兔出血症病毒VPg蛋白的原核表达及抗体制备

Prokaryotes expression and antibody preparation of viral genome-linked protein VPg for rabbit hemorrhagic disease virus

【目的】构建重组兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)VPg(Viral Protein Ge-nome-Linked,VPg)蛋白基因的原核表达质粒,在大肠杆菌BL21(DE3)中表达和纯化VPg蛋白,制备多克隆抗体,并检测抗体的基本特性。【方法】用PCR方法扩增RHDVVPg基因,将其克隆至原核表达载体pET-30a(+)中,转化大肠杆菌BL21(DE3)菌株,用IPTG诱导,使之重组表达VPg蛋白,并用镍柱亲和层析法纯化重组蛋白。PCR扩增获得了345bp的VPg基因,用纯化的重组VPg蛋白免疫试验兔,制备抗VPg的多克隆抗体,用Western blot检测其特异性。【结果】构建了pET-30a/VPg原核表达质粒,转化大肠杆菌BL21(DE3)菌株后,成功表达了VPg蛋白。用该蛋白免疫试验兔后,制备的多克隆抗体能与VPg蛋白特异性反应。【结论】成功表达了RHDV VPg蛋白,并制备了特异性良好的多克隆抗体。

【Objective】 Base on the genome of the Rabbit hemorrhagic disease virus（RHDV）JX/97 strain,we constructed Prokaryotes expression vector of VPg（Viral Protein Genome-Linked）,and the expression of recombinant proteins VPg in the cytoplasm of Escherichia coli was purified and vaccinated into rabbit to produce polyclonal antibodies.【Method】 The VPg gene was generated by PCR and cloned into expression vector pET-30a,and the recombinant plasmid pET-30a/VPg was transfected into E.coil BL21 cells before being induced by IPTG.RHDV VPg was purified by affinity chromatography on NiCam-agarose and vaccinated into rabbit to produce polyclonal antibodies.The anti-serum was collected and detected by western blot.【Result】 We constructed the Prokaryotes expression vector of pET-30a/VPg.The protein was expressed in E.coil BL21 cells and the western blot showed the specificity of the antibody.【Conclusion】 This research provided information for further study on the biological characteristics of VPg.