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Antibody-targeted recombinant fusion proteins and their application in epigenetics

A fusion protein and sequence technology, applied in the biological field, can solve the problems of large sequencing depth, long time consumption, and limited scope of ChIP use.

Active Publication Date: 2019-11-01
VAZYME BIOTECH NANJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the traditional ChIP technique, it is necessary to fix the proteins interacting with DNA with formaldehyde, which may cause some unrelated proteins to be cross-linked, forming a non-specific background (false positive)
There are some transcription factors with relatively small force or due to insufficient formaldehyde cross-linking, the interaction between protein and DNA is destroyed during the subsequent ultrasonic fragmentation of DNA, resulting in false negatives, and traditional ChIP takes a long time (4 days) and requires additional High-affinity antibodies, because it is difficult for low-affinity antibodies to effectively "drag down" protein and DNA complexes, which further limits the scope of ChIP
In addition, traditional ChIP-seq requires formaldehyde to cross-link cells and separate complexes of antibodies and chromosome fragments, followed by library construction and sequencing, requiring a large number of sample cells, high-affinity antibodies, long time and complicated operations , the resolution of sequencing is not high, the sequencing depth is large, and the cost is high
And it is not suitable for epigenetic research on precious samples such as embryonic development, stem cells, and pathological samples

Method used

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  • Antibody-targeted recombinant fusion proteins and their application in epigenetics
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  • Antibody-targeted recombinant fusion proteins and their application in epigenetics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 115

[0076] Example 1 Cloning construction of 15 different ABP-L-DFEs and construction of two DFE-L-ABPs

[0077] In this example, 17 ABP-L-DFE and DFE-L-ABP with different structures were prepared, named ABP-L-DFE-1, ABP-L-DFE-2, ABP-L-DFE-3, ABP respectively -L-DFE-4, ABP-L-DFE-5, ABP-L-DFE-6, ABP-L-DFE-7, ABP-L-DFE-8, ABP-L-DFE-9, ABP-L -DFE-10, ABP-L-DFE-11, ABP-L-DFE-12, ABP-L-DFE-13, ABP-L-DFE-14, ABP-L-DFE-15, DFE-L-ABP -1 and DFE-L-ABP-2.

[0078] 1. All the gene sequences were artificially synthesized, and the gene fragments were respectively cloned into the pET28a vector using a homologous recombination kit (ClonExpress II One StepCloning Kit, Vazyme, C112) to construct a recombinant expression vector.

[0079] (1) A fusion protein called ABP-L-DFE-1, its N-terminal domain is a part of streptococcal G protein (Protein G), and the C-terminal domain is DNAse I, and the two domains are passed through a short Rigid Linker Peptide Linker 1 Ligation.

[0080] The coding nuc...

Embodiment 2

[0201] Example 2 Verification of Antibody Binding Activity of Modified Protein

[0202] Apply the ELISA (enzyme-linked immunosorbent assay) principle to detect the antibody (Ig) binding activity of the fusion protein, dilute the fusion enzyme to be tested to 1-10 μg / mL with coating buffer (50mM carbonate buffer, pH 9.6), The molar concentrations of different fusion enzymes are the same, mix well, add to the microtiter plate, 100 μL / well, each fusion protein has three replicate wells, the negative control does not contain the fusion protein, and incubate overnight at 2-8°C; wash with washing solution (1 *PBST) and washed 3 times; add blocking solution (1*PBS, 5% BSA), 200μL / well, and incubate at 37°C for 2-3h; dilute the enzyme-labeled antibody (1*PBST, 5%BSA) Goat anti-rabbit or goat anti-mouse) diluted to 5 μg / mL, added to each well, 200 μL / well, incubated in a 37°C incubator for 1 hour, then discarded the reaction solution; washed 4 times with washing solution (1*PBST); each...

Embodiment 3A

[0203] The DNA fragmentation activity comparison of embodiment 3ABP-L-DFE-1 (GsLDNase I) and ABP-L-DFE-3 (AsLDNase I)

[0204] Take 5-10ng equimolar concentration of the protein to be tested and 500ng BL21 genomic DNA, mix well in Buffer A (20mM Tris-HCl, 10mM Ca2Cl, pH 8.0), incubate at 37°C for 10min, and immediately run the sample on 1% agarose gel Electrophoresis to verify the effect of DNA fragmentation. Depend on Figure 6 It can be seen that the DNA fragmentation activity of the same DFE fused with different ABPs is equivalent. Specifically, the DNA fragmentation activity of DNase I fused with the Protein A part and the Protein G part is equivalent at equimolar concentrations.

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Abstract

The invention discloses recombinant fusion protein in antibody targeting and application thereof in epigenetics. The recombinant fusion protein comprises an ABP-L-DFE structure, wherein ABP is locatedin the N terminal of the recombinant fusion protein and represents a structure domain with a binding antibody function, L represents a connecting peptide, and DFE represents a structure domain with aDNA (Deoxyribonucleic Acid) fragmentation function. The recombinant fusion protein disclosed by the invention is capable of improving a ChIP-seq (Chromatin Immunoprecipitation-sequencing) technology,simplifying operation, increasing data quality, reducing operation difficulty and expanding the application range, particularly the application in a small amount of precious cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody-targeted recombinant fusion protein and its application in epigenetics. Background technique [0002] For multicellular organisms, such as humans, the genetic material (DNA) of each cell is derived from the same fertilized egg, so the genetic information carried, that is, the DNA sequence is the same, but cells in different tissues, Such as skin and liver cells, but also show different shapes and properties. Different types of cells develop from the same genetic information, which is caused by differences in gene expression patterns in different cells. These gene expression patterns are influenced by the binding of specific transcription factors, chromatin and DNA state in the cell. Epigenetics is a branch of genetics that studies the heritable changes in gene expression without changing the nucleotide sequence of the gene. Epigenetics research includes DNA methylation...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/68
CPCC07K14/31C07K14/315C07K2319/00C12N9/22C12N9/90G01N33/6803
Inventor 冯速郑芳园徐晓昱张力军曹林
Owner VAZYME BIOTECH NANJING
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