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《Journal of Southern Medical University》 2009-06
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Expression of human Id-2 gene in Escherichia coli and preparation of the antisera against human Id-2

TONG Tie-gang1, LIN Yan1,2, MU Dan-mei3,4, BAI Yu1, YANG Mu-lei3, ZHENG Min3, WU Dong-lai1 1National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China; 2College of Life Sciences, Heilongjiang University, Harbin 150080, China; 3First Clinical Medical College of Harbin Medical University, Harbin 150001, China; 4Department of Abdominal Ultrasonagraphy, Daqing Oilfield General Hospital, Daqing 163001, China  
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). Conclusion 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.
【Fund】: 黑龙江省自然科学基金重点项目(ZJY-0603-02)
【CateGory Index】: R392.1
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