A kind of polypeptide with immunogenicity to anaphylaxis-like specific receptor mrgprx2 protein and application thereof

An immunogenic and specific technology, applied in the field of biomedicine, can solve the problem of MRGPRX2 without a kit, etc.

Active Publication Date: 2021-05-28
于向东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are still gaps in the antibodies and proteins for laboratory research against MRGPRX2, and there is no relevant kit for the detection of MRGPRX2

Method used

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  • A kind of polypeptide with immunogenicity to anaphylaxis-like specific receptor mrgprx2 protein and application thereof
  • A kind of polypeptide with immunogenicity to anaphylaxis-like specific receptor mrgprx2 protein and application thereof
  • A kind of polypeptide with immunogenicity to anaphylaxis-like specific receptor mrgprx2 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Synthesis of the Antigen Peptide of the Class 1 Anaphylaxis Specific Receptor MRGPRX2 Protein

[0028] DNAstar analysis software was used to predict and analyze the epitope of human MRGPRX2 amino acid sequence, such as protein hydrophilicity, sequence flexibility, protein surface accessibility, and protein antigenic index, and finally determined the 250th-264th position as the target, amino acid The sequence is WKDSDVLFCHIHPVS. The manual solid-phase Fmoc method is used to synthesize from the C-terminal to the N-terminal direction to obtain the crude product of the target polypeptide. Purify the target polypeptide by using reversed-phase high-performance liquid chromatography (HPLC) method to separate different polypeptide molecules according to the difference in hydrophobicity. After freeze-drying the solvent, the pure polypeptide in a fluffy state was obtained. Its chemical structure was characterized by MALDI-TOF mass spectrometry, and its purity was deter...

Embodiment 2

[0030] Embodiment 2 Preparation of anti-polypeptide mouse monoclonal antibody

[0031] Prepare the conjugated KLH-polypeptide into an emulsified injection for immunization, and inject subcutaneously in points, spray alcohol on the back midline of the mouse, avoid the parts with immune swellings, and inject one injection into four times, respectively. 4 different points. After five immunizations, blood was collected from the tail vein of the mice to detect the titer of antiserum by indirect ELISA. The titer of ELISA antiserum reached 1:50000, indicating that the immunity was qualified. Select the mouse with the highest titer for fusion screening, and then carry out subcloning, and screen pure monoclonal cell lines according to the results of subcloning. The supernatant of the monoclonal cell line was injected into mice to prepare ascites, and the ascites was subjected to protein G affinity purification to obtain purified monoclonal antibodies.

Embodiment 3

[0032] Example 3 Antibody identification using indirect ELISA method to detect antibody titer

[0033] 1) Microtiter plate preparation: After assembling the microtiter plate, prepare for antigen coating.

[0034] 2) Antigen dilution and coating: Add the prepared antigen working solution into the concave sampling tank, take 100 μL of the antigen working solution with a 300 μL range 8-hole pipette and add it to the bottom of the microplate well. After adding the sample, cover it Aluminum foil paper, incubate in an oven at 37°C for 2h or overnight at 4°C.

[0035] 3) Plate washing: Take out the ELISA microplate plate coated with antigen, turn it upside down to remove the liquid in the well, and then place it on a clean absorbent towel to drain; add 200ml TBST to each well with a drain gun until it is full but not If it overflows, after adding the whole plate and let it stand for 3 minutes, shake out the TBST for 3 times, then place it on a clean absorbent towel to drain, and pat...

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Abstract

The invention discloses a polypeptide with immunogenicity for anaphylactoid specific receptor MRGPRX2 protein and application thereof, belonging to the technical field of biomedicine. The polypeptide consists of 15 amino acids, and its polypeptide sequence is: WKDSDVLFCHIHPVS. The epitope of the polypeptide is the 250-264th amino acid sequence of MRGPRX2 protein. It is found through research that it has strong immunogenicity. Therefore, it can be used for the preparation of mouse monoclonal antibody and rabbit polyclonal antibody, and successfully prepared a double-antibody sandwich ELISA method, in which the monoclonal antibody is the coating antibody and the polyclonal antibody is the detection antibody, which verifies that the established method can be used in clinical testing. This will help the research work on the specific receptor MRGPRX2 of the anaphylactoid response, and is of great significance for guiding the safety of drug use in clinical practice.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polypeptide with immunogenicity for anaphylactoid specific receptor MRGPRX2 protein and application thereof. Background technique [0002] Drug-induced allergic reactions are mainly divided into anaphylactoid reactions. The clinical symptoms of anaphylactoid reactions are similar to anaphylaxis. But the mechanism of its reaction is not the same as type I allergic reaction. When the drug enters the body, it can directly cause mast cells to release their contents, triggering an allergic reaction, and the clinical symptoms are severe. Hypersensitivity reactions have nothing to do with antigen-specific immune responses, and occur when they are first exposed to such drugs, and have a certain correlation with the dose and injection speed. As the main effector of allergies, mast cells can not only be attached and activated by specific IgE to cause degranulation, causing type I al...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/72C07K16/28G01N33/68
CPCC07K14/723C07K16/2869G01N33/6854G01N2333/726
Inventor 张涛丁园园刘瑞贺浪冲马维娜韩省力李超美
Owner 于向东
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