β-synuclein蛋白原核表达载体的构建与表达

Construction of Prokaryotic Expression Vector of β-Synuclein and Expression in E. coli BL21

目的构建人β-synuclein基因的原核表达载体,分析其在大肠杆菌中的表达。方法从人脑组织中提取总RNA,用RT-PCR方法获得人β-synuclein基因,克隆至pCUm-T载体中,PCR筛选阳性克隆并测序。将酶切后的目的片段克隆至原核表达载体pGEX-6P-1中,转化大肠杆菌BL21。IPTG诱导后,经SDS-PAGE电泳分析目的蛋白的表达。结果 RT-PCR扩增出人β-synuclein基因,将其亚克隆至pGEX-6P-1构建成重组表达质粒,并在BL21中表达了β-synuclein蛋白。结论成功构建人β-synuclein的原核表达载体,并在大肠杆菌中表达了人β-synuclein融合蛋白,为进一步研究β-synuclein在帕金森病中的作用奠定了良好的基础。

Objective To construct a prokaryotic expression vector of β-synuclein and analyze its expression in E.coli BL21.Methods Total RNA was isolated from human fetal brain tissue.The full-length human β-synuclein cDNA was obtained by RT-PCR and then inserted into pCUm-T vector for sequencing.The target gene fragment was subcloned into pGEX-6P-1 vector correctly and then transformed into E.coli BL21.The expression of β-synuclein was induced with IPTG,and analyzed by SDS-PAGE.Results β-synuclein was amplified by RT-PCR and subcloned into pGEX-6P-1 properly.The recombinant fusion protein was expressed in E.coli BL21.Conclusion The prokaryotic expression vector of β-synuclein has been successfully constructed and expressed in E.coli BL21,providing a foundation for the further study of β-synuclein in Parkinson＇s disease.