摘要
目的 :MCPR1是我室利用消减杂交技术克隆出来的新基因 ,本研究利用融合蛋白制备MCPR1的多克隆抗体。方法 :采用多聚酶链式反应 (PCR)扩增出MCPR1基因蛋白编码序列 ,进一步将该基因片段克隆到中间载体pGEX -T -easy ,再酶切得到MCPR1片段定向克隆到 pGEX -4T -1载体中 ,构建融合表达载体pGEX -4T-MCPR1,在大肠杆菌中用IPTG进行诱导表达 ,得到GST -MCPR1融合蛋白 ,进行初步纯化及SDS -PAGE电泳 ,直接割胶 ,将其作为抗原免疫兔子。结果 :成功构建融合表达载体pGEX -4T -MCPR1,得到初步纯化的GST-MCPR1融合蛋白 ,免疫兔子 1个月后 ,获得免疫血清 ,初步纯化得到多克隆抗体。Western印迹分析表明所得到的抗体具有较强的特异性 ,ELISA分析证实其效价为 10 6。结论 :MCPR1多克隆抗体的获得性为在蛋白水平研究新MCPR1的功能提供了实验基础 ,后续实验可以利用免疫分析技术研究MCPR1蛋白与其它蛋白质的相互作用 ,从而为阐明MCPR1在胚胎发育及腭裂形成中所发挥的作用提供实验手段。
Objective:To prepare polyclonal antibodies of MCPR1 ,which is a novel Mouse Cleft Palate Relate gene cloned by subtractive hybridization method at our department. Method:The segment of open read frame of MCPR1was amplified by PCR from the cloning vector, pMD 18-T-MCPR1, and was further cloned into the pGEM-T-easy vector to produce the new construct, pGEM-T-MCPR1. The cloned MCPR1segment was cut out again with two restriction enzymes and was cloned into the prokaryotic expression vector, pGEX-4T-1, to produce the expression vector pGEX-4T-MCPR1. The recombinant plasmid was transformed into E. coli DH5α.GST-MCPR1 fusion protein was obtained after the induction of IPTG . The fusion protein was extracted and purified by SDS-PAGE, and the new protein band was cut as antigens, injected into the rabbit to produce polyclonal antibodies.Result: The expression vector pEGX-4T-MCPR1 was consturcted successfully,and GST-MCPR1 fusion protein was obtained and injected into the rabbit to acquire the immunoserum , Western blot analyses showed that the antibody reacted specifically in vitro to the produced fusion protein..ELISA analysis showed that the titer for this antibody is 10 6.Conclusion: The antibody made it possible to analyze the role of MCPR1 on the protein level.Such as the interaction between MCPR1 and other proteins with immunological methods, and laid the foundation to elucidate its function during the embryo development and formation of cleft palate.
出处
《临床口腔医学杂志》
2004年第8期469-472,共4页
Journal of Clinical Stomatology
