重组蛋白PACAP-PTD的制备及其活性鉴定

[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。

Preparation and Activity Identification of Recombinant PACAP-PTD

[Objective] This study aimed to prepare recombinant protein PACAP-PTD and identify its activity.[Method] The gene that encodes 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 express.Finally,the target fusion protein PACAP-PTD was purified with purification system mediated by IMPACT(Intein Mediated Purification with an Affinity 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.