A human cyr61 protein ser167 site phosphorylated antigen, antibody and its preparation method and application

A technology of ser167 and phosphorylation, applied in chemical instruments and methods, anti-animal/human immunoglobulins, antibodies, etc.

Active Publication Date: 2021-09-28
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 Ser167 site of human CYR61 protein

Method used

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  • A human cyr61 protein ser167 site phosphorylated antigen, antibody and its preparation method and application
  • A human cyr61 protein ser167 site phosphorylated antigen, antibody and its preparation method and application
  • A human cyr61 protein ser167 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 Ser167 site of human CYR61 protein provided by the embodiment of the present invention generally includes the following steps:

[0044] Step 1: first screen the possible phosphorylated sites in the human CYR61 amino acid sequence by bioinformatics software, and determine the Ser167 site of the 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: Synthesis of antigenic peptides containing Ser167 phosphorylation sites: using peptide synthesis technology to synthesize the 10 amino acid antigenic peptides designed in Step 1 containing Ser167 phosphorylation sites, wherein amino acid Ser167 is added with a phosphorylation group, and combined with the carrier protein Coupling with hemocyanin (KLH) as an immunogen to prepare...

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 ), it was confirmed that Ser167 is a phosphorylation site in the amino acid sequence of human CYR61; meanwhile, 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 Ser167 of human CYR61 protein.

[0051] 1.2 Design the phosphorylated antigen peptide at Ser167 site of human CYR61 protein. Centering on the Ser167 site of human CYR61 protein, the N-terminus is adjacent to four CYR61 amino acid sequences, and the C-terminus is connected to five CYR61 amino acid sequences. The designed synthetic peptide (antig...

Embodiment 2

[0052] Example 2 Synthesis of synthetic peptides (antigenic peptides) including Ser167 phosphorylation sites

[0053] According to the design of hapten synthetic peptides, a phosphorylation group was added to the Ser167 site to obtain phosphorylated synthetic peptides (antigenic peptides), which were coupled with hemocyanin (KLH) to obtain whole antigens for rabbit immunization (pSer167 -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 column (pSer167-BSA). Correspondingly, a non-phosphorylated synthetic peptide corresponding to Ser167 was synthesized and coupled with bovine serum albumin (BSA) to be used as a filler for an affinity separation chromatography column (Ser167-BSA).

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Abstract

The invention discloses a human CYR61 protein Ser167 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 during the occurrence and development of the disease can also be used to detect the difference in the expression of tumor-associated proteins after medication, providing a potential target 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 Ser167 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): C07K14/47C07K16/18C07K16/44C07K16/06G01N33/68G01N33/574A61K39/395A61P35/00A61P7/00A61P9/00
CPCA61P7/00A61P9/00A61P35/00C07K14/4702C07K16/06C07K16/065C07K16/18C07K16/44C07K2317/34C07K2317/76G01N33/57484G01N33/6893G01N2800/22G01N2800/32
Inventor 尹东胡开顺李瑜林乐航吴雯静
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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