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| Cloning of Porcine Granulocyte-macrophage Colony Stimulating Factor （pGM-CSF） Gene and Its Prokaryotic Expression |
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Abstract The porcine granulocyte-macrophage colony stimulating factor (pGM-CSF) full-length gene was cloned from porcine peripheral blood lymphocyte using RT-PCR and ligated into pMD19-T vector. Sequence analysis showed that the cDNA of pGM-CSF was 435 bp in length and encodes 144 amino acids. The gene of mature pGM-CSF deleting a 17 aa signal sequence at N-terminus was cloned by PCR and inserted into pET-32a(+). The recombinant plasmid pET-GM was transformed into Escherichia coli BL21(DE3) and induced by IPTG. The results of SDS-PAGE and Western blot showed that the recombinant protein was about 31 kD, which existed mainly in inclusion body, accounts 30.4% of total bacterium proteins. After dilution renaturation and purification procedure in Ni2+-NTA affinity chromatography column, the biological activity of purified protein was analyzed by MTT method using TF-1 cells. The result indicated that the recombinant protein could effectively stimulate the proliferation of TF-1 cells, with a specific bioactivity of 3.43×105 IU/mg.
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Received: 23 June 2008
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