摘要
目的:构建和制备出一种新型速效人胰岛素类似物(PIns),进行其性质研究。方法:采用加端PCR的方法,扩增出在天然人胰岛素原N端添加精氨酸、脯氨酸、赖氨酸、脯氨酸4个氨基酸残基的重构胰岛素原基因片段,将其插入通用表达载体pET28 a后,再将经过改构后的硫氧还蛋白(1-20 aa)的基因片段与重构胰岛素原基因的5′端通过赖氨酸连接,成功构建了速效人胰岛素原类似物融合蛋白(FKPPIns)的表达载体(pET28 a-FKPPIns)。此融合蛋白在大肠杆菌BL-21(DE3)中以包涵体的形式表达,经包涵体洗涤和DEAE阴离子交换树脂纯化后进行复性、酶切。酶切产物经RP-HPLC纯化后,质谱法测定其分子量;等电聚焦法测定此蛋白的等电点;凝胶过滤法测定其自身的缔合性质。对新西兰大白兔肌肉注射纯化后PIns,进行初步的药效学评价。结果:获得了结构为R-P-K-P-Insu lin的PIns纯品,其等电点为7.3,自身缔合性较标准胰岛素显著下降。结论:PIns与标准人胰岛素相比,起效快、达峰时间及持续时间短。
Objective To construct and prepare a novel short-acting human insulin analog and to explore the basic properties of the protein. Methods The nucleotide sequence coding Lys-Arg-Pro-Lys-Pro was added to the 5＇ of human pronsulin gene through add-on PCR. The reconstructed proinsulin gene was cloned into pET28a, and then the mutational gene of Thioredoxin（ 1-20 aa） was also inserted into pET28a connecting to the 5＇ of reconstructed insulin gene. The recombinant plasmid was expressed in E. coli BL21 at a high level. After being purified and refolded, the fusion protein was digested by trypsin and carboxypeptidase B, the product of which was purified by RP-HPLC. Protein identities were confirmed by Autoflex ToF/ToF mass spectrometry. The pI and self-association of the short-acting human insulin analog were studied through an isoelectrofocusing technigue and the size exclusive chromatagraphy respectively. The declining blood-glucose effect was studied by i. m. to New Zealand rabbits. Results Correct and purified short-acting human insulin analog was obtained. The pI of the protein was 7.3, which was approximate the pH in physiological range and the self-association was lower than normal insulin. Conclusion The short-acting human insulin analog has a faster onset shorter duration of action than human insulin.
出处
《东南大学学报（医学版）》
CAS
2009年第5期371-375,共5页
Journal of Southeast University(Medical Science Edition)
基金
国家自然科学基金资助项目(30772570
30672464)
关键词
融合表达
速效人胰岛素类似物
单体胰岛素
药效学活性
fused expression
short-acting human insulin analog
monoricisulin
activity of pharmacodynamics
