摘要
目的构建蛇毒精氨酸酯酶Agkihpin的原核表达载体,并诱导其表达重组Agkihpin,为将来量产Agkihpin提供方法和依据。方法 RT-PCR法扩增Agkihpin基因,构建重组表达质粒p ET30a(+)-Agkihpin和p EASY-E1-His6-Agkihpin,并转化到BL21(DE3)感受态细胞中进行诱导表达,SDS-PAGE电泳观察重组蛋白Agkihpin的表达情况,Western blot检测其特异性。结果成功构建Agkihpin的原核表达载体p EASY-E1-His6-Agkihpin和p ET30a(+)-His6-Agkihpin,并利用p EASY-E1-His6-Agkihpin首次在大肠杆菌BL21(DE3)中表达出分子量约为30k D的His6-Agkihpin重组融合蛋白。结论首次实现了His6-Agkihpin重组融合蛋白在原核系统中的高效表达,为Agkihpin将来量产提供了方法和实验依据。
Objective For providing the methods and evidence for mass product of Agkihpin,a snake venom arginine esterase,to construct prokaryotic expression vectors of Agkihpin, and to induce these vectors to express recombinant Agkihpin by prokaryotic expression system.Methods Agkihpin gene from venom arginine esterase were used as a template for RT-PCR,and the RT-PCR products were recombined into p ET30a( +)-His6-Agkihpin and p EASY-E1-His6- Agkihpin; then these two recombinant plasmids were transformed and induced in BL21( DE3) competent cells,and the recombinant fusion protein His6-Agkihpin would be detected by SDS-PAGE and Western blot. Results Agkihpin gene were successfully constructed in prokaryotic expression vectors,and the recombinant plasmid p EASY-E1-His6-Agkihpin was transformed and expressed in E. coli BL21( DE3) for the first time. A recombinant fusion protein His6-Agkihpin was detected with the molecular weight of 30 k D by SDS-PAGE.Conclusion Recombinant fusion protein His6-Agkihpin highly expresss in prokaryotic systems,which provides experimental evidence and methods for drug preparation in the future.
出处
《中国生化药物杂志》
CAS
北大核心
2014年第9期33-37,共5页
Chinese Journal of Biochemical Pharmaceutics
基金
广西自然科学基金资助项目(2014GXNSFAA118172)
广西高校科研项目(2013YB047)
