Transposase-antibody binding protein fusion protein and preparation and application thereof

A fusion protein, transposase technology, applied in transferase, fusion polypeptide, antibody mimic/scaffold, etc., can solve the problems of complicated operation, loss of sequencing information, and many steps.

Inactive Publication Date: 2018-06-29
SHANGHAI SINOBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Similarly, ChIP-Seq inherits the same technical difficulties as ChIP. In addition, in the library construction-sequencing link, the end of the DNA fragment is filled first, then A' is added to the end, and a Y-shaped adapter is added to A', and finally The sequencing method is also cumbersome and has many steps, and each step will lose precious samples and

Method used

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  • Transposase-antibody binding protein fusion protein and preparation and application thereof
  • Transposase-antibody binding protein fusion protein and preparation and application thereof
  • Transposase-antibody binding protein fusion protein and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preparation of fusion protein of transposase-antibody binding protein

[0108] 1. Fusion protein structure

[0109] The structure of the fusion protein of this embodiment includes a first domain with a transposition function and a second domain with a function of binding to the Fc fragment of an antibody. The transposition function refers to the function of inserting gene sequence by transposition. The function of binding to the Fc segment of an antibody refers to binding to the function of the Fc segment of an IgG molecule. The first structural domain and the second structural domain are connected by a connecting fragment, ie Linker. Further, the first domain may be a transposase protein, and the second domain may be an antibody binding protein.

[0110] The specific construction scheme of the fusion protein is: the linking fragment between the transposase protein and the antibody binding protein, that is, the structural formula is (GS) a (GGS) b (GGGS) ...

Embodiment 2

[0197] Example 2 Functional detection of the fusion protein of transposase-antibody binding protein

[0198] 1. To detect the function of the fusion protein of the transposase-antibody binding protein obtained in Example 1, firstly verify the randomly interrupted genome of the fusion protein, insert a tag sequence, and construct a sequencing library function after PCR.

[0199] The principle and schematic diagram of random insertion of fusion protein are as follows: Figure 11 Shown: Transposomes formed by fusion proteins and adapters can fragment double-stranded DNA and add adapters to both ends of the fragmented DNA.

[0200]We used fusion proteins 1-5 (i.e. Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5-ProteinA-5) and Tn5 transposase (positive control) respectively When dealing with human genomic DNA, fragmented short DNAs of different sizes and lengths are expected to be obtained, and linker sequences are connected to both ends of these fragmented ...

Embodiment 3

[0275] Example 3 The fusion protein of transposase-antibody binding protein brings a new method for studying protein-DNA interaction: ChT-Seq

[0276] The principle of the traditional ChIP-Seq method is to first specifically enrich the DNA fragments bound by the target protein through chromatin immunoprecipitation (ChIP), then purify these fragments and construct a library, and then perform high-throughput sequencing on them. The experimental procedure is as follows:

[0277] (1) Formaldehyde cross-links the entire cell line (tissue), linking the target protein with chromatin;

[0278] (2) Separating genomic DNA and breaking it into small fragments of a certain length with ultrasound;

[0279] (3) Add a specific antibody that binds to the target protein, and the antibody forms an immunoprecipitation immunobinding complex with the target protein;

[0280] (4) Remove cross-linking and purify DNA to obtain DNA samples of chromatin immunoprecipitation;

[0281] (5) Library cons...

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Abstract

The invention belongs to the technical fields of molecular biology, genomics and biotechnology, and particularly relates to a transposase-antibody binding protein fusion protein and preparation and application thereof. The fusion protein comprises a transposase portion, a linker peptide portion, and a portion capable of binding to an Fc segment of an antibody, and thus, the fusion protein has botha transposition function and an antibody Fc segment binding function. The fusion protein is used to create a new in-vivo protein-chromatin DNA interaction studying method, namely chromatin-transposition-sequencing (ChT-Seq) method. Compared with traditional ChIP-Seq, the ChT-Seq method has the following advantages: fewer steps, high efficiency, saving, good repeatability, less required sample DNA, and greatly-reduced sample and information lost in the process. The method is a powerful tool for studying in-vivo protein-chromatin DNA interaction, and has great significance for research on generegulation and epigenetics and the like.

Description

technical field [0001] The invention belongs to the fields of molecular biology, genomics and biotechnology, and specifically relates to a fusion protein of a transposase-antibody binding protein and its preparation and application. Background technique [0002] Transposon sequences can be inserted and integrated into random positions in the genome under the action of transposases. Because of its random insertion into DNA, transposases are often used in mutant library construction and sequencing library construction. In the library construction link of high-throughput sequencing, transposase can randomly interrupt the sequence to be tested, and add adapters to both ends of the fragmented sequence. After PCR amplification, the next step of sequencing can be directly performed, which is compared with traditional ultrasonic disruption The method has the great advantage that fewer steps save costs and time. [0003] Antibody binding proteins such as ProteinA, ProteinG or Prote...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/12C12N15/62C12Q1/6869C12Q1/6804
CPCC07K14/31C07K14/315C07K2319/70C07K2319/705C12N9/1241C12Q1/6804C12Q1/6869C12Q2535/122C12Q2563/131C12Q2525/191C12N9/22
Inventor 朱化星王米李科何翼
Owner SHANGHAI SINOBIO BIOTECH
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