CN109400714A

CN109400714A - The recombination fusion protein of antibody target and its application in epigenetics

Info

Publication number: CN109400714A
Application number: CN201811283285.9A
Authority: CN
Inventors: 冯速; 郑芳园; 徐晓昱; 张力军; 曹林
Original assignee: VAZYME BIOTECH (NANJING) Co Ltd
Current assignee: Nanjing novozan Biotechnology Co., Ltd
Priority date: 2018-10-26
Filing date: 2018-10-31
Publication date: 2019-03-01
Anticipated expiration: 2038-10-31
Also published as: CN109400714B

Abstract

The present invention discloses recombination fusion protein and its application in epigenetics of antibody target, fusion protein of the present invention includes the structure of ABP-L-DFE, wherein ABP is located at the N-terminal of fusion protein, indicate the structural domain with binding antibody function, L indicates link peptide, and DFE indicates the structural domain with DNA fragmentation function.Recombination fusion protein of the present invention can improve ChIP-seq technology, simplify operation, improve the quality of data, reduce operation difficulty, expanded application range, the especially application on precious, a small amount of cell.

Description

The recombination fusion protein of antibody target and its application in epigenetics

Technical field

The present invention relates to field of biotechnology, and in particular to the recombination fusion protein of antibody target and its in epigenetic Application in.

Background technique

For multicellular organism, such as the mankind, the inhereditary material (DNA) of each of which cell is all from identical Fertilized eggs, so its hereditary information for carrying, i.e. DNA sequence dna is all identical, but the cell in different tissues, such as skin and Liver cell, and show different form and property.Identical hereditary information but develops different types of cell, be due to Caused by the gene expression pattern difference of different cells.And these gene expression patterns are the knots by intracellular specific transcription factor What the state of conjunction, chromatin and DNA influenced.Epigenetics is exactly to study the case where nucleotide sequence of gene does not change Under, a science of heredity subdiscipline of heritable variation of gene expression.The content of epigenetics research includes DNA methyl Change (DNA methylation), genomic imprinting (genomic imprinting), maternal effect (maternal Effects), gene silencing (gene silencing), suspend mode transposon activation etc..

Chromatin immune co-precipitation (Chromatin Immunoprecipitation, ChIP) is a kind of common research The technology of epigenetics.The basic principle is that chromatin and the transcription factor interacted therewith and histone are passed through formaldehyde Equal substances crosslinking is got up, and chromatin is then broken into lesser segment by ultrasound, is added and is directed to specific transcription factor or spy The antibody for the histone very modified, by protein A/protein G microballoon or magnetic bead by antibody-transcription factor-dyeing Matter compound drags, and detects the DNA sequence dna combined with destination protein by PCR or the method for sequencing, and then study this A little action sites of the transcription factor in cell development or growth.Fast development recent years two generation sequencing technologies (NGS, Next generation sequencing) it is combined with tradition ChIP technology, the ChIP-sequencing developed is (referred to as ChIP-seq) technology has further expanded research application of the ChIP technology in terms of epigenetics.

However in traditional ChIP technology, the proteopexy that will first be interacted with DNA with formaldehyde is needed, at this Process is likely to result in some irrelevant proteins and is crosslinked, and forms nonspecific background (false positive).There are some force ratios Lesser transcription factor or since formaldehyde crosslinking is insufficient, effect when subsequent ultrasonic fragmentation DNA between albumen and DNA are broken It is bad, false negative is caused, and time-consuming (4 days) by tradition ChIP, it is also necessary to have the antibody of high-affinity, because low-affinity is anti- Body is difficult effectively by albumen and DNA compound " dragging ", this also further limits the use scope of ChIP.In addition, traditional ChIP-seq needs the compound of formaldehyde crosslinking cell and separation antibody and chromosome segment, then carries out building library and sequencing again, More sample cell is needed, the antibody of high-affinity is needed, needs long period and complicated operation, the resolution ratio of sequencing is not Height, sequencing depth is larger, at high cost.And the table for the precious sample such as be not suitable for embryonic development, stem cell and pathology sample See genetics research.

