子质量一致。Western-blot分析表明,表达产物与HPS阳性血清能发生反应,显示表达产物具有良好的免疫活性。结果表明,成功表达的TbpA为研制HPS新型疫苗和诊断试剂奠定了基础。

To gain the expression product from tbpA gene of Haemophilus parasuis（HPS）,the gene was amplified from HPS serotype 13 Anhui strain（LJ3） by PCR,with primers based on the tbpA sequence of HPS SH0165 strain in GenBank,then it was cloned into pET-SUMO vector.The recombinant plasmid was certified with PCR identification and DNA sequencing and was then transformed into competent Rosetta（DE3） bacteria.The TbpA protein was expressed in resultant strains induced with IPTG and purified with His-Ni-resin.The product was confirmed to be about 110 ku in size by SDS-PAGE.Immunogenicity of the product was also identified by Western-blot test.The result showed that the successful expression of TbpA protein established a platform for development of new vaccines or diagnostic reagent.