[Objective] This study aimed to prepare recombinant protein PACAP-PTD and measure its activity. [Method] The gene that encodes fusion protein PACAP-PTD was cloned into the expression vector pKYB to construct recombina...[Objective] This study aimed to prepare recombinant protein PACAP-PTD and measure 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 expressed under the induction of IPTG. Finally, the target fusion protein PACAP-PTD was purified with IMPACT system （ Intein Mediated Purification with an Affinity of chitin-binding Tag）, and its activities to cross blood-brain barrier and to promote cell proliferation were measured. [ Result~ The molecular weight of the fusion protein PACAP-PTD determined with laser time-of-flight mass spectrometry was con- sistent with the theoretical value. In addition, the protein could effectively cross the blood-brain barrier and promote cell proliferation as well. [ 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.展开更多
[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...[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。展开更多
基金Supported by Science and Technology Program of Dongguan City ( 2008108101036)
文摘[Objective] This study aimed to prepare recombinant protein PACAP-PTD and measure 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 expressed under the induction of IPTG. Finally, the target fusion protein PACAP-PTD was purified with IMPACT system （ Intein Mediated Purification with an Affinity of chitin-binding Tag）, and its activities to cross blood-brain barrier and to promote cell proliferation were measured. [ Result~ The molecular weight of the fusion protein PACAP-PTD determined with laser time-of-flight mass spectrometry was con- sistent with the theoretical value. In addition, the protein could effectively cross the blood-brain barrier and promote cell proliferation as well. [ 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.
文摘[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。
