P277多肽的表达、纯化及初步药效学分析

Expression and purification of peptide P277 and its primary pharmacology

通过PCR方法得到P277多肽基因,插入到含门冬酰胺酶C端第199~326氨基酸残基片段的后面,形成融合蛋白,中间预留酸水解位点,并在P277多肽和融合蛋白之间加入一段碱性序列,构建pET28AnsB-C-KR-P277高效表达载体。克隆的基因在大肠杆菌中高效表达,通过Triton X-100洗涤、乙醇分级沉淀纯化融合蛋白AnsB-C-KR-P277,酸水解后经等电点沉淀去除杂蛋白,再经阴离子交换柱层析,进一步分子筛脱盐,可得到纯化的目的多肽P277。这种多肽生产平台,为多肽生物合成提供了一个很好的方法。纯化后的P277免疫型糖尿病模型动物NOD(non-obese diabetic)小鼠,可明显降低NOD小鼠自发性糖尿病的产生。

The peptide P277 gene was attained by PCR and successfully expressed as inclusion bodies in Escherichia coli by fusing it with the C-terminus of asparaginase and a basic amino acid-rich peptide(KR).After partially purified by washing with 5 mL·L-1 Triton X-100 in 10 mmol·L-1 PB,the pellet was solubilized in 8 mol·L-1 urea.The solution was precipitated with suitable volumes of cold ethanol for removing impurities.The fusion protein in solution was precipitated with triple volumes of ethanol to increase purit...