Neuritin毕赤酵母表达系统的构建及其对PC12细胞的影响

生物技术通报 ›› 2013, Vol. 0 ›› Issue (10): 148-152.

Neuritin毕赤酵母表达系统的构建及其对PC12细胞的影响

张树军^1,2,, 赵臣³, 狄建军^1,2,, 穆莎茉莉^1,2^,, 仙玲玲⁴, 于娜⁴, 黄瑾⁴

（1. 内蒙古民族大学生命科学学院，通辽 028000 ；2. 内蒙古自治区高校蓖麻产业工程研究中心，通辽 028000；3. 包头北方医院普外科，包头 014000 ；4. 石河子大学医学院新疆地方与民族高发病重点实验室，石河子 832002）

收稿日期:2013-03-27 修回日期:2013-10-15 出版日期:2013-10-14 发布日期:2013-10-15
作者简介:张树军，男，讲师，博士研究生，研究方向：生物化学与分子生物学；E-mail ：zh-shujun123456@163.com
基金资助:
内蒙古自治区高校蓖麻产业工程研究中心资助项目（BMYJ2010007）

Constuction the Pichia pastoris Expression System of Neuritin and Its Impacts on PC12 Cell

Zhang Shujun^1,2,, Zhao Chen³, Di Jianjun^1,2,, Mushamoli^1,2,, Xian Lingling⁴, Yu Na⁴, Huang Jin⁴,

（1. College of Life Science，Inner Mongolia Uniity for Nationalities，Tongliao 028000 ；2. Inner Mongolia Industril Engineering Research Center of Universities for Castor，Tongliao 028vers000 ；3. Department of General Surgery，Baotou Northern Hospital，aBaotou 014000 ； 4. Key Laboratory of Xinjiang Endemic and Ethnic Disease University，Shihezi 832002）

Received:2013-03-27 Revised:2013-10-15 Published:2013-10-14 Online:2013-10-15

摘要/Abstract

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Sal Ⅰ 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。

关键词: Neuritin pPIC9k, 毕赤酵母表达系统, PC12 细胞

Abstract:

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.

Key words: Neuritin pPIC9K, Pichia pastoris expression system, PC12 cell

张树军,赵臣,狄建军,穆莎茉莉,仙玲玲,于娜,黄瑾. Neuritin毕赤酵母表达系统的构建及其对PC12细胞的影响[J]. 生物技术通报, 2013, 0(10): 148-152.

Zhang Shujun, Zhao Chen, Di Jianjun, Mushamoli, Xian Lingling, Yu Na, Huang Jin. Constuction the Pichia pastoris Expression System of Neuritin and Its Impacts on PC12 Cell[J]. Biotechnology Bulletin, 2013, 0(10): 148-152.