Summary of the invention

In response to the problems existing in the prior art, the purpose of the present invention is to provide a kind of recombination fusion protein of antibody target with And its application in epigenetics, recombination fusion protein of the present invention can improve ChIP-seq technology, simplify behaviour Make, improve the quality of data, reduces operation difficulty, expanded application range, the especially application on precious, a small amount of cell.

The purpose of the present invention can be achieved by the following measures:

The first aspect of the present invention provides a kind of fusion protein, and the fusion protein includes the structure of ABP-L-DFE, Middle ABP is located at the N-terminal of fusion protein, indicates that the structural domain with binding antibody function, L indicate link peptide, DFE expression has The structural domain of DNA fragmentation function.

Inventor has found the order of connection between DNA fragmentation enzyme (DFE) and antibody binding proteins (ABP) to two functions The effect of unit has larger impact.For example the antibody of ABP-L-Tn5m and Tn5m-L-ABP is bright in conjunction with just having with DNA cleavage activity Significant difference is different.Inventor is better than DFE-L-ABP by many experiments screening discovery, the general activity of ABP-L-DFE, it may be possible to because DNA fragmentation enzyme needs free C-terminal to play activity, as the C-terminal of Tn5m have to its dimerization and activation plays it is important Effect.So the present invention selects ABP-L-DFE fusion protein form.

Preferably, ABP of the present invention indicates the structural domain with the area binding antibody FC function.

In one embodiment, ABP indicates antibody binding proteins (antibody binding protein), preferably golden Staphylococcus aureus A albumen (ProteinA), streptococcal protein G (ProteinG), streptococcus L albumen (ProteinL) or other Albumen or polypeptide analog with binding antibody function.

Staphylococcal protein A (ProteinA) of the present invention can for overall length staphylococcal protein A, The partial function domain of staphylococcal protein A, staphylococcal protein A mutant, the overall length of tape label are golden yellow Staphylococcal protein A, tape label staphylococcal protein A partial function domain or tape label staphylococcus aureus A protein mutant.

Streptococcal protein G (ProteinG) of the present invention can be overall length streptococcal protein G, streptococcal protein G Partial function domain, streptococcal protein G mutant, the overall length streptococcal protein G of tape label, tape label streptococcal protein G portion Divide the streptococcal protein G mutant of functional domain or tape label.

In a kind of preferred embodiment, the amino acid sequence of ABP is selected from SEQ ID No.2 or SEQ ID No.10.

L of the present invention indicates link peptide (linker), and the length of L will affect the shape of fusion protein so as to right Fusion protein is equipped with significant impact to the DNA validity cut and cleavage, and the preferred L length of the present invention is 0-50 amino Acid, further preferred 2-50 amino acid.When L length is 0, indicate that ABP structural domain and DFE structural domain are connected directly.

L of the present invention can be the short linker (short linker, abbreviation sL) of 1-6 amino acid, can also be with For the moderate-length linker (medium linker, abbreviation mL) of the 7-20 amino acid or long linker of > 20 amino acid (longlinker, abbreviation lL).

L of the present invention can have found that flexible peptide linker may for flexible peptide linker or rigid connection peptide, inventor Cause to interact between two structural domains, influence respective function, preferably rigid connection peptide.

In a preferred embodiment, L sequence of the present invention is EAAAK (SEQ ID No.6), preferably just Property link peptide.

In another preferred embodiment, L sequence of the present invention is AEAAAKEAAAKEAAAKEAAAKALE AEAAAKEAAAKEAAAKEAAAKA (SEQ ID No.8), preferably rigid connection peptide.

In another preferred embodiment, the amino acid sequence of L of the present invention such as SEQ ID No.14 or SEQ Shown in ID No.18.

