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| Cloning of Cattle Adipose-specific phospholipase A2 Gene(AdPLA) by Overlap PCR and Its Prokaryotic Expression |
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Abstract Adipose-specific phospholipase A2 gene(AdPLA) is a major regulator of adipocyte lipolysis, and in order to construct the prokaryotic expression system of cattle AdPLA gene, the coding sequence of AdPLA gene was obtained by Overlap PCR method from cattle(Bos taurus) genomic DNA and cloned into pGM-T vector, the positive clone sequencing result was same as the sequence in GenBank (GenBank No. NM_001075280). The positive plasmid was digested with BamHⅠ and HindⅢ, then the fragment was ligated with the expression vector pET28a(+), the recombined plasmid was transformed into Escherichia coli BL21(DE3) and induced with IPTG. SDS-PAGE result showed that the fusion protein His-AdPLA was expressed in E.coli, which indicated the prokaryotic expression system of recombined vector pET28a(+)-AdPLA was constructed successfully. This study provides a good foundation for further research of cattle AdPLA gene and lays a pathway for developing the AdPLA protein product in the future.
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Received: 11 April 2011
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