Chimeric molecules mediating tau protein degradation based on autophagy mechanism and their applications
A chimeric molecule and protein degradation technology, applied in the fields of hybrid peptides, organic chemistry, fusion polypeptides, etc., can solve the problem that it is difficult to efficiently degrade Tau protein aggregates, etc., to overcome the inability to efficiently degrade intracellular macromolecular components. Effect
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[0037] 1. Construction of EGFP-Tau and EGFP-TauP301L fusion protein (target protein) expression vector
[0038] (1) PCR amplification of the full-length nucleic acid fragment of the Tau protein, extracting the DNA of the plasmid pET28a-Tau (containing the full sequence of the wild-type Tau protein) as an amplification template: The PCR reaction system is: 2×PCR Bestaq TM MasterMix with dye12.5μl, upstream primer Tau-F 1μl, downstream primer Tau-R 1μl, DNA template 1μl, add 9.5μl ddH 2 O to a final volume of 25 μl. Refer to Table 1 for primer sequences. The full-length nucleic acid sequence of the wild-type Tau protein is shown in SEQ ID NO:1.
[0039] Table 1. Tau protein full-length nucleic acid sequence PCR amplification primers
[0040]
[0041] The PCR reaction program was: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, and 32 cycles of reaction; extension at 72°C for 10 min, and the PCR pr...
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