摘要
旨在构建Neuritin的毕赤酵母表达系统获得大量有活性的Neuritin蛋白,研究其神经生物学功能。PCR扩增编码neuritin基因cDNA序列,并纯化回收,通过酶切和连接插入穿梭载体pPIC9K中,转化大肠杆菌DH5α,neuritin-pPIC9k重组质粒经PCR与测序鉴定后,使用SalⅠ酶切使其线性化,电击转化酵母菌GS115,经筛选与鉴定,成功构建了Neuritin的毕赤酵母表达系统。结果表明,使用甲醇诱导其表达,SDS-PAGE和Western blot分析得到11 kD的Neuritin蛋白,纯化的Neuritin蛋白加入PC12细胞培养液中,能促进其突起的生长。
It was to intend to construct Pichia pastoris expression system of neuritin, to obtain large amount of active Neuritin protein and study its neurobiological function. Encoding cDNA sequence of neuritin was amplified by PCR, then the fragment was digested and ligated into shuttle vector pPIC9K. The neuritin-pPIC9K recombinant was transformed into E. coli DH5α, and was confirmed by PCR and sequencing. The recombinant was linearized by restriction enzyme Sal Ⅰ , and transformed into yeast GS115 by electric shock. After screening and identification, the Pichia pastoris expression system of neuritin was successfully constructed. Using methanol to induce the expression of neuritin, through SDSPAGE and Western blot analysis, we obtained Neuritin protein about 11 kD. The purified Neuritin protein was added into PC12 cell culture medium and it can promote the neurite growth of PC12 cell.
出处
《生物技术通报》
CAS
CSCD
北大核心
2013年第10期148-152,共5页
Biotechnology Bulletin
基金
内蒙古自治区高校蓖麻产业工程研究中心资助项目(BMYJ2010007)
