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目的 应用大肠杆菌表达系统进行小鼠瘦素基因的克隆、表达、产物纯化与鉴定.方法应用聚合酶链扩增技术,以含有小鼠瘦素cDNA序列的质粒pMET mouse leptin为模板,扩增后克隆至载体pET-28a(+)构建重组表达载体pET-28a-lep,酶切、测序正确后,转化大肠杆菌E. coli BL21,构建工程菌株BL21-pET-lep.使用0.1mmol/L异丙基-硫代半乳糖苷,在不同的时间段诱导蛋白表达,利用十二烷基磺酸钠-聚丙烯酰胺电泳和Western Blotting检测瘦素蛋白的分子质量以及最佳表达时间.使用0.5%十二烷基肌氨酸钠溶解包涵体后通过Ni2+亲和层析柱纯化.结果 正确构建了表达载体pET-28a-lep.0.1 mmol/L异丙基硫代半乳糖苷诱导BL21-pET-lep 6 h具有最高蛋白表达量,含量占菌体总蛋白的39.2%.SDS-PAGE以及Western Blotting检测显示所表达的小鼠瘦素为携带组氨酸标签的融合蛋白,分子质量约为22.5 kDa.使用Ni2+层析柱纯化后的蛋白纯度为1.086 g/L.结论 应用大肠杆菌原核表达系统成功进行了小鼠瘦素基因的克隆、表达、产物纯化与鉴定.“,”Aim To prokaryotic express mouse obese gene in E. coli expression system and purify the fused mouse leptin. Methods Mouse obesity gene leptin was amplified from pMET mouse leptin and subcloned in pET-28a (+) to construct recombinant vector pET-28a-lep. The sequence of pET-28a-lep was checked by sequencing and restric-tion analysis. Expression of BL21-pET-Iep transformed with pET-28a-lep was induced with 0. 1 mmol/L isopropylthiogal-actoside and analyzed with Sodium dodecylsulfonate-polyacrylamide gel electrophoresis and Western Blotting to identify the mouse leptin molecular weight and optimum time for purpose protein expression. The maximum amount of the fused pro-tein expressed was examined with thin layer chromatography. Mouse leptin dissolved with 0.5% Sarkosyl was purified by Ni2+ affinity chromatography column. Results Sequencing and restriction analysis confirmed the right sequence of pET-28a-lep. Sodium dodecylsulfonate-polyacrylamide gel electrophoresis and Western Blotting indicated a 22.5 kDa fused mouse leptin, in the form of inclusion body, was expressed with high efficiency in BL21-pET-Iep. The maximum amount of the fused protein produced was 39.2% of the total cellular protein. The concentration of purified protein is 1.086 g/L.Conclusion BL21-pET-lep could highly express recombinant mouse leptin. This provides a basis for further resear-ches on biological activity of mouse leptin and its role on the developing process of arthresclerosis.