摘要
目的:在大肠杆菌中重组表达斑马鱼p8蛋白并纯化。方法:PCR扩增斑马鱼p8蛋白基因编码区,连接到带有6×His标签的原核表达载体pET-28a中,构建重组表达质粒pET-28a-p8并转化大肠杆菌BL21(DE3),用IPTG诱导表达;优化表达条件后用Ni2+柱纯化重组蛋白。结果:构建了pET-28a-p8重组质粒;目的蛋白在大肠杆菌中获得表达,亲和纯化后,SDS-PAGE显示相对分子质量为预期的12.8×103。结论:获得了斑马鱼p8融合蛋白,为其生物学功能研究奠定了基础。
Objective: To express and purify the recombinant p8 protein of zebrafish in E.coli BL21(DE3). Meth- ods: The coding sequence of p8 protein of zebrafish was amplified by PCR and inserted into the prokaryotic expression vector pET-28a with 6×His tag to construct the recombinant plasmid pET-28a-pS. The recombinant plas- mid was transformed into E.coli BL21(DE3), and the expression of fusion protein was induced by IPTG. Ni2+ met- al chelating column was utilized for the purification of the fusion protein after the expression conditions were opti- mized. Results: Recombinant plasmid pET-28a-p8 was constructed and the recombinant protein was expressed in E.coli successfully. After being purified by affinity chromatography, SDS-PAGE showed a clear protein band with a relative molecular weight of 12.8 kDa expectedly. Conclusion: The fusion p8 protein of zebrafish was successful- ly expressed and purified, which lays the foundation of further study on its biological function.
出处
《生物技术通讯》
CAS
2012年第6期804-808,共5页
Letters in Biotechnology
基金
国家863计划(2008AA092603)
教育部新世纪人才支持计划(NCET-08-0501)
关键词
斑马鱼
p8蛋白
原核表达
纯化
zebrafish
p8 protein
prokaryotic expression
purification