牛病毒性腹泻病毒NS3蛋白原核表达纯化及多克隆抗体的制备

Prokaryotic Expression and Purification of Bovine Viral Diarrhea Virus NS3 Protein and Preparation of Polyclonal Antibody

原核表达牛病毒性腹泻病毒(BVDV)NS3蛋白,并以此免疫动物制备多克隆抗体。对BVDV NS3蛋白的抗原决定簇氨基酸序列分析,构建重组质粒pET30a-NS3,转化至E.coli BL21(DE3)感受态细胞中,使用IPTG诱导目的蛋白表达。选择最优诱导条件对目的蛋白进行亲和纯化,目的蛋白主要以包涵体形式表达,从而获得NS3蛋白。使用NS3蛋白免疫新西兰大白兔,制备兔抗NS3蛋白多克隆抗体,采用ELISA方法检测多克隆抗体效价。结果显示,成功表达并纯化出BVDV NS3蛋白,制备的兔抗NS3蛋白多克隆抗体效价大于1∶128000,为建立BVDV抗体/抗原检测方法及疫苗的研究奠定了基础。

The bovine viral diarrhea virus(BVDV)NS3 protein is expressed in prokaryotic cells,and a polyclonal antibody is prepared by immunizing the animal.The amino acid sequence of the epitope of NS3 protein was analyzed,and the recombinant plasmid pET30a-NS3 was constructed and transformed into E.coli BL21(DE3)competent cells,and the expression of the target protein was induced by IPTG.The optimal inducing conditions were selected to purify the target protein,and the target protein was expressed in the highest amount and mainly existed in the form of inclusion bodies,thereby obtaining NS3 protein.Rabbit anti-NS3 protein polyclonal antibody was prepared by immunizing New Zealand white rabbit with NS3 protein,and polyclonal antibody titer was detected by ELISA.The results showed that the BVDV NS3 protein was successfully expressed in this experiment,and the polyclonal antibody against rabbit anti-NS3 protein was prepared.The antibody titer was greater than 1∶128000,which laid a foundation for the establishment of a novel BVDV antibody/antigen diagnostic method and vaccine.