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目的 研制基因工程胰高血糖素。方法 利用pCYB1表达载体,亚克隆胰高血糖素基因后转化于BL21（DE3）菌种,并对阳性克隆的质粒进行DNA测序。检测不同条件下,IPTG诱导胰高血糖素-intein-几丁质结合结构域（CBD）组成的融合蛋白表达量,确定工程菌表达最适条件。利用几丁质结合柱进行亲和层析,在DTT作用下由intein在柱上进行自我切割,纯化胰高血糖素。SDS-PAGE检测融合蛋白表达量,SDS-PAGE在Tris-Tricine缓冲系统中电泳检测纯度,Lowry法检测蛋白质含量,Western blot检测免疫原性,升血糖实验检测活性并进行 N-末端测序。结果DNA测序显示表达产物基因序列正确,纯化胰高血糖素具有免疫原性和生物学活性,N-端15个氨基酸与天然产品相同,电泳呈单一谱带,产率为1.25mg/L。结论 获得分泌胰高血糖素基因工程菌,适于快速大规模生产。
Objective To develop genetic engineering glucagon. Methods The pCYB1 expression vector was used to subclone the glucagon gene into BL21 (DE3) strain. The positive clones were sequenced by DNA sequencing. Under different conditions, IPTG was used to induce the expression of fusion protein composed of glucagon -intein-chitin binding domain (CBD) to determine the optimal conditions for engineering bacteria expression. Chitin binding column affinity chromatography, in the DTT by intein in the column self-cleavage, purification of glucagon. SDS-PAGE was used to detect the expression of the fusion protein. The purity of the fusion protein was detected by SDS-PAGE electrophoresis in Tris-Tricine buffer system. The protein content was determined by Lowry method. The immunogenicity was detected by Western blot. Results DNA sequencing showed that the sequence of the expressed product was correct. The purified glucagon was immunogenic and biological. The N-terminal 15 amino acids were the same as those of natural products. The single band showed a single band with a yield of 1.25 mg / L. Conclusion Obtain glucagon gene engineering bacteria, suitable for rapid large-scale production.