In one embodiment, DFE of the present invention indicates DNA fragmentation enzyme (DNA fragmenting enzyme)。

Preferably, DFE of the present invention is micrococcal nuclease (Mnase), DNA enzymatic 1 (DNAseI) or TnA transposase Family.

TnA swivel base enzyme family of the present invention is selected from overall length or the following transposase for partial function domain: Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9 or Tn10；The transposase can be with tape label or not tape label, can be with For mutant or non-mutant.

In a kind of preferred embodiment, TnA swivel base enzyme family of the present invention is Tn5m (5 variant of transposons), institute The Tn5m stated refers to saltant type Tn5 transposase disclosed in CN106754811A, such as any one of claim 1 or 3 or 5~7 institute The saltant type Tn5 transposase stated.Compared to the ChIP-seq that other DNA enzymatics mediate, the currently preferred fusion with Tn5m The additional advantage of albumen is to can be omitted the tedious steps such as segment separation, end reparation, the sorting of adjunction head, segment, simplifies and builds The complexity of library operation, further improves precision and resolution ratio.

In a kind of preferred embodiment, the amino acid sequence of DFE of the present invention such as SEQ ID No.4, SEQ ID Shown in No.12 or SEQ ID No.16.

In a kind of preferred embodiment, fusion protein of the present invention is selected from following any: ABP-L-DFE1, ABP- L-DFE2、ABP-L-DFE3、ABP-L-DFE4、ABP-L-DFE5、ABP-L-DFE6、ABP-L-DFE7、ABP-L-DFE8、ABP- L-DFE9、ABP-L-DFE10、ABP-L-DFE11、ABP-L-DFE12、ABP-L-DFE13、ABP-L-DFE14、ABP-L- DFE15。

The second aspect of the present invention provides a kind of for encoding the polynucleotides of above-mentioned fusion protein.

The polynucleotides of the above-mentioned fusion protein of coding of the present invention, can be DNA form or rna form.The present invention The polynucleotides can be prepared by any technology appropriate well known to those skilled in the art.The technology sees this The general description in field, such as " Molecular Cloning:A Laboratory guide " (J. Pehanorm Brooker, Science Press, 1995).Including but not It is limited to the methods of recombinant DNA technology, chemical synthesis.

Preferably, by optimization, the nucleotide sequence of the ABP of SEQ ID No.2 is encoded as shown in SEQ ID No.1.

Preferably, by optimization, the nucleotide sequence of the ABP of SEQ ID No.10 is encoded as shown in SEQ ID No.9.

Preferably, by optimization, the nucleotide sequence of the DFE of SEQ ID No.4 is encoded as shown in SEQ ID No.3.

Preferably, by optimization, the nucleotide sequence such as SEQ ID No.11 institute of the DFE of SEQ ID No.12 is encoded Show.

Preferably, by optimization, the nucleotide sequence such as SEQ ID No.15 institute of the DFE of SEQ ID No.16 is encoded Show.

Preferably, by optimization, the nucleotide sequence of the L of SEQ ID No.6 is encoded as shown in SEQ ID No.5.

Preferably, by optimization, the nucleotide sequence of the L of SEQ ID No.8 is encoded as shown in SEQ ID No.7.

Preferably, by optimization, the nucleotide sequence of the L of SEQ ID No.14 is encoded as shown in SEQ ID No.13.

Preferably, by optimization, the nucleotide sequence of the L of SEQ ID No.18 is encoded as shown in SEQ ID No.17.

The third aspect of the present invention provides a kind of expression vector, contains foregoing polynucleotides.

Expression vector of the present invention can be constructed using method well-known to those having ordinary skill in the art.These method packets Include recombinant DNA technology, DNA synthetic technology etc..The DNA of encoding said fusion protein can be effectively connected to polyclonal in carrier On site, to instruct mRNA to synthesize and then express albumen, or it to be used for homologous recombination.In preferable case of the invention, expression is carried PET28a can be used in body.

The fourth aspect of the present invention provides a kind of host cell, is converted by foregoing expression vectors or cloning vector.

