人肥胖基因在大肠杆菌中的表达及生物学活性检测

肥胖基因(ob)编码的瘦蛋白可以反映机体脂肪含量信息,在发育、繁殖、造血及体重调节等方面具有重要功能。利用PCR方法扩增去除信号肽的人肥胖基因cDNA序列,并将位于基因5!端的CCC转变为大肠杆菌(Escherichiacoli)常用密码子CCG,扩增片段经测序证实后,克隆到原核表达载体pET5a,重组质粒转化大肠杆菌BL21(DE3),经0.1mmol/LIPTG诱导,获得分子量约为16kD的特异蛋白表达带,表达量最高占菌体蛋白总含量的55%。重组蛋白纯化后,注射昆明白小白鼠,小白鼠体重明显下降,说明纯化的重组蛋白具有生物学活性。

Expression of Human Obese Gene in Escherichia coli and Determination of the Recombinant Protein Bioactivity

Leptin has very important functions in regulation of mammals' development, propagation, blood reproduction and body weight. Here, a 456-bp fragment of human ob cDNA encoding ob mature protein without signal peptide part was amplified. A codon CCC in 5' end of the gene was replaced with CCG, which was used frequently in Escherichia coli. And then the fragment was cloned into an expression vector named as pET5a and the proper ligation of the vector was confirmed with sequencing. The aimed protein was expressed with the induction of 0.1 mmol/L IPTG, which was about 55% of total protein in bacteria. The purified recombinant protein was proved to be biologically activity, which reduced body weight on the testing in Kunming mice.