Method for screening autophagy degradation helper protein

A screening method and auxiliary protein technology, applied in the field of protein research, can solve the problems of unclear degradation of MHC-I by autophagy, tumor immune escape, weakening anti-tumor immunotherapy, etc.

Pending Publication Date: 2022-03-18
SHENZHEN INST OF ADVANCED TECH +1
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  • Application Information

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Problems solved by technology

Studies have clarified that autophagic degradation of MHC-I leads to tumor immune escape and weakens anti-tumor immunotherapy, but the process of MHC-I degradation by autophagy is still not very clear

Method used

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  • Method for screening autophagy degradation helper protein

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Experimental program
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Effect test

Embodiment 1

[0035] 1. Induction of Autophagy and Blocking of Autophagic Flux in Vivo

[0036]In tissues and organs such as liver and heart, induction of autophagy can be achieved by starvation. To block autophagic flux in vivo, mice were intraperitoneally injected with 40 mg / kg of interleukin 4 to 5 hours before tissue cell collection.

[0037] 2. Homogenization of GFP-LC3 mouse tissue cells

[0038] 1) Dissect 3-6 mg of histiocytes from euthanized mice into 1 mL of isolation buffer and homogenize the histiocytes in a Dounes homogenizer; transfer the homogenate to a 1.5 mL microcentrifuge tube , and refrigerate the sample; inject the lysate into the tissue cells through a 1mL syringe, if you choose a 25-gauge needle, you can inject 3-5 times, if you choose a smaller needle, you can inject 3-5 times, depending on Specific cell size; continue centrifugation at 1000 (xg) for 10 minutes at 4°C to eliminate nuclei.

[0039] 2) Transfer the denucleated tissue supernatant to a 1.5 mL microcen...

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Abstract

The pancreatic cancer cells overexpressing GFP-LC3 and MHC-I-His protein are constructed by adopting lentivirus, autophagosome is separated by a magnetic separation technology under the condition of inhibiting autophagy degradation, an MHC-I-His protein binding compound in the autophagosome is separated by a nickel column separation technology, and after trypsin hydrolysis, the pancreatic cancer cells are separated by using the MHC-I-His protein binding compound. And carrying out mass spectrometry on the purified hydrolysis peptide fragment by using Nano-LC-Q-TOF-MS, and finally determining the type of the separated protein by searching a database, thereby realizing the screening of the MHC-I autophagy degradation auxiliary protein. The auxiliary protein for MHC-I autophagy degradation can be accurately screened out, and a new target spot is provided for blocking MHC-I autophagy degradation and improving body anti-tumor immunity research.

Description

technical field [0001] The invention belongs to the technical field of protein research, and in particular relates to a method for screening autophagy degradation auxiliary proteins. Background technique [0002] Immune escape is a major obstacle in cancer immunotherapy. The common escape mechanisms mainly include: immune recognition disorder caused by tumor antigen blockage and down-regulation of expression; low expression or deletion of major histocompatibility complex class I (MHC- I) Impairing tumor self-antigen presentation; the tumor microenvironment inhibits the anti-cancer activity of infiltrating immune cells, etc., among which MHC-I mediates the presentation and recognition process of tumor antigens and plays an important role in the anti-tumor immune response. Tumor cells down-regulate the expression level of their own MHC-I through the loss of MHC-I gene heterozygosity, autophagic degradation of MHC-I protein, etc., thereby reducing the probability of antigen pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/62G01N33/68
CPCC12N15/86C12N5/0693C07K14/70539G01N33/6848C12N2740/15043C07K2319/21
Inventor 夏厚军唐秋菊
Owner SHENZHEN INST OF ADVANCED TECH
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