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目的 研制基因工程胰高血糖素，用于治疗因注射胰岛素或口服抗糖尿病药物而引起的严重低血 糖反应。方法 依据人胰高血糖素氨基酸组成序列，采用大肠杆菌出现频率高的氨基酸密码子，体外合成胰高血 糖素基因序列，克隆于ｐＧＥＸ－１Ｎ质粒，转化人ＢＬ２１（ＤＥ３）菌中，对阳性克隆转化质粒进行ＤＮＡ测序。用ＩＰＴＧ诱导 转化的胰高血糖素工程菌，产生谷胱苷肽转移酶的融合蛋白，经谷胱苷肽Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析后，由 Ｆａｃｔｏｒ Ｘａ酶 进行柱上切割，最后用ＲＰ－ＨＰＬＣ得到纯化产物。产品用质谱法测相对分子质量，并对Ｎ末端１５个氨基酸测序。 结果ＤＮＡ测序显示克隆基因序列正确，产品相对分子质量为３４８６Ｊ末端１５个氨基酸序列与理论值相同，产率为 １．９ｍｇ／Ｌ。结论 获得以可溶性融合蛋白方式表达的胰高血糖素基因工程菌，适于快速生产天然序列重组胰高血 糖素。
Objective To develop a genetically engineered glucagon for the treatment of severe hypoglycemia caused by insulin injections or oral antidiabetic drugs. Methods Based on the amino acid sequence of human glucagon, the amino acid codon of Escherichia coli was used to synthesize glucagon gene sequence in vitro. The glucagon gene was cloned into pGEX-1N plasmid and transformed into human BL21 (DE3) Plasmids were transformed for DNA sequencing. After induced by IPTG, the glucagon-engineered bacteria produce the glutathione transferase fusion protein, which is cleaved by Factor Xa enzyme after affinity chromatography with glutathione Sepharose 4B and finally obtained by RP-HPLC Purified product. The product was measured by mass spectrometry relative molecular mass, and N-terminal 15 amino acids were sequenced. Results DNA sequencing showed that the sequence of the cloned gene was correct. The relative molecular mass of the product was 3486J and the 15 amino acid sequences were the same as the theoretical value, and the yield was 1.9mg / L. Conclusion The glucagon genetically engineered bacteria expressed by soluble fusion protein are suitable for the rapid production of natural sequence recombinant glucagon.