中国生物工程杂志

objective: To prepare recombinant human polyamine oxidase (PAO) and polyclonal antibody of rabbit-anti-human PAO by gene recombination techniques. Methods: Human PAO cDNA was amplified by RT-PCR from total RNA of A549 cells. PAO cDNA was then cloned into pET-15b to construct PAO prokaryotic expression vector. The vector was transformed into E. coli. BL21(DE3) and induced to express PAO recombinant protein by IPTG. PAO was purified by Ni-NTA resin under denature condition and dialyzed to recover its native structure. After that, the enzyme activity was analyzed with chemical fluorescent method. PAO polyclonal antibody was prepared by using recombinant human PAO protein which was purified by polyacrylamide gel electrophoresis as antigen to intradermally inoculate and immunize rabbit. The antibody titer and specificity were determined by ELISA, Western Blot and Immune Cell Chemistry. Results: Purified and dialyzed recombinant human PAO has the enzymatic activity of quickly oxidizing N1-acetylspermine. The antibody prepared by recombinant PAO has high titer and specificity against human PAO. Conclusion: The methods for prokaryotic expression, purification of human PAO and preparing specific anti-PAO antibody were successfully established. These methods laid a foundation for the succedent functional research of PAO as an anti-tumor target.