人脂联素球状结构域基因的原核表达及多克隆抗体的制备

Prokaryotic expression of human gAd gene and preparation of its polyclonal antibody

目的:构建人脂联素球状结构域(gAd)基因的原核表达载体PET-28a(+)-gAd,诱导表达并纯化重组gAd蛋白,制备多克隆抗体。方法:以人基因组为模板,用PCR方法扩增人脂联素球状结构域(gAd)基因,构建原核表达载体PET-28a(+)-gAd,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经SDS-PAGE,免疫印迹法检测并鉴定表达产物,表达的目的蛋白用镍亲和层析柱纯化后免疫新西兰大白兔,制备多克隆抗体。结果:原核表达载体PET-28a(+)-gAd转化大肠杆菌BL21(DE3),经IPTG诱导表达,获得gAd重组蛋白,免疫印迹证实能与抗his标签检测抗体结合,制备的多克隆抗体经间接ELISA法测得效价为1∶32 000。免疫印迹证实,抗血清不仅能特异性地识别gAd蛋白,还能识别脂联素蛋白,而不与非特异性蛋白结合。结论:成功构建原核表达载体PET-28a(+)-gAd,并表达、纯化重组蛋白gAd,制备的多克隆抗体特异性好,效价较高,为进一步的研究奠定了基础。

Objective：To construct prokaryotic expression vector PET-28a（＋）-gAd of human gAd gene,induce its expression and purify recombinant protein,then prepare polyclonal antibody.Methods：Human gAd gene was amplified by PCR method using human genome as a template,prokaryotic expression vector PET-28a（＋）-gAd was constructed and transposed it into BL21（DE3）,its expression induced by IPTG,the expression product was checked by SDS-PAGE and Western blot,then to purify the target protein by Ni2＋ affinity column.The purified protein was used as the antigen to immune the New Zealand White rabbits for preparing the polyclonal antibody.Results：The expression protein was obtained from the BL21（DE3） which has been transposed by PET-28a（＋）-gAd vector by IPTG,Western blot proved that the expression protein can combine with the anti-his antibody,the titer of polyclonal antibody was over 1∶320 000 by the methods of ELISA-ABC.Western bolt confirmed that antiserum can specific recognized to combine with gAd protein as well as adiponectin,but no binding with other non-specific protein.Conclusion：Prokaryotic expression vector PET-28a（＋）-gAd was constructed successfully.The expression protein has been obtained and purified to prepare polyclonal antibody,and the antiserum has high titer and specificity.