【摘要】 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。更多还原

【Abstract】 Objective To express and purify the fusion protein of extracellular domain of human Ig domain-containing,neurite outgrowth inhibitor(Nogo) receptor-interacting protein-1(LINGO-1aa76-319) in prokaryotic cells and prepare the rabbit anti-LINGO-1 polyclonal antibody(pAb).Methods The 732 bp DNA sequence of hLINGO-1aa76-319 was obtained from pCMV-SPORT6 by PCR and inserted into pET30a(+) plasmid to construct the prokaryotic expression plasmid pET30a(+)-hLINGO-1aa76-319,which was subsequently transformed into E.coli.The target fusion protein was expressed with IPTG induction and purified by Ni2+-NTA affinity chromatography column.The antiserum against hLINGO-1aa76-319 was obtained from the rabbits immunized with hLINGO-1aa76-319,and the titer of the pAb was determined using enzyme linked immunosorbent assay(ELISA) and its specificity identified using Western blotting.Results The prokaryotic expression plasmid pET30a(+)-hLINGO-1aa76-319 was constructed successfully.Efficient expression of the target fusion protein was achieved with IPTG induction at the optimal concentration of 0.4 mmol/L and culture temperature at 37 ℃ for 2.5 h.The hLINGO-1aa76-319 fusion protein was effectively expressed in E.coli as inclusion bodies,and the soluble protein was obtained through denaturation and refolding procedures,and the purified fusion protein showed a purity above 90%.The titer of the anti-hLINGO-1aa76-319 pAb obtained by immunizing the rabbits with the purified protein reached 1:1.6×106,and Western blotting confirmed its good specificity.Conclusion The fusion protein hLINGO-1aa76-319 with high purity has been obtained and the anti-hLINGO-1aa76-319 pAb obtained shows a high titer and good specificity,which provide important experimental basis for further functional investigation of LINGO-1.更多还原