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A kind of polypeptide for preparing rabbit polyclonal antibody and its application

A polyclonal antibody and receptor technology, applied in the field of biomedicine, can solve the problem of blank clinical detection kits

Active Publication Date: 2021-04-20
于向东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is still a gap in the antibody and protein for laboratory research against MRGPRX2, even more so than the clinical detection kit for MRGPRX2.

Method used

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  • A kind of polypeptide for preparing rabbit polyclonal antibody and its application
  • A kind of polypeptide for preparing rabbit polyclonal antibody and its application
  • A kind of polypeptide for preparing rabbit polyclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Synthesis of the Antigen Peptide of the Class 1 Anaphylaxis Specific Receptor MRGPRX2 Protein

[0025] 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 286-300th position as the target, amino acid The sequence is RKQWRLQQPILKLAL. 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 determ...

Embodiment 2

[0027] Example 2 Preparation of Anti-polypeptide Rabbit Polyclonal Antibody

[0028] Prepare the conjugated KLH-polypeptide into an emulsified injection for immunization, and inject subcutaneously in points. Spray alcohol on the back midline of New Zealand rabbits, avoiding areas with immune swellings. One injection is divided into four injections, respectively. 4 different points. After five times of immunization, blood was collected from the vein of the rabbit's ear to detect the titer of anti-rabbit serum by indirect Dot blot method. The titer of antiserum reached 1:200000, indicating that the immunity was qualified. Rabbits were collected by carotid artery bleeding, and the rabbit polyclonal antibodies were affinity purified.

Embodiment 3

[0029] Example 3 Identification of Antibody: Detection of Polyclonal Antibody Titer by Dot blot

[0030] 1) Membrane preparation: NC membrane is used for Dot blot detection, cut the NC membrane into 6.5cm wide strips, and draw a square grid with a length and width of 1cm×1cm on it (draw on the blue paper above the membrane, draw The film (white) and the blue paper must completely overlap when the grid is made, so that the small square grid on the blue paper can leave correct scratches on the film). Leave a distance of 0.25cm on each side of the membrane, and draw a corresponding number of small square grids according to one antibody corresponding to a 1cm×6cm strip (when drawing the grid, the force should not be too small, and each antibody should be left on the membrane. A small grid of scratches, but can not scratch the film).

[0031] 2) Antigen coating: uncover the blue protective paper on the membrane, use a 2.5 μL pipette to take 2 μL of the corresponding polypeptide di...

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PUM

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Abstract

The invention discloses a polypeptide for preparing rabbit polyclonal antibody and its application, belonging to the technical field of biomedicine. The polypeptide is an immunogenic polypeptide similar to the allergic response specific receptor MRGPRX2 protein, and can be used to prepare rabbit polyclonal antibodies. The enzyme-linked immunosorbent method (direct method) has been successfully prepared, wherein the polyclonal antibody is a detection antibody. The established method has verified its clinical application, which will help the research work on the specific receptor for anaphylactoid MRGPRX2, and is of great significance for clinically guiding the safety of drug use.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a polypeptide for preparing rabbit polyclonal antibody and its application. Background technique [0002] Drug-induced allergic reactions are mainly divided into type I allergic reactions and pseudoanaphylaxis. Anaphylactoid reactions do not require IgE mediation, and allergic symptoms appear upon first contact with the drug, which is obviously dose-dependent. It is generally believed that the mechanism of anaphylactoid reaction is that the sensitizer directly acts on mast cells and basophils to cause the release of active mediators, or induces mast cells or basophils to release biologically active mediators through complement and other pathways. , causing allergic symptoms. At present, the mechanism of anaphylactoid reaction is not fully understood. In 2015, McNeil et al. found for the first time that secretagogues, peptide drugs, tetrahydroisoquinoline drugs, and quinolone...

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 刘瑞马维娜贺浪冲丁园园张涛曹娇李超美
Owner 于向东
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