摘要
将人工合成的人胰高血糖素样肽-1（human glucagon—like peptide-1，hGLP-1）基因插入质粒栽体pET-32a（＋）中，构建成rhGLP-1与硫氧还蛋白（thioredox）及六聚组氨酸（hexahistidine）的融合表达栽体pET32-GLP-1，转化大肠杆菌BL21（DE3）获得表达菌株，经LPTG诱导发酵的菌体超声破碎后，裂解液用Ni离子亲和层析纯化得到融合蛋白，经肠激酶裂解，再次Ni离子亲和层析，得到rhGLP-1样品。经SDS—PAGE和等电聚焦检测，样品纯度大于90％，等电点介于pH5．2-pH5．85之间。质谱测定rhGLP-1分子量为3355．0kDa，肽图分析得到2097．7kDa和1005．5kDa两个胰蛋白酶酶解片断，均与理论分析结果一致。动物实验表明重组蛋白具有明显的降血糖活性和促胰岛素分泌作用。
The synthesized full-length hGLP-1 gene was cloned into pET-32a（ ＋ ） to get the recombinant plasmid pET32-GLP-1, which could express a fusion protein containing thioredox, hexahistidine, and rhGLP-1 consecutively. The recombinant plasmid containing hGLP-1 was transformed into E. coli BL21 （DE3） and expressed by IPTG induction. The fusion protein was purified from lysates with Ni ~ IDA His· Bind affinity chromatography, rhGLP-1 with the purity of 90% was achieved after enterokinase digestion, Ni · IDA His · Bind affinity chromatography again, then was concentrated by ultrafiltration. The purified rhGLP-1 showed a single band on IEF gel with an isoelectric point between pHS. 2 and pI-IS. 85. ESI mass spectrometry showed that the molecular weight was 3355.0kDa as expected, rhGLP-1 was digested with trypsin followed by mass analysis and the peptide mapping produced two expected fragments with the molecular weights of 2097.7kDa and 1005.5kDa, respectively. The purified rhGLP-1 also showed obvious biological activity for both lowering plasma glucose and stimulating insulin secretion in mice.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2006年第1期50-55,共6页
China Biotechnology
