Method for excavating difference protein of eucaryotic gene transcriptional regulation group

A gene transcription, eukaryotic technology, applied in the biological field, can solve problems such as undetermined proteins

Inactive Publication Date: 2013-01-23
CHINA AGRI UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

The DNaseI method is only for the study of the interaction between a specific protein and nucleic acid, or the interaction between a specific short nucleic acid sequence and protein, so what is obtained is only a single nucleic acid fragment or a single protein; and the yeast one-hybrid system is also To study the interaction between proteins and specific sequences of DNA in cells, but only one promoter of a specific gene can be studied at a time; while the gel retardation test is a qualitative method to study the interaction between proteins and genes, but it cannot determine the specific protein

Method used

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  • Method for excavating difference protein of eucaryotic gene transcriptional regulation group
  • Method for excavating difference protein of eucaryotic gene transcriptional regulation group
  • Method for excavating difference protein of eucaryotic gene transcriptional regulation group

Examples

Experimental program
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Effect test

Embodiment

[0023] Example Exploring the transcriptional regulatory protein of myog gene in the differentiation process of C2C12 cells

[0024] It includes the following steps:

[0025] 1. Template construction: referring to the Myog sequence published by NCBI, the Landrace pig genome was extracted as a template, and the Myog promoter luciferase expression vector was amplified and constructed.

[0026] Primer sequence: F: 5’-TAGGGTAAGTGGGATTGAGTC-3’

[0027] R: 5’-CAGGTCGGAAAAGGCTTG-3’

[0028] PCR system:

[0029]

[0030] PCR conditions: 945min; 94℃30s, 60℃30s, 72℃2min 30s, 30 cycles; 72℃7min; 4℃store 15min.

[0031] The gene fragment about 2.4 kb upstream of the translation initiation site of Myog gene was obtained by PCR method, and the T vector was constructed and digested and sequenced; then the fragment was digested with restriction enzymes Nhe Ⅰ and Xho Ⅰ and cloned into PGL3 -Basic luciferase reporter gene vector. The structure of the obtained Myog promoter luciferase expression ...

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Abstract

The invention provides a method for excavating difference protein of a eucaryotic gene transcriptional regulation group. The method comprises the steps of: combining 5' or 3'-end promoter sequence with biotin with a magnetic bead of which the surface absorbs streptavidin to form magnetic bead-promoter binary complex, then extracting nucleoprotein at a specific period from a tissue or a cell, combining with the magnetic bead-promoter binary complex to form a magnetic bead-promoter-nucleoprotein ternary compound; carrying out SDS-PAGE analysis on the ternary compound; finding out a difference band to determine fragment length of target protein to be selected, then carrying out two-dimensional electrophoresis separation to purify the target protein, and carrying out mass spectrometry on the purified target protein so as to determine the contained protein. A new thought is provided for researching adjustment of gene expression from the transcriptional level, searching new protein and understanding the action on transcription regulation, and the foundation is established for further clarifying pathogenesis of certain diseases and gene treatment.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for mining differential proteins in eukaryotic gene transcription regulation groups. Background technique [0002] With the basic completion of human genome sequencing, the research of functional genomics becomes more important, and the regulation of gene expression is an important research field of functional genomics. (Guo Xiaoqiang, Guo Zhengliang. The structural basis of eukaryotic transcription mechanism. Bulletin of Biology, 2006, 41(11): 60-61) The expression of cellular genes, that is, the process of transcribing DNA into RNA and then translating into protein, is subject to strict The regulation control. In the process of cell growth, development and differentiation, the expression of genetic information can change according to a certain time program, and it can be adjusted as the internal and external environmental conditions of the cell changes. This is the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/00C07K1/26
Inventor 孟庆勇王萌高婧李宁
Owner CHINA AGRI UNIV
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