胰高血糖素衍生物在大肠杆菌的克隆表达和纯化

 目的 通过基因工程途径获得胰高血糖素衍生物。 方法 根据胰高血糖素基因及凝血酶酶切位点序列，分段合成引物行 PCR 扩增，以 PCR 扩增得到的胰高血糖素-甘氨酸基因序列和 pET-30a 质粒转化 E.coli DH5α，获得重组质粒 pET-G。将重组质粒 pET-G 转化至 E.coli BL21(DE3)，重组菌株命名为 E.coli BL21pET-G]。以异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，对表达产物进行SDS-Tricine- PAGE十二烷基硫酸钠-三（羟甲基）甲基甘氨酸-聚丙烯酰胺凝胶电泳]分析和蛋白质印迹分析。对表达产物行Ni2+-NTA亲和层析纯化、凝血酶酶切和高效液相色谱（HPLC）纯化后，行 SDS-Tricine-PAGE分析和飞行质谱分析，并以 ELISA 方法检测其免疫活性。 结果 PCR 扩增获得的条带与预期的DNA表达片段大小一致，重组质粒 pET-G 测序结果与预期完全一致。SDS- Tricine-PAGE 和蛋白质印迹分析显示 E.coli BL21（DE3）的表达产物相对分子质量与预期相符。经亲和层析纯化、凝血酶酶切和 HPLC 纯化后得到了完整的重组胰高血糖素-甘氨酸衍生物，SDS-Tricine-PAGE 分析显示其相对分子质量约为 3500，飞行质谱分析相对分子质量为 3531，二者基本一致。ELISA 检测表明重组胰高血糖素-甘氨酸衍生物具有胰高血糖素免疫活性。 结论 采用基因工程技术在大肠杆菌中成功表达了胰高血糖素-甘氨酸衍生物，为通过体外酰胺化途径研制酰胺化胰高血糖素奠定了基础。

Cloning,expression and purification of glucagon-glycine in E.coli

Objective To produce glucagon-glycine(Gly) by using recombinant DNA method. Methods Based on the sequences of glucagon gene and the recognition site of thrombin,the gene encoding glucagon-Gly was amplified with PCR and inserted into the plasmid pET-30a to construct a recombinant plasmid pET-G. Then,the expression of the glucagon-Gly in E.coli BL21 was induced with IPTG and analyzed with SDS-Tricine-PAGE and Western blotting. After being purified by the Ni2 affinity chromatography,and cleaved by thrombin,and then purified with HPLC,the expressive product was analyzed with SDS-Tricine-PAGE and MALDI-TOF mass spectrum. And the immunoactivity of the production was determined using ELISA. Results The results of PCR and the sequence of recombinant plasmid pET-G were consistent to the expected ones. SDS-Tricine-PAGE and Western blotting showed that the relative molecular weight of the expressive product of E.coli BL21(DE3) was same to the expected value. After being purified with Ni2 affinity chromatograph,cleaved by thrombin,and then purified using HPLC,a integral derivative of glucagon,glucagon-Gly was obtained,with a relative molecular weight of 3500 shown by SDS-Tricine-PAGE,which was similar to that shown on the MALDI-TOF mass spectrum(3531). ELISA found that the derivative had glucagon-like immunoactivity. Conclusions Glucagon-Gly can be expressed in E.coli by using gene engineering technique. The results provide a base for producing amidated glucagon.