抗DAZAP2抗体的制备及在多发性骨髓瘤研究中的应用

Preparation of Antibody of Rabbit Anti Human DAZAP2 and Application in Multiple Myeloma Research

为了进一步从蛋白水平上检测DAZAP2(deletedinazoospermiaassociatedprotein2)在多发性骨髓瘤患者中的表达及研究DAZAP2的功能,以正常人的骨髓单个核细胞的总RNA为模板,RT-PCR扩增DAZAP2完整编码序列,构建原核表达重组质粒pQE30-DAZAP2,转化大肠杆菌JM109后,加IPTG诱导表达4h时,表达蛋白显著增加,Ni-NTA层析柱纯化蛋白,以该纯化蛋白免疫新西兰大白兔,制备抗DAZAP2抗体,ELISA检测抗体的效价在16400以上,Westernblot检测抗体的特异性较好.用该抗体检测出DAZAP2在6例正常人及4例多发性骨髓瘤患者中有表达,其他7例患者中没有表达,与RT-PCR结果一致,该抗体具有一定的临床应用前景,并能进一步用于功能研究.

To further verify DAZAP2 (deleted in azoospermia associated protein 2)expression in multiple myeloma(MM) and try to establish an ancillary method in diagnosis of MM,the cDNA of DAZAP2 coding sequence was amplified by RT PCR,then recombined into prokaryotic expression vector pQE30 and transformed into E.coli JM109 for expression.The purified protein(around 17 kD) was gained by Ni affinity chromatography.The rabbits were immunized with these purified protein.The antibody of rabbit anti human DAZAP2 was successfully prepared,its titer was over 1:6 400.The level of DAZAP2 protein was down regulated in MM patient by Western blotting.The results were matched well with the results of RT PCR.