布鲁氏菌外膜蛋白Omp16的原核表达、纯化及多克隆抗体制备

Prokaryotic expression,purification and polyclonal antibody preparation of outer membrane proteins Omp16 from Brucella

目的克隆、表达、纯化布鲁氏菌Omp16重组蛋白,并制备多克隆抗体,评估其作为布鲁氏菌蛋白质疫苗的可行性。方法 PCR法扩增全长及截短的布鲁氏菌Omp16基因,并将其克隆入p ET28a(+)载体中;经菌液PCR和测序鉴定后,再转入大肠杆菌BL21(DE3)中,经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达及镍柱纯化后,获取高纯度的Omp16目的蛋白,然后将其免疫BALB/c小鼠以制备多克隆抗体,Western blotting检测抗体的特异性免疫反应。结果成功构建了全长及截短表达的Omp16原核表达质粒,经IPTG诱导表达后,获得了全长及截短Omp16蛋白,免疫BALB/c小鼠后获得了高效价的抗Omp16多克隆抗体。Western blotting实验证实2种Omp16重组蛋白(Omp16-1、Omp16-2)均能与其相应抗体发生特异性免疫反应。结论该研究成功克隆、表达、纯化了全长及截短的Omp16重组蛋白,2种Omp16重组蛋白均具有很好的免疫原性,且能与高滴度的小鼠多克隆抗体发生特异性结合,为后续以Omp16蛋白为基础的疫苗研发奠定了实验基础。

Objective Cloning and expressing the purified Omp16recombinant protein of Brucella,producing polyclonal antibodies and evaluating its feasibility as a protein vaccine as well.Methods The fulllength and truncated Brucella Omp16gene was amplified by PCR and cloned into pET28a(+)vector,after the identification of PCR and recombinant plasmid sequencing,it was transferred toE.coli BL21(DE3),which was induced by IPTG to express the fusion protein.After purified by nickel column,high purity Omp16protein was obtained,then BALB/c mices were immunized to prepare polyclonal antibody,and Western blotting was used to detect the specific immune response of the antibody.Results The study successfully constructed fulllength and truncated prokaryotic expression plasmid of Omp16.After induction of IPTG,two corresponding proteins were obtained.At the same time,BALB/c mices were immunized with high titer polyclonal antibodies against Omp16.Finally,Western blotting verified that the two Omp16recombinant proteins(Omp16-1,Omp16-2)could react specifically with their corresponding antibodies.Conclusion In this study,the fulllength and truncated Omp16recombinant proteins are cloned,expressed and purified successfully,all of them have good immunogenicity and can specifically bind to high titer mouse polyclonal antibodies,This experiment laid the experimental foundation for the follow?up research and development of the vaccines based on Omp16protein.