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A human cyr61 protein ser188 site phosphorylated antigen, antibody and its preparation method and application

A phosphorylation and protein technology, applied in chemical instruments and methods, anti-animal/human immunoglobulin, immunoglobulin, etc.

Active Publication Date: 2021-07-20
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problem of effective research on the phosphorylation modification of CYR61 protein, the primary purpose of the present invention is to provide a phosphorylated antigenic peptide specific for the Ser188 site of human CYR61 protein

Method used

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  • A human cyr61 protein ser188 site phosphorylated antigen, antibody and its preparation method and application
  • A human cyr61 protein ser188 site phosphorylated antigen, antibody and its preparation method and application
  • A human cyr61 protein ser188 site phosphorylated antigen, antibody and its preparation method and application

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preparation example Construction

[0043] see figure 2 , the preparation method and application of the phosphorylated antibody against the Ser188 site of human CYR61 protein provided by the embodiment of the present invention generally includes the following steps:

[0044] Step 1: First, screen possible phosphorylated sites in the amino acid sequence of human CYR61 by bioinformatics software, and determine the Ser188 site of human CYR61 protein by mass spectrometry and database ( image 3 ), to analyze the antigenicity of the site ( Figure 4 ), design the corresponding antigenic peptide, and analyze its homology;

[0045] Step 2: Synthesizing the antigenic peptide containing the Ser188 phosphorylation site. Using peptide synthesis technology to synthesize 10 amino acid antigenic peptides designed in Step 1 containing Ser188 phosphorylation sites, in which amino acid Ser188 adds a phosphorylation group, and is coupled with the carrier protein hemocyanin (KLH) to prepare phosphate as an immunogen Antibodies...

Embodiment 1

[0049] Example 1 Determine the phosphorylation site of human CYR61 protein

[0050] 1.1 Firstly, the possible phosphorylation sites in human CYR61 protein were screened by bioinformatics software NetPhorest 2.0 and NetPhos 2.0, followed by mass spectrometry literature review and protein phosphorylation site database PhosohoSitePlus( image 3 ), confirmed that Ser188 is a phosphorylation site in the amino acid sequence of human CYR61; at the same time, the antigenicity of the amino acid sequence of human CYR61 was calculated using the software CLC Protein Workbench 5 ( Figure 4 ), and finally determine the specific phosphorylation site Ser188 of human CYR61 protein.

[0051] 1.2 Design the phosphorylated antigen peptide at Ser188 site of human CYR61 protein. Centering on the Ser188 site of human CYR61 protein, the N-terminus is adjacent to four CYR61 amino acid sequences, and the C-terminus is connected to five amino acid sequences. The designed synthetic peptide (antigenic p...

Embodiment 2

[0052] Example 2 Synthesis of synthetic peptides (antigenic peptides) including Ser188 phosphorylation sites

[0053] According to the design of hapten synthetic peptides, a phosphorylation group was added to the Ser188 site to obtain phosphorylated synthetic peptides (antigenic peptides), which were coupled with hemocyanin (KLH) to obtain whole antigens for rabbit immunization (pSer188 -KLH). In addition, the phosphorylated synthetic peptide (antigenic peptide) was coupled with bovine serum albumin (BSA) by the glutaraldehyde method and used as the filler of the affinity purification chromatography column (pSer188-BSA). Correspondingly, a non-phosphorylated synthetic peptide corresponding to Ser188 was synthesized and coupled with bovine serum albumin (BSA) to be used as a filler for an affinity separation chromatography column (Ser188-BSA).

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Abstract

The invention discloses a human CYR61 protein Ser188 site phosphorylated antigen, an antibody, a preparation method and application thereof, and relates to the technical field of the antibody and its preparation. The antibody is prepared by immunizing animals with the active amino acid sequence shown in SEQ ID NO: 1 as the antigen peptide. The phosphorylated antibody provided by the invention helps to study the kinases that mediate its phosphorylation, and to explore the various biological functions of the CYR61 protein; in practical applications, it is convenient to study the phosphorylation modification of specific sites of the human CYR61 protein in specific biological events Or the mechanism of action in the process of disease occurrence and development, and can also be used to detect differences in the expression of tumor-related proteins after medication, providing potential targets for the diagnosis and treatment of clinical tumor diseases.

Description

technical field [0001] The invention relates to the technical field of antibodies and their preparation, in particular to a phosphorylated antigen specific to the Ser188 site of human CYR61 protein, an antibody, its preparation method and application. Background technique [0002] The CCN family consists of 6 proteins with similar functional domains, and CYR61 is the first protein in this family to be discovered. This family of proteins has long been considered as secreted proteins and has been intensively studied as cell matrix proteins. CCN family proteins are synthesized in cells and secreted out of cells under the guidance of signal peptides. They are accompanied by a series of changes in the internal and external environment of tumors during the occurrence and development of tumors, and can function both inside and outside cells. This family of proteins is involved in physiological processes such as cell adhesion, division, migration, drug resistance, survival, differe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K16/30C07K16/06G01N33/574G01N33/68
CPCC07K14/47C07K16/30C07K2317/10G01N33/574G01N33/6893G01N2800/22G01N2800/32
Inventor 尹东李瑜胡开顺陈恒星肖星
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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