大鼠Shh-N融合蛋白的表达、纯化及功能研究

Cloning,Expression, Purification and Biological Activity Analysis of Rat Sonic Hedgehog N-terminal Genes

为了获得重组SonichedgehogN端蛋白 (Shh N)并研究其功能 ,应用PCR技术扩增Shh NcDNA ,然后克隆至原核表达质粒中 ,转化E .coli后获得表达菌株 ,经 1mmol/LIPTG诱导高效表达出带有His tag的融合蛋白 ,其中大部分为可溶性蛋白 ,少量为包涵体 .用His tag特异性结合树脂纯化可溶性融合蛋白 ,经SDS 聚丙烯酰胺凝胶电泳鉴定为单一区带 ,凝胶自动扫描分析表明 ,Shh N的纯度达 85%以上 .纯化后的Shh N在成纤维生长因子 8(FGF8)的协同作用下 ,能诱导神经前体细胞 (NPC)向酪氨酸羟化酶阳性神经元 (TH+ )发育 .在此基础上可以诱导不同类型的人类干细胞定向分化为多巴胺 (DA)神经元 ,从而为临床治疗帕金森病 (PD)

To obtain recombinant sonic hedgehog N-terminal protein (Shh-N) and study its biological activity, the cDNA encoding the functional rat Shh-N was isolated using PCR. The expression plasmid was constructed by inserting Shh-N cDNA into plasmid pET28a ( +) and transformed into E. coli. Then an expression strain was selected. SDS-PAGE and Western blotting analysis revealed that the rat Shh-N protein was highly expressed in the form of a large quantity of soluble protein and a small amount of inclusion body being induced by the IPTG. After Ni-NTA resin affinity chromatography, the purity of the final Shh-N protein was more than 85%. Purified protein being added to neural progenitor cells during developmental stages could induce the phenotype of tyrosine hydroxylase-immunopositive neurons. Based on this study, sufficient donor cells would be available to treat Parkinson's disease in clinical therapy via inducing different kinds of stem cells from human being into dopaminergic neurons.