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| Cloning and Prokaryotic Expression of Goat c-Myc Proto-oncogene, and Purification of GST-Myc Fusion Protein |
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2. 广西壮族自治区南宁市大学路100号 广西大学东校园动物科技学院 邮编：530004
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Abstract This paper was to obtain goat(Capra hircus) GST-Myc fusion protein by means of molecular cloning, prokaryotic expression and protein purification. The c-Myc cDNA of goat was amplified by RT-PCR from intestine tissues, and plasmid pMD18-T-Myc was constructed by T-A cloning. DNA sequencing showed that the cDNA was 1 320 bp，with a complete open reading frame that encoded 439 amino acids, sharing higher nucleotide homology(99.2%) with that of sheep(Ovis aries). The cDNA fragment was subcloned to pGEX-KG, and recombinant plasmid pGEX-KG-Myc was constructed, which was verified by restriction endonuclease analysis and DNA sequencing. The recombinant plasmid was transformed into E.coli BL21, and GST-Myc fusion protein was expressed with induction of IPTG, and then purified with Glutathione-Sepharose 4B argrose. Illustrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), the purified fusion protein was about 75 kD, a clear, and single band, which was expected. Western blot assay revealed that fusion protein could be identified by GST antibody. In conclution, we have cloned goat c-Myc gene, obtained GST-Myc fusion protein with higher purity, which will lead to preparation of polyclonal or monoclonal anti-Sox2 antibody, and further to application in identification of goat iPS cells (induced pluripotent stem cells).
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Received: 06 March 2009
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