胞内劳森菌0710蛋白的表达及免疫活性分析

Immunological activity of the recombinant Lawsonia intracellularis protein LI0710

为了评价胞内劳森菌(Lawsonia intracellularis)鞭毛相关蛋白LI0710的免疫原性,将LI0710基因插入到pET-32a(+)原核表达载体,构建重组表达质粒pET-32a-LI0710,并转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导目的蛋白表达,获得重组目的蛋白(rLI0710),通过Western-blot验证rLI0710的反应原性,同时用rLI0710免疫新西兰大白兔,获得多克隆抗体,用间接ELISA和间接免疫荧光(IFA)方法验证rLI0710的免疫原性。结果显示,重组LI0710蛋白在大肠杆菌中以包涵体形式表达;Western-blot揭示rLI-0710能够与LI试验感染小鼠血清反应,表明rLI0710具有反应原性;ELISA检测表明,rLI0710能诱导家兔产生高效价抗体,且IFA分析表明此抗体能与培养细胞中的LI反应,证明rLI0710具有良好的反应原性和免疫原性。本试验为基于LI0710蛋白的猪增生性回肠炎免疫学诊断方法的建立奠定了基础。

To probe the antigenicity of LI0710,which is a flagella-related protein of an obligate intracellular pathogenic bacteria Lawsonia intracellularis,a recombinant expression plasmid p ET-32 aLI0710 was constructed by inserting the LI0710 gene into p ET-32 a.Then p ET-32 a-LI0710 was transformed into competent E.coli BL21(DE3)and induced by IPTG for expression to obtain the recombinant protein(r LI-0710).For evaluating the reactogenicity and immunogenicity of r LI0710,Western-blot and ELISA/IFA tests were run using sera from mice experimentally infected with L.intracellularis and rabbits vaccinated with r LI0710,respectively.The results showed that the r LI0710 protein in E.coli was expressed in inclusion bodies and could react with the sera from infected mice.Furthermore,the specific antibodies against r LI0710 from r LI0710-vaccinated rabbits were been detected with indirect ELISA and IFA methods.These results suggested that r LI0710 could be used as an antigen for immunological diagnosis of swine proliferative ileitis.