人Leptin基因在大肠杆菌中的高效表达、纯化及其生物学活性研究

将人Leptin表达质粒pBV220-OB转化E.coliJM109，经热诱导获得了目的蛋白的表达。经SDS-PAGE鉴定分析，表达产物以包涵体形式存在，目的蛋白表达量占菌体总蛋白的40%以上。通过包涵体分离，Sephacryl S200HR凝胶和DEAE52离子交换层析及Hypersil C18柱反相色谱纯化，获得纯度在95%以上，内毒素含量小于10EU/mg的高纯度的重组人Leptin。Western-blot鉴定表明，纯化表达产物能和抗Leptin抗体特异性结合；蛋白质N端15个氨基酸序列分析结果和预期的序列一致。纯化产物经复性处理，其分子中Cys96和Cys146形成二硫键。体内活性检测显示，纯化和复性的rh-Leptin明显抑制BALB/c小鼠的进食和体重增长，提示其具有明显的生物学活性。

Purification and Biological Activity of rh-leptin Expressed in Escherichia coli

The human leptin was successfully expressed with high level in E.coli under the control of P L promotor.The yield of recombinant protein was over 40% of total cellular protein and expressed as inclusion bodies.The recombinant human leptin (rh|leptin) was purified with gel filtration,anion|exchange and reverse chromatography.Refolding was achieved by gradually reducing denaturant using a diafiltration method.The refolded rh|leptin was characterized by SDS|PAGE,Western|blotting and its first 15 amino acid residues sequence of the N|terminal.The purified product was found to be biologically active,reducing the food intake and body weight gain upon testing in BALB/c mice.