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目的:纯化毕赤酵母表达的Neuritin蛋白,通过观察其对PC12细胞和鸡胚背根神经节(dorsal root ganglion,DRG)的作用,研究其神经生物学功能。方法:采用镍离子金属螯合亲和层析法纯化Neuritin蛋白,Bradford法对纯化的Neuritin蛋白进行定量,然后加入到PC12细胞和DRG的培养液中,通过观察PC12细胞突起的生长情况和细胞形态的变化,研究其促进细胞突起生长的功能;通过观察鸡胚DRG的神经纤维生长情况,研究其促进DRG神经纤维生长活性;通过观察鸡胚背根神经节的裂解时间,研究其阻止细胞退化和凋亡的活性。结果:纯化后的Neuritin蛋白经SDS-PAGE电泳显示,获得11 kDa左右的唯一蛋白条带,即Neuritin蛋白;Bradford定量法计算出其浓度为490μg/ml。纯化的Neuritin蛋白加入到PC12细胞培养液中,PC12细胞长出突起,发生类神经元样改变;鸡胚DRG培养液中加入Neuritin蛋白后,长出神经纤维,裂解时间也延长,并与浓度呈正相关。结论:毕赤酵母表达的Neuritin蛋白得到有效纯化,纯化的Neuritin蛋白能促进细胞突起的生长,阻止细胞的退化和凋亡,延长细胞的存活时间,这为进一步研究neuritin的功能及作用机制奠定了良好的实验基础。
OBJECTIVE: To purify Neuritin protein expressed in Pichia pastoris and study its neurobiological function by observing its effect on PC12 cells and chick embryo dorsal root ganglion (DRG). Methods: The Neuritin protein was purified by nickel ion chelate affinity chromatography. The purified Neuritin protein was quantified by Bradford method and then added to the culture solution of PC12 cells and DRG. The growth and cell morphology of PC12 cells were observed To study the function of promoting neurite outgrowth; to observe the growth of nerve fiber of DRG in chicken embryo, to study the promotion of DRG nerve fiber growth activity; to observe the lytic time of chick embryo’s dorsal root ganglion to study its function of preventing cell degeneration and Apoptosis activity. Results: The purified Neuritin protein showed SDS-PAGE electrophoresis. The unique protein band of about 11 kDa was obtained, namely Neuritin protein. The concentration of purified Neuritin protein was 490μg / ml by Bradford method. The purified Neuritin protein was added to the culture solution of PC12 cells, PC12 cells were protuberant, the occurrence of neuron-like changes; the addition of Neuritin protein in chicken embryo DRG culture medium, the growth of nerve fibers, lysis time also extended, and with a positive concentration Related. Conclusion: The Neuritin protein expressed by Pichia pastoris can be effectively purified. The purified Neuritin protein can promote the process of neurite outgrowth, prevent the cell from degeneration and apoptosis and prolong the cell survival time, which lays a foundation for further research on the function and mechanism of neuritin Good experimental foundation.