Human NOTCH1 NICD protein antigen and antibody as well as preparation methods and application thereof

A protein and antibody technology, applied in the direction of animal/human protein, anti-animal/human immunoglobulin, antibody, etc., can solve the problems of abnormal phosphorylation modification, half-life extension, etc., and achieve high affinity, good recovery rate, and short preparation cycle short effect

Inactive Publication Date: 2017-05-31
SOUTHERN MEDICAL UNIVERSITY +1
View PDF5 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Abnormal phosphorylation modification, resulting in the prolongation of the half-l

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Human NOTCH1 NICD protein antigen and antibody as well as preparation methods and application thereof
  • Human NOTCH1 NICD protein antigen and antibody as well as preparation methods and application thereof
  • Human NOTCH1 NICD protein antigen and antibody as well as preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0041] see figure 1 , the preparation method and application of the phosphorylated antibody against human NOTCH1 NICD protein Thr1861 site provided by the embodiment of the present invention generally includes the following steps:

[0042] Step 1: Antigen epitope design and screening: The inventor selected the SEQ ID of the intracellular segment protein (NICD) in the human NOTCH1 protein through long-term accumulated experience, which has the ability to induce animals to produce highly antigenic and highly hydrophilic antibodies NO: 1 polypeptide, and the threonine (Thr) at position 1861 was phosphorylated and modified.

[0043] Step 2: Preparation of synthetic peptides and coupled proteins: Synthesize the antigenic peptide shown in SEQ ID NO: 1 using peptide synthesis technology, add a phosphorylated group to the amino acid Thr1861, and a cysteine ​​is connected to the C-terminus (obtained said antigen peptide), after HPLC purification and mass spectrometry (MS) identificati...

Embodiment 1

[0048] Example 1 Antigen epitope design and screening

[0049] 1.1 The inventors of the present invention have screened for the ability of highly specific and highly hydrophilic antibodies phosphorylated at the Thr1861 site of the human NOTCH1 NICD protein. This epitope contains 9 amino acids (aa), and the sequence is as shown in SEQ ID NO: Shown in 1: N-Ala Met Ala Pro p(Thr)Pro Pro Gln Gly-C; Finally, the homology analysis of the synthetic peptide was carried out using blastp on the NCBI website, and the results showed that the peptide sequence of the antigenic epitope was no different from that of rabbits origin, and calculate the antigenicity and hydrophilicity of the human NOTCH1 amino acid sequence ( image 3 , Figure 4 ).

Embodiment 2

[0050] Embodiment 2 Synthetic peptide and coupling protein preparation

[0051] According to the design of the epitope peptide in Example 1, a phosphorylation group was added to the Thr1861 site and a cysteine ​​was added to the C-terminus of the sequence to obtain a phosphorylated synthetic peptide (SEQ ID NO: 2), which was analyzed by HPLC. After purification, a synthetic peptide with a purity of 96% was obtained, which was identified by mass spectrometry and was consistent with the design ( Figure 5 ), and then through the C-terminal cysteine ​​and hemocyanin (KLH) coupling to obtain the whole antigen (pT1861-KLH, Thr referred to as T) will be used for animal immunization; at the same time, the synthetic peptide through the C-terminal cysteine ​​and After coupling with bovine serum albumin (BSA), it was used for the isolation of phosphorylated antibody (pT1861-BSA). Similarly, a synthetic peptide as shown in SEQ ID NO: 1 is synthesized by using peptide synthesis technolog...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a specific Thr1861 site phosphorylated antigen peptide aiming at a human NOTCH1 NICD protein, an antibody, application of the antigen peptide and the antibody, and a preparation method of the antibody. The antibody is shown as SEQ ID NO:1 through an active amino acid sequence, Thr amino acid is phosphorylated and prepared as an antigen peptide immunized animal. When the antibody is prepared, antiserum is purified by utilizing an affinity purification-affinity separation technology. The preparation method has the advantages of being short in preparation period, high in antibody potency, good in recovery rate, and the like; the obtained antibody has high specificity and high affinity; the phosphorylated antibody is helpful to reveal an action mechanism of phosphorylation modification of the NOTCH1 NICD protein in the occurrence and development of tumors and other diseases, and has wide clinical application prospect in accepts of tumor diagnosis, treatment, prognosis judgment and the like.

Description

technical field [0001] The invention relates to the technical field of antibodies and their preparation, and in particular to a phosphorylated antigen specific for the Thr1861 site of human NOTCH1 NICD protein, an antibody, and a preparation method and application thereof. Background technique [0002] NOTCH signal is a highly conserved signaling pathway in the evolution process, widely exists in multiple species of invertebrates and vertebrates, it is one of the main signaling pathways that mediate direct contact between cells, regulates Apoptosis, proliferation and differentiation in multicellular organisms. The genetic characteristics of the NOTCH signaling pathway are highly conserved from Drosophila to humans, and its regulatory process is particularly complex in mammals. [0003] NOTCH receptors, NOTCH ligands and intracellular effector molecule CSL together constitute the NOTCH signaling pathway. NOTCH receptor is a single-chain transmembrane protein, and its molecu...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C07K14/705C07K16/28C07K1/22G01N33/68G01N33/574A61K38/17A61K39/395A61P35/00
CPCA61K38/00A61K2039/505C07K14/705C07K16/28G01N33/574G01N33/57411G01N33/57426G01N33/68G01N33/6854G01N2333/705
Inventor 宁云山邱晓媚李妍薛杨
Owner SOUTHERN MEDICAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap