人肥胖基因的克隆与原核表达载体的构建

目的：克隆人肥胖基因瘦素蛋白（leptin）编码区cDNA序列，并构建其原核表达载体。 方法：在人体脂肪组织中提取mRNA，利用RT-PCR扩增人肥胖基因，将其插入pMD18-T载体，经双酶切、鉴定后将肥胖基因片段亚克隆入pET-28a表达载体，提取质粒。将阳性重组质粒转化表达受体菌E.coliBL21(DE3)，经IPTG诱导肥胖基因表达产物,以聚丙烯酰胺凝胶电泳（SDS-PAGE）检测产物蛋白。 结果：经测序证实获得的肥胖基因序列与相关文献报道一致，其表达产物的相对分子质量约为20 000。 结论：利用pMD18-T克隆载体和pET-28a表达载体可以有效地在原核生物中表达重组人类瘦素蛋白。

Cloning of human obese gene and construction of its prokaryotic expression vector

Objective To clone the sequence of cDNA of human obese gene (leptin) coding area and construct a prokaryotic expression vector. Methods Human obese gene was amplified from human fat tissue by RT-PCR and subcloned in pMD18-T ,a clone vector. The sequence of pMD18-T was checked by sequencing and restriction analysis. Expression of E.coli BL21(DE3) and expression products of obese gene induced by IPTG were analyzed with SDS-PAGE. Results It was proved that the cloned leptin cDNA had been constructed with gene recombinant technique. Sequencing and restriction analysis confirmed the sequence of pMD18-T. A 20 000 leptin fusion protein was expressed with high effeciency in recombinant E.coli BL21. Conclusion pMD18-T and pET-28a could highly express recombinant human leptin.