中国人肥胖抑素cDNA的克隆和在大肠杆菌中的初步表达

Cloning of human leptin cDNA and its expression in E. coli

目的为获得大量人肥胖抑素（ｌｅｐｔｉｎ）以便对其理化特性、生物学功能及其在肥胖发生中的作用进行深入的探讨。方法作者从人脂肪细胞中提取总ＲＮＡ，经逆转录聚合酶链反应（ＲＴ－ＰＣＲ）获得了人ｌｅｐｔｉｎｃＤＮＡ；通过体外ＤＮＡ重组技术，以ｐＢＶ２２０为表达载体构建重组表达质粒ｐＢＶ２２０－ｌｅｐｔｉｎ，经酶切及序列分析所证实。然后将重组表达质粒转化大肠杆菌ＤＨ５α（Ｅ．ｃｏｌｉＤＨ５α）进行表达。结果经十二烷基硫酸钠－聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）分析，含有重组表达质粒的菌株表达出了特异性的１６ｋｕ蛋白质，其产量占总菌体蛋白的３１％～４７％，重组蛋白主要以包涵体形式存在。结论人ｌｅｐｔｉｎ重组表达系统的构建是成功的。

Objective To obtain human leptin and to investigate its characteristics, biological function and its relation to obesity. Methods Human leptin cDNA fragment was obtained by reverse transcription polymerase chain reaction (RT PCR) with total RNA extracted from human adipocytes and the recombinant expressing plasmid pBV220 Leptin was constructed. The recombinant plasmid was confirmed by restriction endonuclease digestion and DNA sequencing. Results SDS PAGE analysis showed that E. coli DH5α containing the recombinant plasmid could produce a kind of protein of 16ku which amounted to 31%～47% of total cellular proteins. Conclusion The results suggest that the construction of human leptin expressing plasmid is successful.