降钙素基因相关肽衍生物的优化表达及纯化.doc

降钙素基因相关肽衍生物的优化表达及纯化 作者：肖莉杰 林平 陈文 姜颖 刘紫君 于晓光 【摘要】 目的 优化重组降钙素基因相关肽衍生物(CGRPVY)蛋白的表达体系，并利用亲和层析获得高纯度的重组蛋白。方法 在前期研究的基础上，将构建的pET32a(+)rhCGRPVY在大肠埃希菌中表达，分别对时间、温度和IPTG的浓度进行优化。采用NiNTA亲和层析柱分离纯化可溶性蛋白，YLN凝胶影像分析系统和Bradford法检测纯化蛋白的纯度与含量。结果 重组蛋白的最佳表达条件为诱导时间4 h、诱导温度37℃、IPTG浓度0.75 mmol/L，重组蛋白的最高表达量占菌体总蛋白的70%～80%，高效表达的CGRPVY融合蛋白大部分可溶，纯化后蛋白纯度可达90%以上，蛋白浓度约为0.09 mg/ml。结论 成功优化了蛋白表达条件，并用亲和层析法纯化出大量可溶性TrxArhCGRPVY。 【关键词】 降钙素基因相关肽衍生物;VY;优化;纯化 　　【Abstract】 Objective To optimize the expression level of engineering strain of calcitonin generelated peptide derivant(CGRPVY), then use affinity chromatograph to obtain recombinant protein of high purity. Methods Based on the groundwork of former research, an expression vector pET32a(+)rhCGRPVY which was constructed by this group was used to express TrxArhCGRPVY in E coli. Expression of TrxArhCGRPVY was optimized on the induction time, temperature, and the concentration of IPTG respectively. The soluble fusion protein was purified by NiNTA chromatography column and its purity and content were detected by YLN2000 gel image analysis and Bradford assay. Results The optimized expression condition of TrxArhCGRPVY was obtained which was 4 h of induction, 37℃ of incubation, 1.0 mmol/L IPTG of final concentration. Expression conditions was optimized, the maximum yield of fusion protein was about 70% ～ 80% of the total mass of bacterial proteins. The purity reached at least 90% and about 0.09 mg/ml protein was acquired. Conclusions To optimize the expression condition of TrxArhCGRPVY in E coli successfully and obtain soluble TrxArhCGRPVY by affinity chromatograph. 　　【Key words】 CGRPVY ;VY;Optimization;Purification 　　降钙素基因相关肽(calcitonin generelated peptide alpha，CGRP)是迄今所知体内较强的扩张血管的多肽成分，具有舒张血管、降低血压等生理功能，同时兼有治疗心衰和增强心输出量的作用。降血压肽(Angiotensinconverting enzyme inhibitory peptides，ACEIP)是一类能够降低人体血压的小分子多肽的总称，是血管紧张素转化酶(Angiotensinconverting enzyme，ACE)的抑制剂，可通过抑制人体血管紧张素Ⅱ的生成而达到降低血压的目的〔1〕。其突出优点是对高血压患者具有降血压作用，而对血压正常的人无降压作用，降压作用持久、无副作用〔2，3〕。二肽