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Preparation of Bioactive Recombinant Chicken Leptin Fusion Protein
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| LIU Ying;LIU Zhi;SHI Zhen-dan;LI Xiao-wei;ZHONG Zi-sui |
| College of Animal Science, South China Agricultural University, Guangzhou 510642, China |
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Abstract The cDNA sequence of chicken Leptin mature peptide was cloned into Nhe Ⅰ and Hind Ⅲ sites of expression vector pRSET A to construct recombinant plasmid pLepSCAU2 which was transfected the Escherichia coli BL21(DE3). After OD600 of the recombinant plasmid which grow in LB medium supplemented ampicillin 100 μg/mL was more than 0.6, the recombinant chicken Leptin fusion protein (including a 6×His tag) of molecular mass of 17.5 kD was expressed with the induction of IPTG 0.05 mol/L and the maximal expression which was 25% of the total protein in bacteria was achieved after 4 h induction and existed in inclusion body. The soluble part was achieved after purified by 50% Ni-NTA agrose and renaturated and folded by dialyze with changes of urea solutions of specific concentrations and pH. Such obtained soluble Leptin fusion protein was characterized of its biological activities in 35 d old growth yellow broilers. Following intraperitoneal injection of 2 mg/kg body weight of Leptin, feed intakes between 80 to 120 min post-injection were all significantly depressed in both pullets (P <0.01, n =10) and cockerels (P <0.05, n =10) compared respectively with PBS treated control pullets (n =10) and cockerels (n =10). Thereafter the accumulative feed intake in Leptin treated cockerels increased to the same level of the PBS treated control cockerels at 330 min post-injection. However in pullets, the depression of accumulative feed intake by Leptin fusion protein was maintained from 120 min to 330 min post-injection. The above results demonstrated that soluble and biologically active recombinant chicken Leptin was obtained, and there was a sex difference in depression of appetite by Leptin treatment.
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Received: 14 December 2005
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Corresponding Authors:
SHI Zhen-dan
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