Prokaryotic expression,purification of human LINGO-1_(aa76-319) and preparation of its polyclonal antibody
2009
目的表达和纯化带多聚组氨酸(6×His)标签的人LINGO-1胞外段(hLINGO-1aa76-319)融合蛋白,并制备兔源性抗hLINGO-1aa76-319的多克隆抗体(pAb)。方法利用PCR从pCMV-SPORT6获得hLINGO-1aa76-319编码序列,将其亚克隆至原核表达载体pET30a(+),构建重组表达质粒pET30a(+)-hLINGO-1aa76-319;将阳性重组质粒转化大肠杆菌,IPTG诱导表达6×His-hLINGO-1aa76-319融合蛋白,经Ni-NTA螯合树脂纯化,纯化蛋白免疫新西兰大白兔制备多克隆抗血清,Protein A Sepharose柱纯化获得多抗,ELISA法检测抗体效价,Western blot法检测抗体特异性。结果成功构建了pET30a(+)-hLINGO-1aa76-319原核表达载体,原核蛋白hLINGO-1aa76-319以包涵体形式在大肠杆菌高水平表达,通过复性与亲和层析获得纯度在90%以上的hLINGO-1aa76-319蛋白,蛋白浓度为4600mg/L,制备的抗hLINGO-1aa76-319多抗效价高达1:1.6×106,Western blotting鉴定其具有良好的特异性。结论获得高纯度hLINGO-1aa76-319蛋白并成功制备特异性pAb,为进一步研究LINGO-1的生物学功能提供实验基础。
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