Exendin-4的表达及其降糖活性研究

Study on the Expression of Exendin-4 and Its activity to Decrease Plasma Glucose

根据GeneBank中登录的Exendin-4的氨基酸序列(AAB22006),结合大肠杆菌的密码子偏嗜性,对原有基因70%的密码子进行改造,使其在大肠杆菌中高效表达;同时添加KpnI和NcoI酶切位点和肠激酶识别位点,构建成重组表达载体pET-32a(+)-Exendin-4,转化大肠杆菌BL21(DE3)。在30℃条件下1mmol/L的IPTG诱导表达6h,融合蛋白表达量占细菌蛋白总量的32%。利用亲和层析纯化融合蛋白,每克湿菌体可获得纯度98%的融合蛋白6.44mg。利用肠激酶切除融合蛋白的N端融合部分,用亲和层析吸附N端融合肽,每克湿菌体可获得纯度99%的Exendin-41.59mg。腹腔注射糖尿病基因小鼠(BKS.Cg-m+/+Leprdb/J鼠),血糖浓度下降极显著,最佳用量为7μg/kg,注射后4h血糖降低达51.93%。

For the sake of a high expression of Exendin-4 in Escherichia coli, the original Exendin-4 gene was modified in light of its amino acid sequence （GeneBank： AAB22006） and the code bias of E. coli. Not only 70% of the codes were changed, but also the sites of KpnI, NcoI and enterokinase were added to the two terminals of Exendin-4 if the gene respectively. The recombinant expression vector of pET- 32a（＋）- Exendin-4 was constructed and transformed into E. coli BL21（DE3）. The fusion protein was 32 percent of total protein in E. coli after induction with 1 mmol/L IPTG for 6 hours at 30℃. With Ni-NTA chromatography, 6.44 mg exendin-4 fusion protein from one gram wet bacteria was obtained and the purity was up to 98%. After the fusion protein was digested by enterokinase, affinity chromatography was used to bind the N terminals of 6× His tag and to purify Exendin-4. The yield of Exendin-4 was up to 1.59 mg per gram wet bacteria with the purity of 99%. The experiment in BKS. Cg-m＋/＋ Lepr db/J mice revealed that intraperitoneal injection of Exendin-4 had prominent glucose-lowering effects. The optimal dose was 7μg/kg and the maximal decrease in plasma glucose was up to 51.93% at 4 h after intraperitoneal injection.