Application of nicotinamide phosphoribosyltransferase in preparation of neuroprotective drugs
A technology of phosphoribosyl and nicotinamide is used in the preparation of neuroprotective and rehabilitation medicines, in the field of preparation of medicines for treating central nervous system diseases, and can solve the problems of insertional mutation and only effect, and achieve the effect of enhancing angiogenesis.
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[0044] 1. Experimental materials and methods
[0045] 1.1 Synthesis of NAMPT recombinant protein
[0046] Since glycosylation is the necessary way for NAMPT protein secretion, the present invention uses eukaryotic expression plasmids to express NAMPT protein, and uses dihydrofolate dehydrogenase-methotrexate system to selectively amplify in CHO cell lines . In order to increase the concentration of NAMPT protein in the culture medium, in this embodiment, the secretion signal peptide of the Ig Kappa chain is connected to the N-terminus of NAMPT, and the His6 tag is connected to the C-terminus of NAMPT for purification and separation. The synthetic cDNA is inserted into the cDNA containing Dihydrofolate dehydrogenase expression plasmid system (plgK-NAMPT-His6), and finally this vector was transfected into Chinese hamster cell line DG44 (DHFR- / -) without dihydrofolate dehydrogenase, using α-MEM medium +10% nucleoside-free fetal bovine serum for culture, adding methotrexate (5Nm...
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