GII.4型人诺如病毒VP2蛋白的原核表达与多克隆抗体制备

Prokaryotic expression of GII.4 human norovirus VP2 protein and preparation of anti-VP2 polyclonal antibody

目的原核表达GII.4基因型人诺如病毒(human norovirus, HuNoV)次要衣壳蛋白VP2并制备多克隆抗体。方法设计特异性引物扩增GII.4 HuNoV的VP2全基因, 酶切连接至原核表达载体pGEX-6P-1, 将鉴定正确的重组质粒转化BL21(DE3)感受态细胞。挑取单克隆摇菌, 加入IPTG诱导重组GST-VP2融合蛋白的表达, 经GST亲和层析纯化和酶切, 获得GII.4 HuNoV VP2蛋白。通过SDS-PAGE分析纯化后HuNoV VP2蛋白相对分子质量。将纯化的GII.4 HuNoV VP2蛋白(0.5 mg/ml)免疫BALB/c小鼠, 制备多克隆抗体。结果 GII.4 HuNoV VP2蛋白被成功表达和纯化, 相对分子质量(Mr.×103)约29。将GII.4 HuNoV VP2蛋白免疫BALB/c小鼠制备的多克隆抗体滴度高达1∶1 280 000。Western blot与间接ELISA分析显示该多克隆抗体能和GII.4 HuNoV VP2抗原特异性结合。结论利用原核表达系统成功表达了GII.4 HuNoV VP2蛋白, 并成功制备出高效价GII.4 HuNoV VP2多克隆抗体。

Objective To express prokaryotically GII.4 human norovirus(HuNoV)VP2 protein and to prepare polyclonal antibody against VP2.Methods Design specific primers to amplify the VP2 gene of GII.4 HuNoV,digest and connect to the prokaryotic expression vector pGEX-6P-1,transform the correctly identified recombinant plasmid into BL21(DE3)competent cells.Pick out and shake the monoclonal bacteria,and add IPTG to induce recombinant GST-VP2.The fusion protein was expressed,purified by GST affinity chromatography and digested to obtain GII.4 HuNoV VP2 protein.The relative molecular mass(Mr.×103)of the purified HuNoV VP2 protein was analyzed by SDS-PAGE.BALB/c mice were immunized with purified GII.4 HuNoV VP2 protein(0.5 mg/ml)to prepare polyclonal antibodies.Results The VP2 protein of GII.4 HuNoV was successfully expressed and purified,with a relative molecular mass(Mr.×103)of about 29;the VP2 polyclonal antibody of GII.4 HuNoV was successfully prepared and its titer was as high as 1∶1280000.Western blot and indirect ELISA analysis showed that the polyclonal antibody could specifically bind to the VP2 antigen of GII.4 HuNoV.Conclusions The purified GII.4 HuNoV VP2 after prokaryotic fusion expression can be used to prepare high titer polyclonal antibody.