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Cell Journal، جلد ۱۵، شماره Suppl ۱، صفحات ۶۹-۶۹
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عنوان انگلیسی Ps-113: Production of Recombinant hGDNF in HEK293t Cell Line
چکیده انگلیسی مقاله Objective: Human glial cell line-derived neurotrophic factor (hGDNF) is a disulfide linked homodimeric glycoprotein. This protein is able to protect the dopaminergic neurons and it can also induce regeneration of injured neurons in the central nervous system in vivo. So, it is a very promising protein for the treatment of Parkinson’s disease and other neurodegenerative disorders. GDNF has further critical roles outside the nervous system in the regulation of kidney morphogenesis and spermatogenesis. GDNF needs a multicomponent receptor system, composed of RET and GFRα1 receptors. GDNF family ligands also signal through the neural cell adhesion molecule NCAM. The present work describes a method to obtain high amount of purified hGDNF using a mammalian cell line. Materials and Methods: The coding sequence of the hGDNF which was fused with the signal sequence at Nterminal and histidine tag at the C-terminal was cloned in the expression vector pBudCE4.1/attB. Then HEK293T cell line was stably transfected with the construction with Lipofectamine. The secreted protein was purified by chromatography and protein expression was analyzed by SDS-PAGE and by Western Blot.The biological activity of the purified GDNF was assessed using the PC12 cell line. For this The expression of the two GDNF receptors, RET and GFR1α, on PC12 cells was confirmed by RT PCR. Then we assayed the ability of purified GDNF to induce neurite outgrowth from this cells. Results: In this study, the expression vector was constructed and it was transfected successfully into HEK293T cell line and the recombinant biologically active GDNF was obtained. Conclusion: In this study, we describes a method to obtain high amount of purified hGDNF using a mammalian cell line.
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نشانی اینترنتی http://celljournal.org/journal/article/abstract/917
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