重组人胰高血糖素样肽-1类似物的分离纯化和鉴定

将合成的人胰高血糖素样肽-1类似物基因插入到原核表达质粒pGEX-4T-3中，构建成rhGLP-1类似物与谷胱甘肽巯基转移酶（glutathione-S-transferases，GST）的融合表达载体pGEX-rhGLP-1类似物，转化大肠杆菌BL21（DE3）获得重组菌株。IPTG诱导表达的菌体经高压均质机破碎后，离心收集包涵体，经尿素变性、Glutathione-Sepharose 4B亲和层析、肠激酶酶切、SP-Sepharose FF层析和反相层析RP-C18脱盐后冻干，得到纯度大于96%的rhGLP-1类似物，经质谱测定，分子量与理论值一致。生物学活性分析表明，rhGLP-1类似物具有促进表达有GLP-1受体的HEK293细胞cAMP增加的活性。

Purification and Characterization of Recombinant Human Glucagon-like Peptide-1 Analogue

The synthetic recombinant human glucagon-like peptide-1(7~37) (rhGLP-1) analogue gene was cloned into prokaryotic expressing vector pGEX-4T-3 fused with glutathione-S-transferases (GST) to construct a recombinant plasmid pGEX-rhGLP-1 analogue, then transformed into E. coli BL21(DE3). The constructed E. coli BL21(DE3)/pGEX-rhGLP-1 analogue was induced by IPTG and proteins were expressed, and then broken to extract inclusion bodies by high pressure homogenizer. The inclusion bodies were collected through centrifugation. The rhGLP-1 analogue was purified by urea denaturation, Glutathione-Sepharose 4B affinity chromatography and digested by enterokinase. By further purification with SP-Sepharose FF chromatography and RP-C18 reverse phase chromatography, the target rhGLP-1 analogue was gained with purity more than 96% by HPLC analysis and molecular weight was consistent with theoretical value by MS analysis. The biological activity analysis showed that rhGLP-1 analogue stimulated cAMP production from HEK293 cells expressing GLP-1 receptors.