Polypeptide for targeted inhibition of MLKL acetylation and/or derivative thereof and application thereof
A technology of acetylation and derivatives, which is applied in the field of peptides and/or derivatives that target the inhibition of MLKL acetylation, to achieve significant curative effect, less toxic and side effects, and promote lung regeneration
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Embodiment 1
[0033] Example 1 Preparation of an animal model of pulmonary fibrosis
[0034] 1.1 Main reagents and experimental animals
[0035] The bleomycin used in the experiment was purchased from Nippon Kayaku, lot number X81040.
[0036]The compounds used in the experiments were purchased from Sigma unless otherwise specified.
[0037] The SPF grade C57BL / 6 mice (male, 6-8 weeks old, 16-18 g) used in the experiment were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd.
[0038] 1.2 Preparation of animal model of pulmonary fibrosis
[0039] Male C57BL / 6 (age 6-8 weeks) mice were fasted overnight, anesthetized with sodium pentobarbital (45mg / kg, i.p.), and injected with bleomycin (1U / kg) intratracheally for a total of 6 days. times, each interval is 14 days.
[0040] The specific plan is: fix the mouse in the prone position after anesthesia, irradiate the neck of the mouse with a cold light source, pull the mouse tongue out with the tweezers in the right hand, and open...
Embodiment 2
[0041] Example 2 Real-time fluorescence quantitative PCR method detects the expression of KAT5 in the lung tissue of mice with pulmonary fibrosis
[0042] 1. Isolate the lung tissue of mice in the normal group and the model group, and extract RNA. Total tissue RNA was extracted by Trizol method, the steps are as follows:
[0043] (1) Weigh the tissue, cut it into small pieces, and place it in a 1.5mL EP tube.
[0044] (2) Add 1 mL of Trizol solution, mix well, and use a tissue homogenizer to fully grind the tissue and lyse the lysed cells.
[0045] (3) Add 200 μL of chloroform to the lysate, vortex thoroughly for 30 seconds, and then let stand at room temperature for 15 minutes.
[0046] (4) Centrifuge at 12,000×g at 4°C for 15 minutes, and pipette 400 μL of the upper colorless aqueous phase into a new EP tube.
[0047] (5) Add 400 μL of isopropanol, turn it upside down, and let stand at room temperature for 10 minutes.
[0048] (6) Centrifuge at 12,000×g at 4°C for 10 min...
Embodiment 3
[0069] Example 3 Verification of the binding of KAT5 and protein MLKL in alveolar macrophages by co-immunoprecipitation
[0070] 3.1 Co-immunoprecipitation reagents
[0071] Lysis solution A: 0.6057g Tris base, 1.7532g NaCl, 0.1017g MgCl 2 ·6H 2 O, 0.0742g EDTA, 10mL glycerin, 10mL 10% NP40, add deionized water to 150mL, adjust the pH value to 7.6 with HCl, adjust the volume to 191mL, mix well, filter through a 0.45μm membrane filter, and store at 4°C.
[0072] Lysis Solution B: 200μL 2M β-glycerophosphate, 4mL 2.5M NaF, 2mL 100mM NaVO 3 , 2mL 100mMPMSF, 20μL 1M DTT, 200μL each of 1mg / mL Leu, Pep, and Apr, the total volume is 9mL. The mother liquor was stored at -20°C. Before use, thaw the mother liquor of each component in liquid B, add it to liquid A according to the above composition ratio and mix well.
[0073] Protein A / G Plus-Agarose was purchased from Santa Cruz, USA.
[0074] 3.2 Specific operation steps
[0075] (1) Collect alveolar macrophages in mouse alveola...
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