In a kind of preferred embodiment, E. coli expression strains BL21 (DE3) is can be used in the host cell.

The fifth aspect of the present invention provides a kind of method for preparing foregoing fusion albumen, includes the following steps:

Synthesis or clone's target DNA sequence, construct the cloning vector containing target DNA sequence, and building contains target DNA The expression vector of the sequence containing target DNA is converted to prokaryotic host cell, is screened in growth medium by the expression vector of sequence Highly expressed high yielding cell sarain cultivates the overexpression cell line screened and expressed fusion protein, purifies and obtain from expression product Obtain the fusion protein.

In preferable case of the invention, pET28a is can be used in the expression vector.Large intestine bar can be used in the host cell Bacterium expresses bacterial strain BL21 (DE3).

The sixth aspect of the present invention provides application of the foregoing fusion albumen in building sequencing library.

The eighth aspect of the present invention provides application of the foregoing fusion albumen in epigenetics research.

The seventh aspect of the present invention provides application of the foregoing fusion albumen in research albumen-chromatin interaction, more specifically Be chromatin immune co-precipitation in application.

The present invention also provides a kind of application of preferred fusion protein in chromatin immune co-precipitation, including walk as follows It is rapid:

(1) it is burrowed on cell with detergent；

(2) antibody is added in conjunction with chromosome knob hop protein or methylation histone；

(3) fusion protein of the present invention is added in conjunction with antibody specificity, is formed ternary complex, is preferably cleaned again The fusion protein and antibody of non-specific binding are removed, to reduce background；

(4) addition metal ion activates fusion protein, fragmentation chromosome；

(5) for release target fragment to extracellularly, recycling continues library and sequencing after carrying out.

Preferably, metal ion described in step (4) is magnesium ion or calcium ion.

The detergent of step (1) of the present invention can be the common reagent for being used for cell perforation, as triton or it is high It is pungent.The concentration of the detergent is preferably 0.05%-0.005%.

In a preferred embodiment, it (such as methylates when for the more close albumen in conjunction with DNA or transcription factor Histone) site specific fragment when, step (3) select contain short linker fusion protein, such as ABP- of the invention L-DFE-1, ABP-L-DFE-3, ABP-L-DFE-5, ABP-L-DFE-7, ABP-L-DFE-10 or ABP-L-DFE-12；When being used for When the fixed point fragmentation of the macromoleculars transcription complex such as pol II, step (3) selects the fusion protein for containing long linker, such as originally ABP-L-DFE-2, ABP-L-DFE-4, ABP-L-DFE-6, ABP-L-DFE-9, ABP-L-DFE-11, ABP-L-DFE- of invention 14 or ABP-L-DFE-15.

With the help of the fusion protein of ABP-L-DFE form of the present invention, realize cell without formaldehyde crosslinking and The method of ChIP-seq is carried out under native state.It is mediated by this ABP-L-DFE fusion protein and ABP-L-DFE ChIP-seq technology (ADEACS, ABP-L-DFE assistant ChIP-seq), when may be implemented traditional ChIP-seq Between shortened to time half a day from 4 days, and since cell does not need the processing such as formaldehyde crosslinking, greatly reduce nonspecific back Scape improves the specificity and resolution ratio of cutting, and cell number required for primary experiment can be traditional down to 100 ChIP-seq needs at least 5,000,000 cells.The scope of application of ChIP-seq can be greatly expanded after reduction cell usage quantity, It such as can be used for studying in development epigenetics in embryo or stem cell and human pathology's sample, these precious samples Cell quantity is seldom, is not suitable for traditional ChIP-seq.

Due to reducing nonspecific background and cell number, sequencing depth needed for ADEACS is greatly reduced, and is reduced The cost of sequencing and requirement to data analysis.Further, since not needing additionally to separate antibody and chromosome segment, confrontation The affine force request of body is greatly reduced, and the target point protein that affinity of antibody was limited to before many can also carry out ChIP-seq Research, greatly expands the application range of ChIP-seq.

