用硫氧还蛋白融合表达系统表达小鼠内皮抑素的方法研究  

To express mouse endostatin by thioredoxin fusion expression system

作　　者：马卫列[1] 刘芳[1] 何承伟[1] 

机构地区：[1]广东医学院生物化学教研室,湛江524023

出　　处：《第三军医大学学报》2006年第14期1476-1478,共3页Journal of Third Military Medical University

基　　金：广东省卫生厅青年科研基金资助项目(B2000118);湛江市科技计划项目(2000072)~~

摘　　要：目的探索用硫氧还蛋白融合表达系统表达小鼠内皮抑素(endostatin)重组蛋白的最佳生产工艺路线。方法用pTh ioH is-endo重组质粒转化不同的大肠杆菌BL21、JM109、DH5α,经几种不同浓度的异丙基硫代半乳糖苷(isopro-pylth io-galactoside,IPTG)诱导表达重组融合蛋白,或诱导表达不同时间后,采用SDS-PAGE分析表达产物;按所优化的条件进行重组内皮抑素的表达、包涵体的提取、洗涤、变性和复性,最后经N i+柱纯化,再通过SDS-PAGE分析纯化的结果。结果3种大肠杆菌表达效果相差无几,最佳IPTG的浓度0.9 mmol/L,最佳诱导时间为5 h,经N i+柱纯化后可获得可溶性的重组内皮抑素。结论利用本实验的优化条件可获得高产量、高纯度、可溶性的小鼠内皮抑素重组蛋白。Objective To explore the best technology to produce recombinant mouse endostatin by thioredoxin fusion expression system. Methods The recombinant plasmid pThioHis-endo was further transformed into different E. colt. , including BL21, JM109, DH5α. After induction with IPTG of different concentration or for different time period, thioredoxin-endo fusion protein was expressed in E. colt. and the product was identified by SDS-PAGE. The recombinant endostatin expression and extraction, wash, degeneration and refolding of inclusion bodies were carried out by the optimized route, and the product was further purified by affinity chromatography through Ni^＋ column and identified by SDS-PAGE. Results No difference of endostatin expression in BL21, JM109, DH5α was found. The optimal concentration of IPTG was 0.9 mmol/L and optimal inductive phase was 5 h. The soluble recombinant endostatin could be obtained by affinity chromatography through Ni^＋ column. Conclusion Soluble endostatin recombinant fusion protein of high purity and yield could be obtained by the optimized technique route.

关 键 词：ENDOSTATIN 硫氧还蛋白融合表达系统 融合蛋白 

分 类 号：Q503[生物学—生物化学] Q51