鲑鱼降钙素基因在大肠杆菌中的表达纯化及药理活性研究-中药学专业论文.docx

中文摘要 中文摘要 降钙素(calcitonin，CT)是一类重要的多肽类激素，在钙、磷 代谢，骨骼的形成、分化和发育等多个环节有重要的作用，特别是鲑 鱼降钙素(salmon calcitonin，sCT)在临床土有十分重要用途。 sCT和许多多肽C末端的酰胺化对其生物学功能有重要的作用， 但是这种酰胺化修饰在原核表达系统很难完成，表达的前体蛋白需要 在细胞外经过酶的修饰才具有生物活性。为了用大肠杆菌制备有生物 活性的sCT，在本研究中我们采用含有内蛋白子(Intein)的表达系 统，在Intein剪切过程中，加入提供NH3的试剂达到酰胺化的目的。 本文利用半化学半酶促法获得大肠杆菌密码子偏嗜的sCT基因， 将sCT基因克隆入含有Intein的大肠杆菌融合型表达载体pTWIN 1 中。重组表达载体转到大肠杆菌ER2566中，表达条件优化后，经发 酵罐流加发酵，破菌，然后将表达的融合蛋白经CBD亲合柱纯化， 并且在亲和柱上一步完成Intein介导的目的蛋白的剪切并口C末端的酰 胺化。获得的sCT进一步的分离纯化得到初级纯品，通过观察其对 破骨细胞活动的抑制和大鼠降血钙试验来初步检测其生物活性。 结果表明大肠杆菌重组的sCT融合蛋白经inein介导的剪切和酰 胺化后，具有与化学合成的sCT相同的理化特征，并具有显著的降 低大鼠血钙的活性，如果进一步优化工艺有望用于x-,．1k化生产。 关键觑鲑鱼降钙素；内蛋白子；酰胺化；大肠杆菌；重组 Expressing Expressing of Salmon Calcitonin in E coli and Its Pharmacological Activity Study Postgraduate：Shou-Sheng Yah Director：Yu—Wang Tian rHunan College ofTCM，Changsha,410007) Abstract Calcitonin is one of the most important peptides in metabolism of calcium and phosphorous and in development and differentiation of bone． We aimed to express recombinant salmon calcitonin“sCT)in ESch盯ichia coli．，however sCT and many polypeptides need carboxy amidation for full activ时or prolonged bioavailability,this modification is not possible in prokaryotes． A method of using self-cleaving Intein expression system was used in this study，the system is able to making amidated sCT and some other peptides by using special buffers with ammonia during the self-cleaving of the C-terminal Intein．In this smdg we cloned sCT gene into pTWIN 1 vector and used E．coli strain ER2566 as the host。Expression experiments were carried out both in shake flasks and a 5L bioreactor． Purifled rsCT was identified with RT-HPLC and isoelectric focusing． Biological assays of rsCT were based on osteoclastic cells responsiveness and blood calcium determination in rats： The results show that most of the rsCT is in form of amidation after self-cleaving by Inteins and has good effect of hypocalcemic bioactivity． Keywords： Salmon calcitonin； Intein； Amidation； E． coil； Recombinantion