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Conjugate of mutant protease 3 and biotin, and preparation method and application of conjugate

A biotin derivative, protease technology, applied in the field of biological detection, can solve the problems of affecting the binding ability of antigen and antibody, unable to achieve site-specific labeling, masking protein epitopes, etc., to improve antibody detection performance, sensitivity and accuracy. , the effect of increasing the distance

Active Publication Date: 2022-01-25
苏州携创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This patent selects proteins with other chemically active functional groups such as carboxyl, amino, and sulfhydryl groups on the surface, and couples these functional groups with the first click chemical reagent that provides an azide reactive group or azide group. The surface of the protein will be coupled with more azide reactive groups or azide groups, so it is impossible to achieve fixed-point labeling, and it may also cover the antigenic epitope of the protein, affecting the binding ability of antigens and antibodies. group or azide reactive group, so when coupled with magnetic beads, a protein molecule may bind to multiple streptavidin magnetic beads, which will also result in the masking of antigenic epitopes and increase the cost of preparation
In addition, using chemical reactions to couple click chemical reagents to target proteins requires many steps, takes a long time, affects protein activity, and even leads to protein denaturation
Therefore, this method is not suitable for conjugation of proteinase 3 to biotin

Method used

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  • Conjugate of mutant protease 3 and biotin, and preparation method and application of conjugate
  • Conjugate of mutant protease 3 and biotin, and preparation method and application of conjugate
  • Conjugate of mutant protease 3 and biotin, and preparation method and application of conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] (1) Construction of antigenic protein expression plasmids for site-directed mutagenesis

[0063] 1. Selection of mutation sites

[0064] The antigenic epitope of protease 3 was predicted by using the online tool Antigenic Peptide Prediction of the website Immunomedicine Group.

[0065] Download the crystal structure of protease 3 from the PDB database, and select amino acid sites that are located on the surface of the protein crystal structure and do not belong to antigenic epitopes as mutation sites. After these amino acid sites are mutated into unnatural amino acids, azide or alkynyl groups can be directly exposure to solvents. The specific mutation sites are shown in Table 1.

[0066] Table 1. Protease 3 mutation sites

[0067]

[0068] 2. Expression plasmid acquisition

[0069] According to the gene sequence of protease 3 published by NCBI Gene Bank, a full-length DNA fragment was obtained through total gene synthesis, which was constructed in the pET28a expr...

Embodiment 2

[0080] It is basically the same as Example 1, except that the unnatural amino acid used is Phe-azido, and the purified protease 3 mutant protein is marked as PR3-R36-2, PR3-H48-2, and PR3-R74-2 respectively , PR3-S115-2.

Embodiment 3

[0081] Example 3, Preparation of Anti-Protease 3 Antibody Detection Reagent

[0082] (1) The mutant proteins obtained in Example 1 and Example 2 were respectively coupled with long-arm alkynyl biotin through a copper-catalyzed click chemical reaction, and the reaction system was as follows:

[0083]

[0084]

[0085] Small pieces of copper wire.

[0086] The reaction conditions were as follows: 4°C, vertical suspension for 30 min, after the reaction, EDTA with a final concentration of 1 mM was added to terminate the reaction, and the long-arm biotin-protease 3 conjugate was obtained.

[0087] (2) Coating the biotin-labeled mutant protein with streptavidin magnetic beads

[0088] Take 200 μL of 10 mg / mL streptavidin magnetic beads, add 1 mL of PBS buffer (pH=7.0), wash three times through a magnetic separator, add 20 μg of point mutant protease 3 molecules, and vertically suspend for 30 min at room temperature, add 1 mL of PBS buffer solution (0.1M, pH=7.0), washed thre...

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Abstract

The invention provides a conjugate of mutant protease 3 and biotin, and a preparation method and application of the conjugate. The amino acid at the 36 site, the 48 site or the 74 site of the amino acid sequence of the mutant protease 3 is mutated into non-natural amino acid with one of an azide group and an alkynyl group; the biotin is a biotin derivative with the other one of the alkynyl group and the azide group; and the conjugate of the mutant protease 3 and the biotin is obtained by carrying out a click chemical reaction on the non-natural amino acid and the biotin derivative. The invention has the advantages of strong operability, high reaction efficiency and mild conditions, improves the biotin labeling efficiency, enhances the antigen-antibody binding reactivity, improves the sensitivity and accuracy of antiproteinase 3 antibody detection, and has wide prospects in clinical application.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a conjugate of mutant protease 3 and biotin, a preparation method and application thereof. Background technique [0002] Protease 3 (PR3) is a serine protease in neutrophil cytoplasmic azurophilic granules, a glycoprotein with a molecular weight of about 29Ka. PR3 can degrade a variety of extracellular matrix such as elastin, hemoglobin, type Ⅳ or Ⅴ collagen and other tissue components. Proteinase 3 also promotes platelet activation through cathepsin G and inactivates C inhibitors. PR3 antibody is a specific antibody for Weigla granulomatosis (SigG). Although its pathogenesis is unclear, anti-PR3 antibodies have been implicated in the pathogenesis of VGD. In addition, the concentration of PR3 antibody is closely related to the activity of other diseases, which can be seen in primary systemic vasculitis, chronic inflammatory enteritis, infection (such as HV and oth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C07K1/107G01N33/573G01N33/543G01N33/533G01N21/76
CPCC12N9/50G01N33/573G01N33/54326G01N33/533G01N21/76Y02A50/30
Inventor 向乾银陈蕊于婷李伟甲秦枫赵婷王健孙雅静向雪花胡江鸿
Owner 苏州携创生物技术有限公司
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