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Enriching method for transcription factor target gene through co-immunoprecititation of protein bead

A technology for transcription factors and proteins, applied in the field of molecular biology, can solve the problem of not being able to determine whether the transcription factor regulates the target gene directly or indirectly, and achieves the effects of high specificity, improved specificity and high cleaning efficiency.

Inactive Publication Date: 2011-11-23
BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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Problems solved by technology

However, the limitation is that it is not possible to determine whether the transcription factor regulates the target gene directly or indirectly.

Method used

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  • Enriching method for transcription factor target gene through co-immunoprecititation of protein bead
  • Enriching method for transcription factor target gene through co-immunoprecititation of protein bead
  • Enriching method for transcription factor target gene through co-immunoprecititation of protein bead

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preparation example Construction

[0032] 6. Preparation of linker and its connection with enzyme-cut DNA

[0033] Different linker sequences were designed for the restriction enzymes used in step 5, and ligated with the digested DNA products. For example, the four oligonucleotide primers synthesized for EcoRI and MseI are: EcoRI linker 1: CTCGTAGACTGCGTACC and EcoRI linker 2: AATTGGTACGCAGTCTAC; and MseI linker 1: GACGATGAGTCCTGAG and MseI linker 2: TACTCAGGACTCAT, and then annealed in an equimolar ratio ( The reaction conditions are: 94°C, 10min; 37°C, 30min) to form EcoR I and Mse Double-stranded linkers AdpterE and AdpterM at I cohesive ends. via T 4 DNA ligase acts to cut the two ends of DNA fragments and anneal to form specific adapters. And use the ligation product as a template for amplification. If yes Eco RI and Mse For the double enzyme-digested ligation product of I, primers EcoRI + A: GACTGCGTACCAATTCA and MseI + C: GATGAGTCCTGAGTAAC can be used to amplify the ligation product.

[0034]...

Embodiment 1

[0038] Example 1: Enrichment of Rice WRKY Transcription Factor SUSIRI Target Genes by Immunomagnetic Beads

[0039] rice gene SUSIRI It is a transcription factor gene of WRKY family cloned from the young panicle of japonica rice Nipponbare. In order to obtain its target gene to elucidate its biological function, the cloned SUSIRI The full-length cDNA of the gene ( SUSIRI For details of the gene, please refer to: Chen Jian, Lin Zhongping, Duan Yuanlin, et al. Cloning and expression analysis of a SUSIBA2-like gene in rice. Molecular Plant Breeding, 2008, 6(3): 579-582) inserted into the PET-28a vector , constructed into a prokaryotic expression vector PET-SU containing a 6XHis antigen tag at the end. Western blot detection showed that the expression vector PET-SU could express the 63kD target protein in Escherichia coli BL21(DE3) plysS after IPTG induction, and the expression of SUSIRI protein was the highest after 2-3 hours of induction. Further use magnetic beads to t...

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Abstract

The invention provides an enriching method for transcription factor target genes through co-immunoprecititation of protein beads. The method comprises the following steps: expressing transcription factor protein containing antigen labels, and subjecting the transcription factor protein containing antigen labels and monoclonal antibodies directed at the antigen labels to an immunoreaction so as toform antigen-antibody compounds; carrying out co-immunoprecititation on protein beads and the antigen-antibody compounds to form antigen-antibody-protein bead compounds which have absorbed transcription factor proteins; binding the antigen-antibody-protein bead compounds to DNA digested segments of genomes so as to remove DNA segments which can not bind to the transcription factors of the compounds, which means candidate target gene segments which can bind to the transcription factors are enriched; further constructing an insertion library of the enriched segments, and carrying out sequencingto obtain target genes of the transcription factors. According to the invention, the target genes capable of directly binding to transcription factor proteins can be obtained effectively from genomesthrough a simplified method thanks to the introduction of the beads and improvement to experimental steps.

Description

technical field [0001] The present invention belongs to the field of molecular biology. More specifically, it relates to a method for in vitro enrichment of transcription factor target genes by using co-immunoprecipitation reaction of magnetic beads. Background technique [0002] With the completion of various biological genome projects, the reading frames of all genes that may exist in the genome will be resolved, and it is very urgent to study the expression regulation of genes, especially the expression regulation at the transcriptional level. Since transcription factors are a specific class of protein molecules that bind to DNA, the study of transcription factors is the study of the molecular mechanism of transcriptional regulation. Therefore, the functional research on transcription factors and their genes has become a hotspot in the field of biological research. For example, 2025 transcription factors have been predicted from the rice genome. But only by annotating ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 陈坚连肖华胡鸢雷林忠平
Owner BIOLOGICAL TECH INST OF FUJIAN ACADEMY OF AGRI SCI
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