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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 a recombinant human leptin mammalian expression vector and express recombinant human leptin in COS 7 cells. Methods The total RNA of adipocytes was extracted. The human leptin cDNA was amplified by RT PCR and cloned into vector pUCm T. The cloned gene was analyzed by DNA sequencing. The cloned human leptin cDNA was used as a template to amplify the leptin gene with specific primers. The recombinant leptin gene was digested with KpnI and BamHI and inserted into the correspondingly digested mammalian expression vector pcDNA3. The recombinant mammalian expression vector was constructed and transfected into COS7 cells. RT-PCR and Western blotting were used to detect the expression in COS 7 cells. Results The DNA fragment amplified by RT PCR was consistent with the expected size of human leptin cDNA. Sequence analysis showed that the cloned gene sequence was identical with the human leptin gene sequence reported in the literature. After identification by RT PCR and Western blotting, the transfected COS 7 Cells can express human leptin. Conclusion The human leptin mammalian cell expression vector was constructed and the secretory expression of recombinant human leptin was successfully obtained in COS 7 cells.