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| Prokaryotic Expression and Polyclonal Preparation of a Novel Transcriptional Factor MDFIC |
1.National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou 510632, China
2.College of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou 510006, China |
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Abstract Mouse MDFIC (MyoD family inhibitor domain containing) protein is a newly found transcriptional factor, which shows high homology with HIC (Human I-mfa domain containing protein) and may play important roles in myogenic differentiation. In this article, cDNA encoding full-length of MDFIC was amplified by PCR and inserted it into prokaryotic expression vector pGEX-4T-3. The recombinant plasmid pGEX-4T-3- MDFIC was then transformed into E. coli BL21 and induced. The expression product was indentified by SDS-PAGE and Western blotting analysis. The fusion protein was purified by affinity chromatography with glutathione sepharose-4B column and then was used to immunize rabbits to generate polyclonal antibody. Indirect ELISA analysis shows that the titer of the antibody is higher than 1:640 000. Besides, Western blotting analysis indicates that the antibody can detect both exogenous and endogenous MDFIC protein. The polyclonal antibody of MDFIC provides an important tool to study the function of MDFIC and further to explore the mechanisms of myogenesis.
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Received: 27 October 2009
Published: 26 February 2010
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| Fund: Research Fund for the Doctoral Program of Higher Education of China |
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| ［1］ Weintraub H. The MyoD family and myogenesis: redundancy, networks and theresholds. Cell, 1993, 75(7): 12411244.
［2］ Chen C M, Kraut N, Groudine M, et al. Imf, a novel myogenic repressor, interacts with members of the MyoD family. Cell, 1996, 86(5): 731741.
［3］ Kusano S, RaabTraub N. Imfa domain proteins interact with axin and affect its regulation of the Wnt and cJun Nterminal kinase signaling pathways. Mol Cell Biol, 2002, 22(18): 63936205.
［4］ Kawai J, Shinagawa A, Shibata K, et al. The RIKEN genome exploration research group phase II team and the FANTOM consortium. Functional annotation of a fulllength mouse cDNA collection. Nature, 2001, 409(6821): 685690.
［5］ Thebault S, Gachon F, Lemasson I, et al. Molecular clonning of a novel human Imfa domaincontaining protein that differently regulates human Tcell leukemia virus type I and HIV1 expression. J Biol Chem, 2000, 275(7): 48484857.
［6］ Snider L, Thirlwell H, Miller J R, et al. Inhibition of Tcf3 binding by Imfa domain proteins. Mol Cell Biol, 2001, 21(5):18661873.
［7］ 罗志文, 郭芬, 李月琴, 等. mPem蛋白相作用分子的筛选与鉴定. 生物工程学报, 2006, 22(1): 125130. Luo Z W, Guo F, Li Y Q, et al. Chin J Biotech, 2006, 22(1): 125130.
［8］ Saluta M, Bell P A. Troubleshooting GST fusion protein expression in E.coli. Life Sci News, 1997, 1: 201209.
［9］ Hu Y X, Guo J Y, Shen L, et al. Get effective polyclonal antisera in one month. Cell Research, 2002, 12(2): 157160.
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