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目的:表达和纯化人GITR胞外段(hGITRaa27-165)融合蛋白,制备hGITRaa27-165融合蛋白的兔源性多克隆抗体(pAb)。方法:利用PCR从pGEMT-hGITR获得hGITRaa27-165编码序列,将其亚克隆至原核表达载体pQE30,构建重组表达质粒pQE30-hGITRaa27-165;将阳性重组质粒转化大肠杆菌,IPTG诱导表达融合蛋白,通过Profinity IMACNi-Charged Resin亲和层析柱进行纯化;纯化蛋白免疫新西兰大白兔,获取多克隆抗血清并利用Protein A Sepharose柱进行纯化,ELISA法检测抗体效价,Western blot法检测抗体特异性。结果:构建了pQE30-hGITRaa27-165原核表达载体,原核蛋白hGI-TRaa27-165蛋白以包涵体形式在大肠杆菌得到高水平表达,通过复性与亲和层析获得纯度在90%以上的hGITRaa27-165蛋白,蛋白浓度为400mg/L,制备的抗hGITRaa27-165多抗效价高达1∶1.6×105,Western blot鉴定其具有良好的特异性。结论:获得高纯度hGITRaa27-165蛋白,并制备特异性pAb,将为进一步研究hGITR的生物学功能提供实验基础。
Objective: To express and purify human GITR extracellular domain (hGITRaa27-165) fusion protein and prepare rabbit polyclonal antibody (pAb) of hGITRaa27-165 fusion protein. Methods: The hGITRaa27-165 coding sequence was obtained from pGEMT-hGITR by PCR and subcloned into prokaryotic expression vector pQE30 to construct recombinant expression plasmid pQE30-hGITRaa27-165. The recombinant plasmid was transformed into E. coli and induced by IPTG. Profinity IMACNi-Charged Resin affinity column. The purified protein was used to immunize New Zealand white rabbits. The polyclonal antiserum was obtained and purified by Protein A Sepharose column. The antibody titer was detected by ELISA and the antibody specificity was detected by Western blot. Results: The prokaryotic expression vector pGE30-hGITRaa27-165 was constructed. The prokaryotic expression vector hGI-TRaa27-165 was highly expressed in E. coli using inclusion bodies. The purity of hGITRaa27-165 was over 90% by renaturation and affinity chromatography. 165 protein with a protein concentration of 400 mg / L. The titer of the anti-hGITRaa27-165 polyclonal antibody was as high as 1: 1.6 × 10 5, and its specificity was confirmed by Western blot. Conclusion: Obtaining high purity hGITRaa27-165 protein and preparing specific pAb will provide the experimental basis for further study on the biological function of hGITR.