摘要
为了获得人重组 persephin( PSP)并研究其生物学活性 ,从人胎脑组织中提取总 RNA,以RT- PCR方法获取编码人 PSP成熟蛋白 c DNA.将人 PSP c DNA插入含 T7启动子的质粒 p ET-2 8a( + ) ,构建表达质粒 p ET- PSP,转化大肠杆菌 BL 2 1 ( DE3)获得表达菌株 BLPSP,经 IPTG诱导表达的 PSP形成包含体 .凝胶自动扫描分析表明 ,PSP表达量约占菌体总蛋白 2 0 %以上 .用Ni2 + - NTA树脂一步法纯化目的蛋白 ,纯度达 85%以上 .纯化和复性的 PSP蛋白能显著促进脊髓神经元的存活 .
The objective is to get human recombinant persephin(PSP)and its biological activity.The cDNA encoding the mature human PSP was isolated using RT PCR with total RNA extracted from fetal human brain.The expression plasmid pET PSP was constructed by inserting PSP cDNA into plasmid pET 28a(+)containing T7 promoter and transformed into E.coli BL21(DE3).An expression strain BLPSP was selected.SDS PAGE analysis revealed that the human PSP protein was highly expressed and accumulated up to above 20% of the total bacterial proteins in the form of inclusion body after the induction.By Ni 2+ chelation affinity chromatography,up to 85% PSP protein was purified by one step.Purified and refolded PSP protein could significantly promote the survival of spinal cord neurons.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
2000年第5期626-630,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家重点基础研究项目!(973课题)
国家自然科学基金资助课题
