摘要
运用PCR方法扩增副猪嗜血杆菌SC-1株荚膜多糖输出蛋白(CPEP)基因,克隆至pMD-19T载体。经鉴定测序验证后,将获得的CPEP基因克隆至pET-32a(+)原核表达载体,构建原核表达质粒pET-32a-CPEP,转化大肠杆菌BL21(DE3),IPTG诱导表达,进行SDS-PAGE以及Western blot分析免疫原性,纯化CPEP,并用纯化的蛋白免疫小鼠,检测其特异性血清IgG抗体水平及攻毒保护率。结果显示,成功构建原核表达质粒pET-32aCPEP;SDS-PAGE分析结果显示,得到约35ku的蛋白;Western blot分析显示,CPEP能与Hps SC-1阳性血清发生抗原抗体的特异性反应,证实该重组CPEP蛋白具有反应原性。以致死剂量的强毒株SC-1攻毒CPEP免疫小鼠,重组蛋白的免疫能够减缓发病,并表现出40%的保护率。重组蛋白CPEP的部分保护作用表明其可以作为免疫候选因子,为未来副猪嗜血杆菌标准化疫苗的研制奠定基础。
Capsular polysaccharide export protein(CPEP)gene of Haemophilus parasuis SC-1 was amplified by PCR and then was cloned into the pMD-19Tvector.After being certified by sequence and digestion with BamHⅠand XhoⅠrespectively,the gene were cloned into the pET-32a(+)vector.The recombinant vector was transformed into BL21(DE3)and then induced by IPTG. The product of expression was studied by SDS-PAGE and Western blot,and then was purified. We further evaluated the immune responses and protective efficacy of purified CPEP in mice models.The results showed that the recombinant vector pET32a-CPEP was constructed successfully. The SDS-PAGE and the western blot results showed that the purified protein was 35kDa and the recombinant CPEP possessed reactogenicity respectively.The protective capacity of the anti-CPEP antibodies was evaluated by the inoculation of highly virulent strain SC-1.Vaccinated animals had a delayed course of disease and 40%of the animals survived the lethal challenge.The partial pro-tection achieved with the recombinant CPEP supports their potential as candidates to be included in future vaccine formulations against H.parasuis.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2014年第4期621-630,共10页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
公益性行业(农业)科研专项(201303034-1)
关键词
副猪嗜血杆菌
荚膜多糖输出蛋白基因
克隆表达
免疫原性
Haemophilus parasuis
capsular polysaccharide export protein gene
expression
immunogenicity analysis
