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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 detect the expression of DAZAP2 in patients with multiple myeloma at the protein level and to study the function of DAZAP2, the complete coding sequence of DAZAP2 was amplified by RT-PCR using the total RNA of normal human bone marrow mononuclear cells as a template. The recombinant plasmid pQE30-DAZAP2 was transformed into E.coli JM109 and then induced with IPTG for 4h. The expression of protein was significantly increased. The purified protein was purified on Ni-NTA column and immunized New Zealand white rabbits to prepare anti-DAZAP2 antibody. The titer of the ELISA antibody was above 16 400, and the specificity of the antibody detected by Western blot was better. The expression of DAZAP2 was detected in 6 normal and 4 multiple myeloma patients by this antibody, but not in the other 7 patients. Consistent with RT-PCR results, the antibody has certain clinical application prospects and can be further used for functional studies.