|
|
|
| Prokaryotic Expression and Purification of Qinchuan Cattle's Natural Resistance-associated Macrophage Protein-1 Gene(Nramp1) N-terminal Fragment |
|
|
|
|
Abstract To explore the function of bovine natural resistance-associated macrophage protein-1(Nramp1) and seek for the objective genes for resistance breeding against intracellular pathogen via transgenic technology, Nramp1 gene N-terminal fragment was amplified from total RNA of Qinchuan cattle's(Bos taurus) peripheral blood by RT-PCR, inserted into pMD18-T simple vector, and secondly subcloned into pGEX-4T-1 vector(named pGEX-N2). And the recombinant plasmid pGEX-N2 was transformed into Escherichia coli BL21(DE3) strain. SDS-PAGE analysis showed that GST-Nramp1-N fusion protein was satisfactorily expressed by optimizing the concentration and induction time of IPTG. Eventually, GST-Nramp1-N fusion protein was purified by glutathione Sepharose 4B. The results demonstrated that Qinchuan Cattle's Nramp1 gene N-terminal fragment was 186 bp; Compared to gene order of Nramp1 published at GenBank(U12862.1), it had a single nucleotide mutation. This nucleotide mutation made the 49th amino acid which encoded by the Qinchuan cattle's Nramp1 gene N-terminal fragment changed to the alanine. Recombinant plasmid pGEX-N2 was expressed efficiently in the Escherichia coli BL21(DE3) under the optimized induction condition (0.1 mmol/mL IPTG for 10 h at 35℃) and its expressive product (33 kD) was soluble; In brief, the findings that depurated GST-Nramp1-N fusion protein obtained in the present study provides a good foundation for further research to determine the biological activity of bovine Nramp1 and lay a pathway for resistance breeding by animal transgenic technology.
|
|
Received: 30 April 2010
|
|
|
|
|
|
|
