重组人球状脂联素在毕赤酵母中的表达和生物学活性 分析

Recombinant human gapM1 expressed in Pichia pastoris and its anti-diabetic effect

摘要:

脂联素是一种重要的脂肪细胞因子, 而球状脂联素(脂联素的球状结构域, gapM1)有望开发为一种新药, 用来治疗Ⅱ型糖尿病。本研究分别通过摇瓶和发酵罐培养, 用毕赤酵母工程菌进行重组人gapM1的表达, 并用凝胶过滤和阴离子交换进行蛋白的纯化。然后, 用高脂饲料和低剂量STZ (链脲佐菌素)相结合的方法建立Ⅱ型糖尿病大鼠模型,鉴定gapM1的生物学活性。SDS-PAGE结果表明gapM1在毕赤酵母中得到了高效表达, Western blotting结果显示表达的蛋白为gapM1, 并从10 L发酵上清中纯化得到200 mg纯度为96%的gapM1。而制备的重组人gapM1能显著降低Ⅱ型糖尿病大鼠的血糖(34.2%)、血甘油三酯(79.6%)和血总胆固醇(62.1%)水平。因此, 重组人gapM1在毕赤酵母中得到了成功表达, 并且通过动物模型证明其有很好的降血糖和降血脂活性。

Abstract:

Adiponectin is an adipokine predominantly synthesized and secreted by adipocytes in the white adipose tissue, and it can lower the blood glucose level and increase free fatty acid oxidation. In the current study, we developed the globular domain of adiponectin (gapM1) to treat type II diabetes. In both flask and fermentor, we cultivated Pichia pastoris expressing recombinant gapM1 and established the purification procedure by using gel filtration and anion exchange chromatography. To evaluate the biological activity of recombinant gapM1, we used rat type II diabetes model fed high-fat diet in combination with low-dose STZ (Streptozocin) induction. We purified 200 mg gapM1 with purity of 96% from 10 liters of supernatant. The recombinant gapM1 significantly lowered blood glucose (34.2%), serum triglyceride (79.6%) and total cholesterol (62.1%) in type II diabetes induced rat. Therefore, the recombinant human gapM1 is successfully expressed in Pichia pastoris and effectively treated type II diabetes in rat models.