Pharmaceutical composition containing ubiquitin-like modified protein and application thereof
A ubiquitin-like and protein-like technology, applied in the field of biomedicine, can solve problems such as unclear mechanisms, and achieve the effects of promoting the growth of neuron axons, increasing the level of microtubule cutting, and promoting repair
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0053] Example 1 Ubiquitin-like modification of Katanin p60
[0054] (1) Construct GST-SUMO1, GST-SUMO2, and GST-SUMO3 plasmids, and transform the constructed plasmids into competent BL21 cells;
[0055] (2) After overnight culture, select monoclonal positive colonies and verify that they are correct, then take 3 mL of the bacterial liquid and culture it in 250 mL of LB medium containing 1% ampicillin resistance, in a shaker at 37°C, 180 rpm, until the OD of the bacterial liquid is 0.8;
[0056] (3) Add 0.1mM IPTG to induce at 30°C for 6-8h;
[0057] (4) Collect the bacterial liquid at 4°C, 12000 rpm, add an appropriate amount of lysate, bathe in ice for 1 hour, oscillate several times every 15 minutes, use an ultrasonic breaker with 20% intensity, 5s×5s, and sonicate 6 times;
[0058] (5) Centrifuge at 12,000 rpm for 15 minutes at 4°C to take the turbid supernatant from the upper layer;
[0059] (6) Add 200 μL beads to the centrifuge tube, add 1 mL of lysate, remove the sup...
Embodiment 2
[0072] Example 2 Co-localization of Katanin p60 and SUMO2 protein
[0073] (1) Culture hippocampal neuron cells, fix with 4% PFA at 4°C for 40 min, and wash with pre-cooled PBS;
[0074] (2) Drill holes with TBST (containing 1% Triton X-100) at room temperature (add 500 μL of cells to each well), 5 min / time, repeat twice;
[0075] (3) Prepare 3% blocking serum (0.3g BSA+10mL TBST), add 200μL blocking serum to each well of cells, and block for 1h at room temperature;
[0076] (4) Transfer the Coverlips to the antibody incubation box, with the cell-containing slide facing up, wash with TBST, add the antibody to the blocking serum for dilution, add 60 μL of the mixed dilution of Katanin p60 antibody and SUMO2 antibody to each Coverlips, and incubate at 4°C overnight;
[0077] (5) After recovering the mixed diluent of Katanin p60 antibody and SUMO2 antibody incubated in step (4), wash with TBST, add secondary antibody and blocking serum, add 60 μL of antibody diluent to each Cov...
Embodiment 3
[0081] Example 3 Effect of Katanin p60 mutant on microtubule cutting function
[0082] (1) Construct GFP-Katanin p60 plasmid (NCBI Reference Sequence: NP_001004217.2);
[0083] (2) Through bioinformatics software analysis, three potential SUMOylation sites in Katanin p60 were determined, namely K77, K157 and K330 (see Figure 4 );
[0084] (3) For the three sites K77, K157 and K330, perform site mutations on the GFP-Katanin p60 plasmid constructed in step (1) to obtain GFP-Katanin p60-K77R, GFP-Katanin p60-K157R, and GFP-Katanin p60-K330R and PAN with three simultaneous mutations;
[0085] (4) After COS7 cells were treated with 10mM Tubacin, COS7 cells were transfected with GFF, the GFP-Katanin p60 plasmid constructed in step (1) and the mutant plasmid obtained in step (3);
[0086](5) Observe the changes of Tubulin by immunofluorescence chemical staining.
[0087] The inventors of the present invention found that Katanin p60 has the ability to cut microtubules, especially...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More