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Regulating and controlling effect of thymine DNA glycosylase on signal transducing path

A technology of thymine and signal transduction, applied in the biological field, can solve problems such as not finding TDG

Inactive Publication Date: 2007-08-22
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there is no report that TDG is involved in the Wnt signal transduction pathway

Method used

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  • Regulating and controlling effect of thymine DNA glycosylase on signal transducing path
  • Regulating and controlling effect of thymine DNA glycosylase on signal transducing path
  • Regulating and controlling effect of thymine DNA glycosylase on signal transducing path

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Cloning of Example 1 TDG

[0055] TDG uses LEF1 (GenBank accession number: NM_010703) as "bait" to screen a clone containing a complete reading frame (GenBank accession number: NM_011561.1) obtained from a mouse 10.5-day embryo bank (purchased from GIBCO) by yeast two-hybrid , the specific sequence is as follows (SEQ ID NO: 1):

[0056] ATGATGGCAG AAGTTCCCTAA CATGGCAGTC ACGACTGGAC AGCAGGTGCC AGCAGTAGCT 60

[0057] CCTAACATGG CAACCGTGAC AGAACAGCAG GTGCCCGCAG ACGCTCCTGT CCAGGAACCT 120

[0058] GCACCAGAAG CTCCAAAGAG AAGGAAAAGG AAACCCAGAG CAGCAGAGCC CCAGGAACCA 180

[0059] GTGGAGCCCA AAAAACCTGC TACGTCGAAG AAATCCGGCA AGTCTACAAA ATCAAAGGAA 240

[0060]AAGCAGGAGA AAATCACAGA CGCGTTTAAA GTGAAAAGGA AAGTGGACCG CTTCAACGGC 300

[0061] GTCTCTGAAG CTGAGCTTCT GACCAAGACT CTTCCTGACA TTTTGACCTT CAATCTGGAT 360

[0062] ATTGTGATCA TTGGCATTAA CCCGGGATTA ATGGCTGCTT ACAAAGGACA TCACTACCCT 420

[0063] GGGCCTGGAA ATCACTTCTG GAAGTGTCTG TTCATGTCGG GGCTGAGTGA GGTGCAGCTG 480

[0064] AATCACA...

Embodiment 2

[0078] Example 2. TDG and LEF1 protein interaction

[0079] 1. Interaction between TDG and LEF1

[0080] TDG was obtained by yeast two-hybrid screening using LEF1 as "bait", suggesting that there may be protein interaction between TDG and LEF1.

[0081] In order to confirm this interaction between TDG and LEF1, first the inventors transiently transfected exogenous TDG with GG small peptide tag in human embryonic kidney cells HEK293T cells (carrier with GG (pGG) was remodeled from STRATAGENE's pCMV plasmid: synthesize a GG small peptide sequence (atgtctctagaagaagaagaatacatgccgatggaagcc (SEQ ID NO: 2)) with XbaI / XhoI restriction sites at both ends, and connect it to the pCMV vector purchased from STRATAGENE after XbaI / XhoI digestion; then The aforementioned recombinant vector was digested with SalI / NotI, and the TDG insert inserted with the same restriction enzyme was cloned into the vector to obtain a recombinant vector with GG small peptide tag and TDG) and HA-tagged LEF1 (ve...

Embodiment 3

[0092] Example 3. Function of TDG in the canonical Wnt signal transduction pathway

[0093] 1. TDG plays a positive regulatory role in the canonical Wnt signal transduction pathway

[0094] In this example, the function of TDG in the canonical Wnt signal transduction pathway was mainly detected by the LEF1 reporter gene activity system.

[0095] In conventional human embryonic kidney cell 293 cells and cervical cancer HeLa cells, transfect the reporter gene with LEF1 binding site in the luciferase promoter region such as TOPflash / FOPflash (purchased from upstate company) and LEF-luc (purchased from Panomics Company), green fluorescent protein GFP (purchased from BD Company), TDG and ΔN-β-catenin (deleting 45 amino acids at the N-terminal, not regulated by Wnt signal, and constitutively active β-catenin). After about 18 hours of transfection, the culture medium was replaced with Wnt3a glycoprotein (commonly known as Willert K, Nature, 2003, 423: 448-452) for stimulation for ab...

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Abstract

The present invention discloses the use of thymine DNA glycosylase or its excitant or antagonist in preparing medicine for regulating classical Wnt signal transduction path. The present invention also discloses method of screening abnormally activated matter for regulating Wnt signal transduction path. The present invention reveals the participation of thymine DNA glycosylase to classical Wnt signal transduction path for the first time, and provides excellent target point for screening Wnt signal transduction path regulating medicine or tumor preventing and treating medicine.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to the new function of thymine DNA glycosylase (TDG) involved in regulating the classic Wnt signal transduction pathway. Background technique [0002] Signal transduction is one of the frontier fields of biological research. In recent years, it has been found that there is a very conserved signaling pathway among individuals of different races. Its signaling molecule is a type of glycoprotein superfamily-Wnt protein, so it is called Wnt Signal transduction pathway. The Wnt signaling molecule family includes more than a dozen members. At present, it is found that different Wnt molecules cause different signal responses, which can be divided into the classic Wnt signal transduction pathway (Wnt / β-catenin), such as Wnt3 and Wnt8, and non-classical Wnt signal transduction pathway (Wnt / Ca 2+ and Wnt / JNK pathway, wherein the Wnt / JNK pathway has not yet found a con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61K38/55A61K48/00A61P35/00A61P25/28A61P13/12G01N33/68C12Q1/25
Inventor 李林贾莹莹黄涛王伟叶央儿
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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