Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T73 site

A technique for preparing tudor-sn and antibodies, applied in the direction of anti-enzyme immunoglobulin, immunoglobulin from serum, etc.

Inactive Publication Date: 2015-01-14
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problem of effectively studying the epigenetic modification of human Tudor-SN protein, and to provide a method for preparing a stress phosphorylated antibody against the T73 site of human Tudor-SN protein

Method used

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  • Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T73 site
  • Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T73 site
  • Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T73 site

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1, first determine the stress phosphorylation site of Tudor-SN protein

[0035] 1) We passed LI-COR Odyssey ? Near-infrared two-color laser imaging system ( figure 2 ) found that when HeLa cells were given arsenite oxidative stress, 45°C heat shock and 4°C cold shock treatment, the overall level of threonine (Thr) phosphorylation of endogenous Tudor-SN protein increased.

[0036] 2) Subsequent LC-MS / MS results showed that when HeLa cells were given arsenite oxidative stress, threonine at position 73 in the SN1 domain of Tudor-SN protein would be phosphorylated ( image 3). Therefore, we prepared a rabbit-derived polyclonal phosphorylated antibody against the T73 site.

[0037]

Embodiment 2

[0038] Example 2, Synthesis of Hapten Polypeptides Including T73 Phosphorylation Sites

[0039] (1) First, we predict the secondary structure of the functional region of the Tudor-SN protein containing the T73 site, including accessibility, flexibility, surface probability, antigenicity, affinity Hydrophilicity, Dipole, etc., to analyze the antigenicity of Tudor-SN. The results show that( Figure 4 ), the total antigenic score of the N-terminal part around T73 is higher (Total red line, arrow), which is predicted to be easily exposed on the molecular surface.

[0040] (2) According to the secondary structure prediction, determine the candidate amino acid sequence of the polypeptide containing T73 phosphorylation site. In order to ensure that the phosphorylation site can be recognized as much as possible, the length of the antigen polypeptide is designed to be the minimum length of the antibody, that is, 5 amino acids are selected around T73 as the center. At the same time, ...

Embodiment 3

[0044] Embodiment 3, carrying out antigen immunization animal and serum acquisition

[0045] The synthesized phosphorylated hapten polypeptide at T73 site was coupled with carrier protein (KLH) respectively to form a complete antigen. SPF grade New Zealand white rabbits were immunized four times with complete antigen and adjuvant, and a total of two New Zealand white rabbits were immunized. Note: Before immunizing animals, 1ml of pre-immune serum should be taken from each rabbit as a negative control. Day 0: Initial immunization (complete Freund's adjuvant + antigen, 600~800ug / cause); Day 21: first booster immunization (incomplete Freund's adjuvant + antigen, 400~500ug / cause); Day 35 Day: second booster immunization (incomplete Freund's adjuvant + antigen, 400~500ug / monkey); day 49: third booster immunization (incomplete Freund's adjuvant + antigen, 200ug / bird); day 56 Whole blood was collected to separate and collect about 75ml of antiserum.

[0046]

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Abstract

The invention discloses a stress phosphorylation antibody aiming at a human Tudor-SN protein T73 site and a preparation method thereof. The preparation method adopts a multifunctional human Tudor-SN protein as a research object and comprises the following steps of determining if threonine at a 73 site of a human Tudor-SN protein under external environment oxidation stress is subjected to phosphorylation modification by near-infrared double-color laser imaging and mass spectrometry, synthesizing a T73 phosphorylation site-containing semiantigen polypeptide according to the secondary structure prediction result, coupling the T73 phosphorylation site-containing semiantigen polypeptide and a carrier protein (KLH) into a complete antigen, carrying out immunization on SPF-level New Zealand white rabbit by the complete antigen combined with an adjuvant four times and collecting, purifying and identifying a T73 specific stress phosphorylation polyclonal antibody. In practical application, Tudor-SN protein epigenetic phosphorylation modification in different stress environments can be detected, the effect mechanism of the Tudor-SN protein epigenetic phosphorylation modification in cell stress tumor propagation and migration can be discussed and a latent action target point for clinical tumor disease diagnosis and treatment is provided.

Description

technical field [0001] The invention relates to the technical field of antibody preparation, in particular to the preparation of a stress phosphorylated antibody directed at the T73 site of human Tudor-SN protein. Background technique [0002] Tudor-SN (tudor staphylococcal nuclease), also known as p100 protein, SND1 (staphylococcal nuclease domain containing 1), is a transcriptional regulatory activator discovered for the first time as EBNA2 (Epstein-Barr virus nuclear protein 2, EB virus nuclear antigen 2) a multifunctional protein. [0003] Human Tudor-SN protein is composed of SN (1~4) domains with 4 repeated staphylococcal nucleases-like (SN-like) domains at the N-terminus and SN5a-Tudor-SN5b (TSN) domains at the C-terminus ( figure 1 ). SN (staphylococcal nuclease), also known as thermostable nuclease, is a Ca 2+ Depending on the enzyme, it can hydrolyze the 5'-phosphodiester bonds of single / double-stranded DNA and RNA to generate 3'-mononucleotides and polynucleoti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C07K16/06
Inventor 杨洁高星杰苏超张毅葛林何津岩
Owner TIANJIN MEDICAL UNIV
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