Preparation of Bioactive Recombinant Chicken Leptin Fusion Protein

将鸡Leptin成熟肽的cDNA片段插入表达载体pRSET A的Nhe Ⅰ和Hind Ⅲ两位点之间，构建重组表达质粒pLep -SCAU2并转化大肠杆菌(Escherichia coli )菌株BL21（DE3）, 将重组菌在含氨苄青霉素100 μg/mL 的LB培养基中培养至OD600大于0.6之后，经IPTG 0.05 mol/L诱导表达出分子量约为17.5 kD的含6×His标记的重组鸡Leptin，诱导4 h后表达量达最高，占总菌体蛋白的25％左右,且以包涵体形式存在。该重组鸡Leptin经50％Ni-NTA树脂纯化和透析复性折叠后分离出可溶性部分。对35日龄的矮脚黄鸡腹腔注射该可溶性重组鸡Leptin（2 mg/kg 体重）；注射后60 min内的采食量与对照组差异不显著 （P > 0.05），但在注射后80~120 min都显著低于对照组（P < 0.05）。Leptin处理公鸡的采食量在注射2 h后逐渐上升并在5.5 h时与对照组公鸡相同。但Leptin处理母鸡的采食量持续受到抑制，在注射后5.5 h时仍然显著低于对照组母鸡（P <0.01）。表明所表达的重组鸡Leptin能显著抑制鸡的采食量（母鸡比公鸡效果更为明显），证明所制备的重组鸡Leptin融合蛋白具有生物学活性。

Preparation of Bioactive Recombinant Chicken Leptin Fusion Protein

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.