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摘要: 为大量获取人胰高血糖素样肽-1，利用基因串联的方法构建了人胰高血糖素样肽-1的一组串联体.将化学合成的人胰高血糖素样肽-1（human glucagonlike peptide-1, hGLP-1）cDNA基因插入质粒载体pET-32a(+)中，构建成硫氧还蛋白（thioredoxin）及六聚组氨酸（hexahistidine）与rhGLP-1的融合表达载体pET32-GLP-1.在此基础上将该融合基因序列进行同向串联，获得二串和三串的表达载体pET32-GLP-1-2 和pET32-GLP-1-3.将该三种表达载体转化大肠杆菌BLR(DE3)后获得相应的基因工程菌.结果表明：三种基因工程菌经发酵和IPTG诱导后均正确表达目的蛋白，目的蛋白表达量随着基因串数的增加而得到一定提高，重组蛋白经分离纯化后进行生物学活性测试具有明显的降低血糖浓度作用.Abstract: To get a large quantity of human glucagon-like peptide-1, different hGLP-1 multimers containing different copies of rhGLP-1 were constructed. The synthetic hGLP-1 cDNA gene was inserted into pET-32a(+) to get the recombinant plasmid pET32-GLP-1, which could express a fusion protein including thioredoxin， hexahistidine, and rhGLP-1. The coding sequence for the fusion protein was repeatedly inserted into the vector in tandem to get pET32-GLP-1-2 and pET32-GLP-1-3 with two or three copies, respectively. Three different plasmids were transformed into E.coli BLR (DE3) and induced by IPTG. The results demonstrated that the three kinds of E.colicells expressed the desired protein and the percentages of the desired protein increased with the numbers of gene copy. The purified rhGLP-1 showed obvious biological activity of lowering plasma glucose.
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Key words:
- hGLP-1multimerexpression /
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