人Leptin的基因克隆及其在大肠杆菌中的表达

用 RT- PCR自脂肪细胞 RNA扩增人瘦素 (leptin)基因的 c DNA片段 (约 50 0 bp)并克隆至克隆载体 p SK(+) ,获得重组质粒 p SK- OB,DNA序列分析显示获得的 Leptin基因和文献报道一致 .用限制酶 Eco R 和 Bam H 从 p SK- OB切下并插入原核表达载体 p BV2 2 0的相应限制酶位点 ,转化大肠杆菌 DH5α.转化菌株经 42℃诱导 ,SDS- PAGE检测和 Western印迹鉴定 ,获得高水平重组人瘦素的表达菌株 ,其表达量达菌体总蛋白的 30 %以上 .

Cloning of Human Leptin Gene and Its Expression in E. coli

Human leptin gene was amplified from human adipocyte RNA with RT PCR and cloned into plasmid pSK(+) to construct recombinant pSK OB,which was then sequenced.The result showed that DNA sequence of cloned human leptin was the same as that reported previously.Leptin DNA fragment was cut from pSK OB with Eco RⅠ and Bam HⅠ and inserted into procaryotic expression vector pBV220.The expression recombinant showed a high level expression of recombinant protein (about 30% of total bacterial protein).SDS PAGE and Western blot analysis indicated that the molecular weight of recombinant protein was about 16 kD and could react specifically with anti leptin antibody.