胰高血糖素基因克隆、表达及鉴定

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。

Cloning, Expression and Characterization of Recombinant Human Glucagon

Objective To prepare a bioengineering product for treating Glucatonia.Methods Glucatonia gene was chemically synthesized according to the amino acid sequence of it with amino acid codens appearing frequentlys in E. coli and cloned into pGEX-1N vector,then transformed to BL21(DE3)cell.The plasmids transformed with positive clones were sequenced. The expressed product was purified by affinity chromatography, digested with factor Xa enzyme,and further purified by RP-HPLC. Results DNA. sequecing showed that the Glucagon gene in Glucagon-pGEX-1N vector was correct. The 15 amino acids at the N-terminal of recombinant Glucagon were identical to those of native Glucagon.The relative molecular weight of it was 3486, and the yield was 1 .9mg/L. Conclusion The engineering strain expressing Glucagon in the form of soluble fusion protein was obtained. It was suitable for the rapid preparation of recombinant Glucagon with native sequence.