[Prokaryotic expression and polyclonal antibody preparation of human spermine oxidase].
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.
Cao
Chun-Yu
CY
Institute of Molecular Biology, Medical College, Three Gorges University, Yichang 443002, China. 407788923@qq.com
Han
Yu
Y
Ren
Yu-Shan
YS
Wang
Yan-Lin
YL
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
D000906:Antibodies; D018076:DNA, Complementary; D000587:Oxidoreductases Acting on CH-NH Group Donors; D000097667:Polyamine Oxidase