In addition, can when cutting DNA when the DFE of fusion protein of the present invention is preferably Tn5m transposase To add connector simultaneously, the target dna of belt lacing is used directly for expanding and building library and sequencing, does not need to hand over cell Connection processing, therefore the background of non-characteristic is lower, whole operation substantially reduced the operating time in 6 hours, made ChIP-seq can It is extensive to carry out with high throughput.

Detailed description of the invention

Fig. 1: DNase I/Mnase fusion protein mediated ChIP-seq technology schematic diagram.

Fig. 2: Tn5m fusion protein mediated ChIP-seq technology schematic diagram.

Fig. 3: fusion protein inducing expression electrophoretogram.

Fig. 4: rabbit igg antibody binding activity compares figure, and ordinate indicates OD450 value, and abscissa indicates different fusion proteins, Wherein, 1:ABP-L-DFE-1；2:ABP-L-DFE-2；3:ABP-L-DFE-3；4:ABP-L-DFE-4；5:ABP-L-DFE-5；6: ABP-L-DFE-6；7:ABP-L-DFE-7；8:ABP-L-DFE-8；9:ABP-L-DFE-1；9:ABP-L-DFE-1；10:ABP-L- DFE-10；11:ABP-L-DFE-11；12:ABP-L-DFE-12；13:ABP-L-DFE-13；14:ABP-L-DFE-14；15: ABP-L-DFE-15；16:DFE-L-ABP-1；17:DFE-L-ABP-2；18: negative control；19: blank control.

Fig. 5: mouse IgG antibody combination expression activitiy figure, ordinate indicate OD450 value, and abscissa indicates different fusion proteins, Wherein, 1:ABP-L-DFE-1；2:ABP-L-DFE-2；3:ABP-L-DFE-3；4:ABP-L-DFE-4；5:ABP-L-DFE-5；6: ABP-L-DFE-6；7:ABP-L-DFE-7；8:ABP-L-DFE-8；9:ABP-L-DFE-1；9:ABP-L-DFE-1；10:ABP-L- DFE-10；11:ABP-L-DFE-11；12:ABP-L-DFE-12；13:ABP-L-DFE-13；14:ABP-L-DFE-14；15: ABP-L-DFE-15；16:DFE-L-ABP-1；17:DFE-L-ABP-2；18: negative control；19: blank control.

The fragmentation activity proof diagram of Fig. 6: ABP-L-DFE-1 and ABP-L-DFE-3, wherein swimming lane 1:DNA molecular weight mark It is quasi-；The DNA that swimming lane 2:ABP-L-DFE-1 is interrupted；The DNA that swimming lane 3:ABP-L-DFE-3 is interrupted.

Fig. 7: ABP-L-DFE-5, the fragmentation expression activitiy figure of ABP-L-DFE-6 and ABP-L-DFE-8, wherein swimming lane 1:DNA molecular weight standard；The DNA that swimming lane 2:ABP-L-DFE-5 is interrupted；The DNA that swimming lane 3:ABP-L-DFE-8 is interrupted；Swimming lane 4: The DNA that ABP-L-DFE-6 is interrupted.

Fig. 8: ABP-L-DFE-11, the fragmentation activity ratio of ABP-L-DFE-14 and DFE-L-ABP-1, DFE-L-ABP-2 Compared with figure, wherein swimming lane 1:DNA molecular weight standard；Swimming lane 2: the DNA that the DFE-L-ABP-2 of embedding Adapter Mix is interrupted；Swimming Road 3: the DNA that the ABP-L-DFE-14 of embedding Adapter Mix is interrupted；Swimming lane 4: the DFE-L-ABP-1 of embedding Adapter Mix The DNA interrupted；Swimming lane 5: the DNA that the ABP-L-DFE-11 of embedding Adapter Mix is interrupted.

