人瘦素的基因克隆及其在COS-7细胞中的表达

Cloning of human leptin gene and its expression in COS-7 cells

目的 构建重组人瘦素哺乳细胞表达载体并在COS 7细胞表达重组人瘦素。方法 提取脂肪细胞总RNA ,用RT PCR扩增人瘦素cDNA并克隆至载体 pUCm T ,并对克隆基因进行DNA序列分析。以克隆的人瘦素cDNA为模板 ,用特异引物扩增瘦素基因 ,经KpnI和BamHI酶切 ,插入相应酶切的哺乳细胞表达载体pcDNA3 ,构建重组哺乳细胞表达载体并转染COS 7细胞 ,RT PCR和Western印迹检测其在COS 7细胞中的表达。结果 RT PCR扩增的DNA片断和预期的人瘦素cDNA大小一致 ;序列分析显示 ,克隆的基因序列和文献报道的人瘦素基因序列一致 ;经RT PCR和Western印迹鉴定 ,转染的COS 7细胞可表达、分泌人瘦素。结论 构建了人瘦素的哺乳动物细胞表达载体 ,并成功地在COS 7细胞中获得重组人瘦素的分泌表达。

Objective To construct recombinant human leptin mammalian cell expression vector, and to express recombinant human leptin in COS-7 cells. Methods Human leptin cDNA was amplified from total RNA of human adipocytes by RT-PCR and cloned into pUCm-T vector to construct recombinant pUCm-T-OB, which was then sequenced. Sequenced human leptin cDNA was used as template to carry out the second round of PCR with upstream primer containing Kozak sequence and KpnI site, and downstream primer containing BamH I site. The PCR product was digested with KpnI and BamH I and inserted into eukaryotic expression vector pcDNA3 to obtain recombinant human leptin expression plasmid pcDNA3-OB which was transfected into COS-7 cells. The transient expression of human leptin was examined with RT-PCR and Western blotting. Results The DNA sequence of cloned human leptin gene was the same as reported previously. Human leptin mRNA was highly expressed in transfected COS-7 cells, and secretive recombinant human leptin was detected in the culture supernatant of transfected COS-7 cells by Western blotting. Conclusion Cloning and expression recombinants of human leptin are obtained and secretive recombinant human leptin is successfully expressed in COS-7 cells.