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目的:采用基因重组技术获得有酶活性的重组人多胺氧化酶(polyamine oxidase,PAO)并制备兔抗人多胺氧化酶多克隆抗体。方法:采用RT-PCR法从人非小细胞肺腺癌A549细胞株总RNA中克隆人多胺氧化酶cDNA,构建PAO原核表达质粒pET-15b/PAO并转化E.coli的BL21(DE3)菌株。IPTG诱导表达的重组PAO经Ni-NTA亲和层析纯化和透析复性后,化学荧光法测定酶活性。用制备性聚丙烯酰胺凝胶电泳分离纯化的重组人PAO蛋白,皮内接种日本大耳白兔以制备多克隆抗体。以ELISA、Western blot和免疫细胞化学法分别检测兔血清中抗体滴度和抗原特异性。结果:纯化并透析复性后的重组人PAO蛋白具备快速氧化特异性底物的酶活性。用此基因重组蛋白制备的抗体具有高抗体滴度和针对人PAO的特异性。结论:本试验建立了人PAO原核表达、纯化系统,获得了有酶活性的高纯度人PAO蛋白并成功制备了兔抗人PAO的多克隆抗体,为以PAO为靶点的抗肿瘤基础和临床研究提供了有力的研究工具。
OBJECTIVE: To obtain recombinant polyamine oxidase (PAO) with enzyme activity and to prepare polyclonal anti-human polyamine oxidase polyclonal antibody by gene recombination technology. METHODS: Human polyamine oxidase cDNA was cloned from the total RNA of human non-small cell lung adenocarcinoma A549 cell line by RT-PCR. The BL21 (DE3) strain of PAO prokaryotic expression plasmid pET-15b / PAO was transformed into E. coli . Recombinant PAO induced by IPTG was purified by Ni-NTA affinity chromatography and dialyzed for refolding. The enzymatic activity was determined by chemical fluorimetry. Purified recombinant human PAO protein was separated by preparative polyacrylamide gel electrophoresis, and Japanese white rabbits were inoculated intradermally to prepare polyclonal antibodies. The antibody titer and antigen specificity of rabbit serum were detected by ELISA, Western blot and immunocytochemistry respectively. Results: Purified and dialyzed recombinant human PAO protein has the enzymatic activity of rapidly oxidizing specific substrates. Antibodies prepared using this gene recombinant protein have high antibody titers and are specific for human PAO. CONCLUSION: In this study, we established a prokaryotic expression system for human PAO, obtained high purity human PAO protein with enzyme activity, and successfully prepared polyclonal antibody against rabbit polyclonal antibody against human PAO. It is an anti-tumor basis and clinical target of PAO Research provides a powerful research tool.