Dominant epitope peptide of anti-Mrgprx2 antibody and application thereof

A dominant antigen and epitope peptide technology, applied in the field of biomedicine, can solve the problem that there are no relevant clinical detection kits and drugs for MRGPRX2 diagnosis and treatment, and achieve strong immunogenicity

Active Publication Date: 2019-09-06
于向东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are still gaps in the antibodies and proteins for laboratory research against MRGP

Method used

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  • Dominant epitope peptide of anti-Mrgprx2 antibody and application thereof
  • Dominant epitope peptide of anti-Mrgprx2 antibody and application thereof
  • Dominant epitope peptide of anti-Mrgprx2 antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0027] Example 1 Synthesis of MRGPRX2 protein antigen peptide

[0028] Use DNAstar analysis software to predict and analyze the human MRGPRX2 amino acid sequence such as protein hydrophilicity, sequence flexibility, protein surface accessibility, protein antigenic index and other antigenic epitopes, and finally determine that the 166-184th position is the target, the amino acid The sequence is KFCGFLFSDGDSGWCQTFD (as shown in SEQ ID NO:1). The manual solid phase Fmoc method was used to synthesize from the C-terminal to the N-terminal direction to obtain the crude target peptide. Purify the target polypeptide by using reversed-phase high performance liquid chromatography (HPLC) method to separate different polypeptide molecules according to their hydrophobicity differences. After lyophilizing the solvent, the pure peptide is obtained in a fluffy state. Its chemical structure is characterized by MALDI-TOF mass spectrometry, and its purity is measured by an analytical high performa...

Example Embodiment

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

[0031] The conjugated KLH-polypeptide was used to prepare an emulsified injection for immunization. The subcutaneous injection was used to spray alcohol on the back and midline of the mouse to avoid the immune swelling part. One injection was injected into four times. 4 different points. After five immunizations, blood was collected from the tail vein of the mice for indirect ELISA to detect the titer of antiserum. The ELISA antiserum titer reaches 1:50,000, indicating that the immunization is qualified. The mouse with the highest titer was selected for fusion screening, and then subcloned, and pure monoclonal cell lines were screened according to the results of subcloning. The supernatant of the monoclonal cell line was injected into mice to prepare ascites, and the ascites were taken for protein G affinity purification to obtain purified monoclonal antibodies.

Example Embodiment

[0032] Example 3 Antibody identification: use indirect ELISA to detect monoclonal antibody titer

[0033] 1) Preparation of the ELISA plate: After the ELISA plate is assembled, prepare for antigen coating.

[0034] 2) Antigen dilution and coating: add the prepared antigen working solution to the concave sample slot, use a 300μL volume 8-well pipette to take 100μL of the antigen working solution and add it to the bottom of the plate well. After the sample is added, cover it Incubate in an oven at 37°C for 2h or overnight at 4°C on aluminum foil.

[0035] 3) Wash the plate: Take out the ELISA plate that has been coated with the antigen, turn it over 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 row gun until it is full but not If it overflows, after adding the whole plate and letting it stand for 3 minutes, shake the TBST out, shake it 3 times, and then place it on a clean absorbent towel to drain it, pat 6 ...

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Abstract

The invention discloses a dominant epitope polypeptide of an anti-MRgprx2 antibody and an application thereof, which belong to the technical field of bio-medicine. A bioinformatics method is used to predict the dominant epitope of MRGPRX2 and screens for a polypeptide sequence with the dominant epitope; the peptide sequence is synthesized by a manual solid-phase Fmoc method, and the purity of thepolypeptide is analyzed by analytical high-performance liquid chromatography; a mouse monoclonal antibody and a rabbit polyclonal antibody are prepared by monoclonal and polyclonal techniques, and theestablished method is validated for clinical detection. The double-anti-sandwich MRGPRX2 clinical test kit is successfully prepared, which is of great significance for clinically guiding the safety of medication.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a dominant antigenic epitope of an anti-Mrgprx2 antibody and an application thereof. Background technique [0002] Drug-induced allergic reactions are mainly divided into type I (immediate), type II (cytotoxic), type III (immune complex type), type IV (delayed) anaphylaxis and pseudoanaphylaxis. 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. Anaphylactic shock occurs in a very small number of individuals who receive therapeutic doses of drugs. There is no sensitization process, and the serum IgE concentration of the patients does not increase, but they show typical symptoms of immediate allergic reactions, so ...

Claims

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Application Information

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IPC IPC(8): C07K14/705C07K16/28G01N33/68
CPCC07K14/705C07K16/28G01N33/6854G01N2333/705
Inventor 贺浪冲韩省力张涛丁园园刘瑞王楠
Owner 于向东
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