Novel brain targeting preparation for preventing and treating neurodegenerative disease
A new type of preparation technology, applied in the field of new brain-targeted preparations for the prevention and treatment of neurodegenerative diseases, can solve the problems of lack of research on differences and immature mechanism of action
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Embodiment 1
[0038] Construction, expression and purification of TAT-aFGF fusion protein
[0039] The DNA fragment encoding TAT-aFGF (SEQ ID No: 8) was cloned into the prokaryotic expression vector pET-3c (the vector was purchased from invitrogen), and transformed into Escherichia coli BL21 (DE3) (the cells were purchased from invitrogen), and the ampicillin resistance was screened Recombinant. The expression was induced with a final concentration of 1 mM IPTG, and then combined with cation exchange (CM-Sepharose FF) chromatography, heparin affinity chromatography (Heparin-Sepharose CL-6B) and gel exclusion chromatography (Sephadex G-25). The fusion protein was isolated and purified using the method. MTT method was used to detect the proliferative activity of the fusion protein on Balb / c 3T3 cells.
Embodiment 2
[0041] Construction, expression and purification of TAT-bFGF / OX26 fusion protein
[0042] Conventional gene cloning and PCR overlapping techniques were used to construct the Pichia pastoris pAO815α-TAT-bFGF / OX26 transfer plasmid containing the αfactor signal peptide gene to express the fusion protein TAT-bFGF / OX26 (the sequence of TAT-bFGF / OX26 is shown in SEQ ID No: 10, plasmid pAO815α was purchased from Invitrogen Company), methanol was used as the only carbon source for yeast growth, and the fusion protein was directly secreted into the yeast in vitro culture medium. The fusion protein was isolated and purified by cation exchange (CM-Sepharose FF) chromatography, heparin affinity chromatography (Heparin-Sepharose CL-6B) and gel exclusion chromatography (Sephadex G-25).
Embodiment 3
[0044] Construction, expression and purification of bio-TAT-aFGF / OX26-SA fusion protein
[0045] Bio (biotin)-TAT-aFGF was expressed in fusion, and bio-TAT-aFGF / OX26-SA was formed by covalently combining it with OX26-SA (purchased from the British abcam company) linked with antibiotics. After purification, it is prepared into a freeze-dried preparation, which is dissolved with physiological saline before use, and administered intravenously or nasally.
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