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A dominant antigenic epitope polypeptide of an anti-mrgprx2 antibody and its application

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

Active Publication Date: 2021-04-20
陕西添一生物科技有限公司
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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 MRGPRX2, and there are no relevant clinical detection kits and drugs for the diagnosis and treatment of MRGPRX2

Method used

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  • A dominant antigenic epitope polypeptide of an anti-mrgprx2 antibody and its application
  • A dominant antigenic epitope polypeptide of an anti-mrgprx2 antibody and its application
  • A dominant antigenic epitope polypeptide of an anti-mrgprx2 antibody and its application

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 epitopes of human MRGPRX2 amino acid sequence, such as protein hydrophilicity, sequence flexibility, protein surface accessibility, and protein antigenic index, and finally determined the 166th-184th position as the target, amino acid The sequence is KFCGFLFSDGDSGWCQTFD (as shown in SEQ ID NO: 1). 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 spectr...

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 Identification of Antibody: Using Indirect ELISA to Detect Monoclonal 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, ...

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PUM

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Abstract

The invention discloses a dominant antigenic epitope polypeptide of an anti-Mrgprx2 antibody and an application thereof, belonging to the technical field of biomedicine. The dominant epitope of MRGPRX2 was predicted by bioinformatics method, and the peptide sequence with the dominant antigenic epitope was screened; the peptide sequence was synthesized by manual solid-phase Fmoc method, and the purity of the peptide was analyzed by analytical high performance liquid chromatography; finally Mouse monoclonal antibody and rabbit polyclonal antibody were prepared by monoclonal technology and polyclonal technology, which verified that the established method could be used in clinical testing. The double-antibody sandwich MRGPRX2 clinical detection kit was successfully prepared, which is of great significance for the safety of clinically guiding 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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Patent Type & Authority Patents(China)
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 陕西添一生物科技有限公司
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