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DNA fragment with E2F1 protein binding property and application to E2F1 activity detection

A technology of E2F1 and protein, applied in the field of molecular biology, can solve problems such as changes in activity and differences in E2F1 protein content

Active Publication Date: 2017-06-20
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Application Information

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Problems solved by technology

However, the current detection of E2F1 endogenous activity still relies on techniques such as Western Blot or RCR. Although these methods have high sensitivity, they only detect the difference in E2F1 protein content and cannot directly reflect the change in activity after it binds to the target sequence.

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  • DNA fragment with E2F1 protein binding property and application to E2F1 activity detection
  • DNA fragment with E2F1 protein binding property and application to E2F1 activity detection

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Embodiment Construction

[0027] The principles and features of the present invention are described below in conjunction with examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0028] 1. Construction of E2F1 protein-binding DNA fragments

[0029] The inventors conducted research and analysis on E2F1 and found that the DNA sequence bound by E2F1 protein is 5'-TTTSSCGS-3' (S means C or G). When synthesizing the DNA core sequence, this DNA core sequence is repeated four times, spaced by 2-4 A and / or T, its sequence is shown in SEQ ID NO: 1, and connected to the DNA carrying firefly luciferase Expression vector (pGL3.0-Basic).

[0030] In order to ensure the success of the vector construction, the cohesive end of the corresponding restriction endonuclease site was added at the end. In this example, the cohesive end of the Kpn I restriction site (CATGG) was added at the 5' end and the 3' end was added Sticky ends of the Hind I...

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Abstract

The invention relates to a DNA fragment with E2F1 protein binding property containing one or more E2F1 protein binding cassettes, application of the DNA fragment, and a method for detecting transcriptional control activity of E2F1 in cells. By the adoption of the DNA fragment and the method, people can directly detect transcriptional control activity of E2F1 in cells in a targeted mode instead of only detecting the content of transcript or protein of E2F1, so that the function of E2F1 as a transcription factor in developmental biology and pathology development can be analyzed more accurately.

Description

technical field [0001] The invention relates to the field of molecular biology, and more particularly relates to a DNA fragment that can be combined with E2F1 protein and its application in detecting the E2F1 transcription regulation activity in cells. Background technique [0002] Transcription factor E2F1 is encoded by the human E2F1 gene and is a member of the E2F family of cell cycle-related transcription factors. Its activation can transmit signals to the nucleus and promote the cell cycle from G1 phase to S phase. The E2F family plays an important role in the control of the cell cycle and the function of tumor suppressor genes, and is also the target of transforming proteins of small DNA oncoviruses. Multiple member proteins in the E2F family contain many evolutionarily conserved domains, namely DNA binding domain, dimerization domain interacting with protein, transactivation domain rich in acidic amino acids and tumor suppressor located in the transactivation domain ...

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12N15/85
CPCC12N15/11C12N15/85C12Q1/68
Inventor 郑丽端童强松洪梅肖文晶王建群叶霖李聃宋华杰
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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