Method for separating specific protein-DNA (Deoxyribose Nucleic Acid) complex in living body, fusion protein and preparation method thereof

A technology of fusion protein and complex, applied in the field of bioengineering

Pending Publication Date: 2021-06-29
NANHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, nuclease targeted cut and release (CUT&RUN) technology, which mainly uses target-specific primary antibody and pAG-micrococcal nuclease (pAG-MNase) to isolate specific protein-DNA complexes in vivo, and uses Ca 2+ Control the activity of the enzyme, build a library and sequence the excised DNA fragments, so as to obtain the DNA fragments specifically bound by the protein, but this method requires a highly specific target protein primary antibody

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  • Method for separating specific protein-DNA (Deoxyribose Nucleic Acid) complex in living body, fusion protein and preparation method thereof
  • Method for separating specific protein-DNA (Deoxyribose Nucleic Acid) complex in living body, fusion protein and preparation method thereof
  • Method for separating specific protein-DNA (Deoxyribose Nucleic Acid) complex in living body, fusion protein and preparation method thereof

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Embodiment Construction

[0031] The key point of the present invention is to use the tagged nanobody to combine with the same tag in the target protein, bring the micrococcal nuclease to the binding site between the target protein and chromatin, and promote the cleavage of chromatin through the activation of the nuclease. The release of the protein-DNA complex does not require additional antigen-specific antibodies, and then the protein in the complex can be digested to sequence the DNA to determine the binding site, and the operation steps are simpler.

[0032] In order to facilitate the understanding of those skilled in the art, the following will take the green fluorescent protein nanobody-micrococcal nuclease fusion protein (nanoGM) as an example to further illustrate the present invention in conjunction with the accompanying drawings. The content mentioned in the examples does not limit the present invention .

[0033] It should be noted in advance that most of the materials used in this example ...

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Abstract

The invention discloses a method for separating a specific protein-DNA (Deoxyribose Nucleic Acid) complex in a living body, a fusion protein and a preparation method thereof, and relates to the technical field of bioengineering. The key point of the invention is that a nano antibody of a tag binds the same tag in a target protein, micrococcus nuclease is brought to a binding site between the target protein and chromatin, and the chromatin is cleaved through activation of nuclease, so that the protein-DNA complex is released, and DNA can be sequenced after the protein is digested, so as to determine the binding site. The method can be applied to research of protein-DNA interaction, when studying the protein specific binding DNA sequence, an antigen-specific antibody does not need to be additionally provided, the steps are simple, and a new technical means is provided for searching for the in-vivo chromatin binding sequence of different proteins containing the same tag.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for separating a specific protein-DNA complex in a living body, a fusion protein and a preparation method thereof. Background technique [0002] Chromatin immunoprecipitation assay (Chromatin immunoprecipitation assay, ChIP) has been the main method for studying protein-DNA interaction in vivo. The bound chromatin is analyzed to finally obtain the binding site of the protein on the genome. ChIP technology also has certain limitations, such as the need for highly specific antibodies, false negative signals caused by cross-linking or invalid antibodies, and false positive signals during formaldehyde fixation. [0003] In recent years, innovations and advances in genomic technology have provided new tools for better understanding the distribution of proteins on chromatin. For example, nuclease targeted cut and release (CUT&RUN) technology, which mainly uses target-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C07K19/00C12N9/22G01N33/531C12R1/19
CPCC12N15/70C07K16/18C12N9/22G01N33/531C07K2319/00C07K2317/569
Inventor 容益康陈涛程琳吴靖
Owner NANHUA UNIV
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