重组蛋白PACAP-PTD的制备及其活性鉴定  

Preparation and Activity Identification of Recombinant PACAP-PTD

作　　者：黄霖[1] 余榕捷[1] 李娟 刘晓飞[1] 王静静 

机构地区：[1]暨南大学生物工程研究所,广东广州510632 [2]鲁南制药集团新时代药业有限公司,山东费县273400

出　　处：《安徽农业科学》2012年第13期7645-7649,共5页Journal of Anhui Agricultural Sciences

基　　金：东莞市科技计划项目(2008108101036)

摘　　要：[目的]制备重组蛋白PACAP-PTD,并对其活性进行鉴定。[方法]设计编码融合蛋白PACAP-PTD基因,克隆到表达载体pKYB,构建重组表达载体pKYB-PACAP-PTD,转化大肠杆菌ER2566中。采用IPTG诱导由PACAP-PTD、内含肽和几丁质组成的融合蛋白表达。利用IMPACT(Intein Mediated Purification with an Affinity Chitin-binding Tag)介导的纯化系统制备目的融合蛋白PACAP-PTD,并对其活性进行鉴定。[结果]试验所得的目的蛋白经测定分子量,结果与理论值相符,且试验中PACAP-PTD能有效的穿越血脑屏障。[结论]融合蛋白PACAP-PTD的构建和制备为其生物学功能的深入研究奠定了基础,同时可用于改善其用药途径,扩大其应用范围。[ Objective ] This study aimed to prepare recombinant protein PACAP-PTD and identify its activity. [ Method ] The gene that en- codes fusion protein PACAP-PTD was cloned into the expression vector pKYB to construct recombinant expression vector pKYB-PACAP-PTD, which was then transformed into E. coli ER2566. The fusion protein consisting of PACAP-PTD, intein and chitin was induced with IPTG to ex- press. Finally, the target fusion protein PACAP-PTD was purified with purification system mediated by IMPACT （Intein Mediated Purification with an AITmity of chitin-binding Tag）, and its activity was identified. [ Result] The molecular weight of target protein determined with laser time-of-flight mass spectrometry was consistent with the theoretical value. The recombinant protein PACAP-PTD could effectively cross the blood-brain barrier. [ Conclusion] The construction and preparation of the fusion protein PACAP-PTD not only lays foundation for further study on its biological function, but also improves the route of PACAP administration, and thus expands its scope of application.

关 键 词：PACAP-PTD 队cAP 内含肽 FITC 血脑屏障 原核表达 

分 类 号：S188[农业科学—农业基础科学]