title: [Prokaryotic expression and polyclonal antibody preparation of human spermine oxidase].
abstract: OBJECTIVE
  To prepare recombinant human spermine oxidase (SMO) and polyclonal antibody against human SMO by gene recombination techniques.
  
  
  METHODS
  Human SMO cDNA was amplified from total RNA of A549 cells through reverse transcription PCR. The cDNA was then cloned into pET-15b to construct SMO prokaryotic expression vector. After transforming, the vector was induced to express recombinant SMO by IPTG in E.coli BL21 (DE(3)). Recombinant SMO was purified by Ni-NTA resin under denaturing condition and then was dialyzed to renature. The enzyme activity of recombinant SMO was analyzed by chemical fluorescent method. SMO polyclonal antibody was prepared by using recombinant human SMO protein purified by polyacrylamide gel electrophoresis as antigen to inoculate rabbit intradermally. The titer and specificity of anti-sera were determined by ELISA, Western blot and Immune Cell Chemistry.
  
  
  RESULTS
  Purified and dialyzed recombinant human SMO has the specificity of oxidizing the spermine. The polyclonal antibody has high titer and specificity against human SMO.
  
  
  CONCLUSIONS
  This research established a method for prokaryotic expression, purification and polyclonal antibody preparation of human SMO. The method lays a foundation for the future functional research of SMO.
authors.affiliation: Institute of Molecular Biology, Medical College, Three Gorges University, Yichang 443002, China. 407788923@qq.com
authors.forename: Chun-Yu
authors.initials: CY
authors.lastname: Cao
authors.forename: Yu
authors.initials: Y
authors.lastname: Han
authors.forename: Yu-Shan
authors.initials: YS
authors.lastname: Ren
authors.forename: Yan-Lin
authors.initials: YL
authors.lastname: Wang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000097667:Polyamine Oxidase
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000097667:Polyamine Oxidase