Prokaryotic expression, purification and preparation of polyclonal antibody for wheat grain peroxidase WP1 gene.
- Author:
Liwei SHAN
1
;
Ruchun TANG
;
Sanyang LIU
;
Sanhong FAN
;
Aiguang GUO
Author Information
1. College of Life Sciences, Northwest A & F University, Yangling 712100, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antibodies;
immunology;
metabolism;
Escherichia coli;
genetics;
metabolism;
Genetic Vectors;
Peroxidases;
biosynthesis;
genetics;
Rabbits;
Recombinant Proteins;
biosynthesis;
genetics;
immunology
- From:
Chinese Journal of Biotechnology
2011;27(1):26-30
- CountryChina
- Language:Chinese
-
Abstract:
Wheat peroxidases 1 (WP1) is the major cationic peroxidase of wheat (Triticum aestivum) grain, which is involved in the development of seeds and an important factor to affect the final processing quality of flour. We constructed a prokaryotic expression vector pET28a-WP1, and transformed it into E. coli host strain T7 Expression. His-tag fused WP1 existed as inclusion body, and the recombinant protein was purified by Ni-NTA resin affinity chromatography under denatured condition. The purity of target protein reached 98%. The recombinant WP1 was refolded by gradient urea dialysis, then used as antigen to immune rabbit to prepare polyclonal antibody. The result of ELISA showed that the titer of rabbit anti-WP1 antiserum was higher than 1:625 000. The result of Western blotting demonstrated that the prepared WP1 polyclonal antibody could be used to detect WP1 with high specificity.
