新型内含肽介导PHB系统表达纯化PoIFN-α的研究  

Research on Expression and Purification of Porcine Interferon Alpha in Escherichia coli with Novel Intein-mediated PHB System

作　　者：宋振威[1,2] 周学章[1] 张仁参[1] 贾芳[1] 王玉炯[1] 

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室,宁夏银川750021 [2]中山大学生命科学学院,广东广州510275

出　　处：《宁夏大学学报（自然科学版）》2010年第3期264-268,共5页Journal of Ningxia University(Natural Science Edition)

基　　金：国家重点基础研究发展计划(973)项目(2006CB504401);宁夏科技攻关项目(2008)

摘　　要：为简化猪α干扰素蛋白（PoIFNα）的纯化步骤,以蛋白质自切割元件内含肽（Intein）替代蛋白酶,并以细菌自身产生的聚β-羟基丁酸酯（PHB）颗粒替代亲和配基,构建了新型内含肽介导PHB纯化PoIFNα的体系.结果显示,构建的基因工程大肠杆菌系统可成功实现PoIFNα的表达和纯化;φ（乳酸钠）=2.7%时,比较适宜工程菌的PHB累积;当诱导自切割反应温度为20℃,pH=6.5,反应时间为24~36 h时,可以实现内含肽C端高效自切割,纯化出的PoIFNα产量为20~30 mg/L.实验为重组猪α干扰素的工业化规模生产奠定了基础.To purify porcine interferon alpha（PoIFNα） in a very simple way,a recombinant gene engineer system was constructed.In this gene engineer E.coli,the self-splicing of intein can take place protease and the endogenous poly-β-hydroxy butyrate（PHB） granules which produced in the same cells can be as an affinity matrix to replace the traditional immobilized ligand.The results showed that PoIFNα can be expressed and purified by this constructed system,and the best concentration of sodium lactate for PHB granules production was 2.7%;when the temperature of solution was 20 ℃,pH was 6.5,and the self-splicing process lasted for 24-36 h,the intein can do well in releasing the target protein PoIFNα from its C-terminus for good yield at 20-30 mg/L.This work might be feasible for industrial-scale production of recombination protein,especially for the gene engineering drug porcine interferon alpha.

关 键 词：内含肽 聚Β-羟基丁酸酯 猪Α干扰素 表达纯化 

分 类 号：Q789[生物学—分子生物学]