[Expression of human Id-2 gene in Escherichia coli and preparation of the antisera against human Id-2].
OBJECTIVE
To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2.


METHODS
The 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.


RESULTS
PCR, 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).


CONCLUSIONS
The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.
Tong
Tie-Gang
TG
National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research institute, Chinese Academy of Agricultural Sciences, Harbin, China. teargone2003@yahoo.com
Lin
Yan
Y
Mu
Dan-Mei
DM
Bai
Yu
Y
Yang
Mu-Lei
ML
Zheng
Min
M
Wu
Dong-Lai
DL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology; D001943:Breast Neoplasms / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000096:biosynthesis; D051797:Inhibitor of Differentiation Protein 2 / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
D000911:Antibodies, Monoclonal; C497450:ID2 protein, human; D007106:Immune Sera; D051797:Inhibitor of Differentiation Protein 2; D011994:Recombinant Proteins