布鲁菌L7/L12蛋白的原核表达及多克隆抗体的制备

Prokaryotic Expression and Polyclonal Antibody Preparation of L7/L12 Protein of Brucella suis S2 Strain

为制备布鲁菌(Brucella)L7/L12蛋白及其小鼠源多克隆抗体,根据猪布鲁菌(Brucella suis)S2株L7/L12基因序列,设计合成特异性引物,PCR扩增L7/L12基因并将其克隆至原核表达载体pET-28a(+)中,构建重组质粒pET-28a-L7/L12,经鉴定正确后转化至大肠埃希菌BL21(DE3)感受态细胞进行诱导表达。SDS-PAGE分析表明,重组菌经IPTG诱导后成功表达出分子质量为17ku的重组蛋白。将诱导表达的蛋白经镍柱亲和层析纯化后免疫小鼠制备多克隆抗体,并测定其抗体效价。Western blot结果表明,原核表达后纯化的L7/L12蛋白能够与制得的小鼠抗L7/L12蛋白多克隆抗体特异性结合。成功获得了纯化的L7/L12重组蛋白和小鼠抗L7/L12多克隆抗体,为进一步研究L7/L12蛋白对布鲁菌感染的诊断方法和基因工程疫苗的研发奠定了基础。

In order to obtain the recombinant L7/L12protein and the polyclonal antibody against L7/L12protein,a pair of specific primers were designed according to the published sequence of L7/L12gene from Brucella suis S2strain.The L7/L12gene was amplified and cloned into pET-28a(+)vector to construct the prokaryotic expression plasmid pET-28a-L7/L12.The expression of recombinant plasmid in E.coli BL21(DE3)competent cell was induced.The recombinant protein with molecular weight of17ku was successfully expressed following IPTG induction by SDS-PAGE analysis.The recombinant protein was purified by Ni-NTA affinity chromatography,and then used to vaccinate Balb/c mice to prepare the anti-L7/L12polyclonal antibody.Western-blot analysis showed that the recombinant protein could react specifically with the anti-L7/L12polyclonal antibody.In conclusion,the purified L7/L12protein and the anti-L7/L12polyclonal antibody were successfully obtained,which lay a foundation for the diagnosis of Brucella infection and the development of genetic engineering vaccine.