人GITRaa27-165蛋白原核表达及其多克隆抗体制备.docVIP

人GITRaa27-165蛋白原核表达及其多克隆抗体制备 　作者：仝佳, 王胜军*, 陈君, 毛朝明, 杨云良, 杨敏, 胡正军, 只棣媛, 许化溪 【摘要】 　　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pGEM T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 IMAC Ni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hGITRaa27-165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400 mg/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。 【关键词】 hGITR 原核表达 多克隆抗体 　　Prokaryotic expression of human GITRaa27-165 and preparation of its polyclonal antibody 　　TONG Jia, WANG Shengjun*, CHEN Jun, MAO Chaoming, YANG Yunliang, YANG Min, HU Zhengjun, ZHI Diyuan, XU Huaxi 　　Department of Immunology and Laboratory Immunology, School of Medical Technology; Department of Ophthalmology, the Affiliated Hospital; Class 3, Grade 2004, Laboratory Medicine of Jiangsu University, Zhenjiang 212013, China 　　[Abstract] AIM: To express and purify the extracellular region of human glucocorticoidinduced tumor necrosis factor (hGITRaa27-165), and to prepare and identify the polyclonal antibody (pAb) against this fusion protein. METHODS: The 429 bp DNA sequence of hGITRaa27-165 was obtained from pGEM ThGITR by PCR and then it was inserted into pQE30 plasmid to construct the prokaryotic expression plasmid pQE30hGITRaa27-165. pQE30hGITRaa27-165 was transformed into E.coli. The target fusion protein was expressed with the induction of Isopropyl βD1thiogalacto pyranoside (IPTG) and purified by Ni2+NTA affinity chromatography column. The antiserum against hGITRaa27-165 was obtained from the rabbits immunized with hGITRaa27-165. The titer of pAb was detected by ELISA and the specificity of pAb was identified by Western blot. RESULTS: The prokaryotic expression