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Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T103 site

A technology of tudor-sn and T103, applied in the field of antibody preparation

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

AI Technical Summary

Benefits of technology

This patented technology describes an improved version of this molecule called Stress Phosphoryla (SPL). It has been found useful during research into how certain genes are involved with cancer development or metastasis. Its activity could be detected through changes caused by these factors. Additionally it was shown that SPLL had some other beneficial functions related to gene regulation and disease detection. Overall, stressed phosphate group analysis makes possible better understanding of the role played by mutations associated with inherited forms of malignancy.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving plants against environmental stresses like saltwater immersion stress caused by drought conditions during winter months when crops have been severely damaged due to snow fall.

Method used

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  • Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T103 site
  • Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T103 site
  • Preparation method of stress phosphorylation antibody aiming at human Tudor-SN protein T103 site

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Experimental program
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Effect test

Embodiment 1

[0033] Example 1, first determine the stress phosphorylation site of Tudor-SN protein

[0034] 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.

[0035] 2) Subsequent LC-MS / MS results showed that when HeLa cells were given arsenite oxidative stress, the threonine at position 103 in the SN1 domain of Tudor-SN protein would be phosphorylated ( image 3 ). Therefore, we prepared a rabbit-derived polyclonal phosphorylated antibody against the T103 site.

[0036]

Embodiment 2

[0037]Example 2 Synthesis of Hapten Polypeptides Comprising T103 Phosphorylation Sites

[0038] (1) First, we predict the secondary structure of the functional region of the Tudor-SN protein containing the T103 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 score around T103 is low, it is possible that in the Native state protein, it is not easy to be exposed on the molecular surface.

[0039] (2) According to the secondary structure prediction, determine the candidate amino acid sequence of the polypeptide containing T103 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 that can obtain the antibody, that is, 5 amino acids are selected around T103 as the center. At the same time, in order to...

Embodiment 3

[0043] Embodiment 3, carrying out antigen immunization animal and serum acquisition

[0044] The synthesized phosphorylated hapten polypeptide at T103 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.

[0045]

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Abstract

The invention discloses a stress phosphorylation antibody aiming at a human Tudor-SN protein T103 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 103 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 T103 phosphorylation site-containing semiantigen polypeptide according to the secondary structure prediction result, coupling the T103 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 T103 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

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Claims

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Application Information

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Owner TIANJIN MEDICAL UNIV
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