Expression of human Id-2 gene in Escherichia coli and preparation of the antisera against human Id-2.
- Author:
Tie-Gang TONG
1
;
Yan LIN
;
Dan-Mei MU
;
Yu BAI
;
Mu-Lei YANG
;
Min ZHENG
;
Dong-Lai WU
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Antibodies, Monoclonal; biosynthesis; immunology; Breast Neoplasms; genetics; Escherichia coli; genetics; metabolism; Female; Humans; Immune Sera; biosynthesis; Inhibitor of Differentiation Protein 2; biosynthesis; genetics; immunology; Rabbits; Recombinant Proteins; biosynthesis; genetics
- From: Journal of Southern Medical University 2009;29(6):1094-1097
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.
METHODSThe coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2.
RESULTSPCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP).
CONCLUSIONThe prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.
