Antibody for detecting R342 site asymmetric dimethylation modification of EZH2

An asymmetric, EZH2 technology, applied in the biological field, can solve the problems of no research reports, no antibody reports for detecting EZH2 asymmetric dimethylation modification, etc., and achieve the effect of enhancing protein stability

Pending Publication Date: 2020-01-14
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Whether EZH2 can undergo asymmetric dimethylation modification of arginine has not been reported so far, nor has there been any antibody report for detecting asymmetric dimethylation modification of EZH2

Method used

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  • Antibody for detecting R342 site asymmetric dimethylation modification of EZH2
  • Antibody for detecting R342 site asymmetric dimethylation modification of EZH2
  • Antibody for detecting R342 site asymmetric dimethylation modification of EZH2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 PRMT1 mediates the asymmetric dimethylation modification of the R342 site of EZH2

[0048] 1. PRMT1 and EZH2 binding detection: the constructed prokaryotic expression GST-PRMT1 plasmid was transformed into BL21 bacteria to prokaryotically express GST-PRMT1, sonicated and enriched with GST-Beads, and MDA-MB-231 breast cancer cells were lysed The solution was incubated, GST pull down experiment was performed, and the anti-EZH2 antibody was used to detect the EZH2 signal by Western Blot, so as to detect the combination of GST-PRMT1 and EZH2.

[0049] The specific operation is as follows:

[0050] GST tagged protein (GST-EZH2 and GST-PRMT1) purification:

[0051] 1) To activate the strain, take 50 μl of the bacterial solution and add it to 3 ml of LB medium, shake at 200 rpm at 37°C overnight.

[0052] 2) Bacteria solution transfer: Transfer the above bacteria solution to 100ml LB medium, culture at 37°C and 170rpm for 1-2h, measure OD 600 At around 0.5, IPTG w...

Embodiment 2

[0087] Example 2 The asymmetric dimethylation modification at the R342 site of EZH2 enhances the stability of EZH2 protein

[0088] Experimental method: EZH2 wild-type (Flag-EZH2-WT) and R342 site mutant (Flag-EZH2-R342K) plasmids were overexpressed in HEK293T cells, and then treated with MG132 (an inhibitor of proteasome degradation) by Western Blot Detection of EZH2 changes.

[0089] The specific methods for overexpressing EZH2 wild-type (Flag-EZH2-WT) and R342 site mutant (Flag-EZH2-R342K) plasmids in HEK293T cells are as follows:

[0090] ⑴ Plate HEK293T cells 24 hours in advance, so that the cell density before transfection reaches about 40%.

[0091] (2) Change to DMEM serum-free medium without double antibody before transfection. The plasmid and transfection reagent should be vector / Flag-EZH2-WT / Flag-EZH2-R342K plasmid (ug): PEI (ul) = 8: 3, according to the culture The amount of transfected plasmid is calculated according to the number of cells. Generally, 4 μg of pl...

Embodiment 3

[0096] The preparation of embodiment 3 antibody

[0097] The preparation method of the antibody (meR342-EZH2 antibody) for detecting the asymmetric dimethylation modification of the R342 position of EZH2 comprises the following steps:

[0098] (1) Using polypeptide synthesis technology to synthesize a polypeptide with an amino acid sequence such as the antibody shown in SEQ ID No.1, so that the purity of the polypeptide is above 85%, and then cross-linking with hemocyanin KLH to obtain the antigen;

[0099] (2) The antigen was mixed with complete adjuvant (CFA) or incomplete adjuvant (IFA) in equal volumes and then injected into the animal body for 8 times of immunization. The whole blood of the animal was collected and purified to obtain the meR342-EZH2 antibody.

[0100] In step (2), the animals are New Zealand white rabbits. Take two adult male New Zealand white rabbits purchased from Weitong Lihua Company (the numbers are RB11081 and RB11082), and immunize them according t...

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Abstract

The invention relates to an antibody for detecting R342 site asymmetric dimethylation modification of EZH2, and the antibody is a polyclonal antibody, wherein the amino acid sequence of the antibody is shown as SEQ ID No. 1. The preparation method of the antibody comprises the following steps: synthesizing polypeptide of the antibody shown as SEQ ID No.1 by adopting the polypeptide synthesis technology, and then performing crosslinking with hemocyanin KLH to obtain an antigen; mixing the antigen with a complete adjuvant or an incomplete adjuvant, injecting the obtained mixture into an animal body, performing immunizing for 8 times, taking animal whole blood, and performing purifying to obtain a product. The research of the inventors indicates that the R342 site of the EZH2 can be subjectedto asymmetric dimethylation modification by the PRMT1; the antibody provided by the invention can effectively and specifically recognize the asymmetric dimethylation modification condition of the R342 site in the EZH2, and plays a crucial role in researching the regulation and control of the post-translational modification of the EZH2 protein in the future.

Description

technical field [0001] The invention relates to an antibody for detecting the asymmetric dimethylation modification of the R342 site of EZH2, which belongs to the field of biotechnology. Background technique [0002] EZH2 (Enhancer of Zeste Homolog 2) is an important histone methyltransferase, which can catalyze the trimethylation modification (H3K27me3) of the 27th lysine of histone H3 in its target gene. Human EZH2 is a gene with 20 exons and a length of about 40kb. The EZH2 gene codes to produce a protein with a size of about 100KD, containing 746 amino acid residues, and its sequence is highly conserved among many species such as human and mouse. With the deepening of research, more and more research reports have found that the post-translational modification (PTM) of EZH2 protein plays an important role in its various biological functions. The currently known post-translational modifications of EZH2 mainly focus on the phosphorylation, acetylation, glycosylation and u...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/6812
Inventor 李中伟白津郑骏年王典典侯平甫李敏乐柴大飞
Owner XUZHOU MEDICAL UNIV
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