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将合成的人胰高血糖素样肽-1[recombinant human glucagon-like peptide-1(7~37),rhGLP-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c AMP增加的活性。
The synthetic human glucagon-like peptide-1 (7 ~ 37), rhGLP-1] analogue gene was inserted into prokaryotic expression plasmid pGEX-4T-3 to construct rhGLP- 1 analogues and glutathione-S-transferases (GST) fusion expression vector pGEX-rhGLP-1 analogues were transformed into E. coli BL21 (DE3) to obtain a recombinant strain. IPTG-induced bacterial cells were disrupted by high pressure homogenizer, and the inclusion body was collected by centrifugation. After urea denaturation, Glutathione-Sepharose 4B affinity chromatography, enterokinase digestion, SP-Sepharose FF chromatography and RP- After desalted, C18 was lyophilized to obtain rhGLP-1 analogues with a purity higher than 96%. The molecular weights were consistent with the theoretical values by mass spectrometry. Biological activity analysis revealed that rhGLP-1 analogs have an activity that increases cAMP in HEK293 cells expressing the GLP-1 receptor.