胶质细胞衍生的神经营养因子成熟肽基因的克隆、表达及纯化的研究  

Glia Cell line Derived Neurotrophic Factor Gene Cloning, Expression and Purification

作　　者：马雪梅[1] 黄秉仁[1] 蔡良琬[1] 

机构地区：[1]中国医学科学院中国协和医科大学基础医学研究所

出　　处：《中国生物化学与分子生物学报》1998年第5期506-511,共6页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：通过ＰＣＲ的方法克隆了胶质细胞衍生的神经营养因子（ｇｌｉａｃｅｌ－ｌｉｎｅｄｅｒｉｖｅｄｎｅｕ－ｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ，ＧＤＮＦ）成熟肽的基因，并将其连接到Ｅ．ｃｏｌｉ高效表达载体ｐＥＴ１６ｂ，在Ｅ．ｃｏｌｉ中获得高效表达．表达蛋白占菌体总蛋白２１％以上，以包涵体形式存在，经体外复性后用金属螯合亲和层析的方法得到具有较高纯度和活性ｒｈＧＤＮＦ．Neurotrophic factors are believed to play an essential role in the proliferation,differentiation and survival of neurons in the nervous system.Glia cell line derived neurotrophic factor (GDNF) is a potent survival factor for embryonic dopaminergic,spinal motor,cranial sensory,sympathetic,and hindbrain noradrenergic neurons,and may repay further evaluation for treating neuron loss in both ALS and Parkinsons disease.Overproduction of this polypeptide will greatly promote the process of basic research,and pave the way for clinical trial.In this work,the gene of GDNF mature peptides was cloned by PCR method,then transferred into the E.coli expression vector pET16b.Recombinant protein was expressed in E.coli host strain BL21(DE3) pLysS in the form of inclusion body,and the expression level was more than 21% of the total cell lysate. In vitro refolding and metal chelate chromatography were used to purify rhGDNF with high purity and activity.In brief,the gene of GDNF was successfully cloned and active rhGDNF was purified.

关 键 词：GDNF E.COLI 基因表达 亲和层析 纯化 

分 类 号：Q953[生物学—动物学]