Fig. 9: ABP-L-DFE-10, the fragmentation activity of ABP-L-DFE-11, ABP-L-DFE-12 and ABP-L-DFE-14 Compare figure, wherein swimming lane 1:DNA molecular weight standard；Swimming lane 2: the DNA that the ABP-L-DFE-11 of embedding Adapter Mix is interrupted； Swimming lane 3: the DNA that the ABP-L-DFE-14 of embedding Adapter Mix is interrupted；Swimming lane 4: the ABP-L- of embedding Adapter Mix The DNA that DFE-12 is interrupted；Swimming lane 5: the DNA that the ABP-L-DFE-10 of embedding Adapter Mix is interrupted.

The fragmentation expression activitiy figure of Figure 10: ABP-L-DFE-13 and ABP-L-DFE-15, wherein swimming lane 1:DNA molecule Amount standard；The DNA that swimming lane 2:ABP-L-DFE-15 is interrupted；The DNA that swimming lane 3:ABP-L-DFE-13 is interrupted.

Figure 11: on 100 and 1000 cellular levels, cutting spectrogram and ENCODE in the site H3K27me3 (Encyclopedia of DNA Elements, DNA element encyclopedia) is referring to map.

Figure 12: cutting spectrogram and ENCODE the reference map on 100 cellular levels, in the site H3K27me3.

Figure 13: on 500 cellular levels, in cutting spectrogram and ENCODE the reference map of CTCF binding site.

Figure 14: on 500 cellular levels, cutting spectrogram and the ENCODE reference in pol II transcription complex site are schemed Spectrum.

Figure 15: on 200 cellular levels, cutting spectrogram and the ENCODE reference in pol II transcription complex site are schemed Spectrum.

Figure 16: cutting spectrogram and ENCODE the reference map on 100 cellular levels, in the site H3K4me3.

Specific embodiment

Below with reference to embodiment, the present invention will be further described, it should be understood that specific embodiment described herein is only To explain the present invention, it is not intended to limit the present invention, all letters under concept thereof of the invention to preparation method of the present invention Single improve belongs within protection scope of the present invention.Following example test method without specific conditions, usually according to The known approaches of this field.

The building of the clone building and two kinds of DFE-L-ABP of 1 15 kinds of difference ABP-L-DFE of embodiment

The present embodiment is prepared for the ABP-L-DFE and DFE-L-ABP of 17 different configurations, is respectively designated as ABP-L-DFE- 1、ABP-L-DFE-2、ABP-L-DFE-3、ABP-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.

1, all gene orders are artificial synthesized, use homologous recombination kit (ClonExpress II One Step Cloning Kit, Vazyme, C112) genetic fragment is cloned into respectively in pET28a carrier, construct recombinant expression carrier.

(1) it is named as the fusion protein of ABP-L-DFE-1, N-terminal structural domain is streptococcal protein G (Protein G) A part, C-terminal structural domain are DNAse I, and described two structural domains pass through the short connection of rigid connection peptide Linker 1.

The coding nucleotide sequence of a part of the Protein G is as shown in SEQ ID No.1.

The amino acid sequence of a part of the Protein G is as shown in SEQ ID No.2.

The coding nucleotide sequence of the DNAse I is as shown in SEQ ID No.3.

The amino acid sequence of the DNAse I is as shown in SEQ ID No.4.

The coding nucleotide sequence of the short rigid connection peptide Linker 1 is as shown in SEQ ID No.5.

The amino acid sequence of the short rigid connection peptide Linker 1 is as shown in SEQ ID No.6.

(2) it is named as the fusion protein of ABP-L-DFE-2, N-terminal structural domain is a part of Protein G, C-terminal structure Domain is DNAse I, and described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The coding nucleotide sequence of a part of the Protein G is the same as SEQ ID No.1.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the DNAse I is the same as SEQ ID No.3.

The amino acid sequence of the DNAse I is the same as SEQ ID No.4.

The length rigid connection peptide Linker 2 coding nucleotide sequence as shown in SEQ ID No.7, specifically:

The length rigid connection peptide Linker 2 amino acid sequence as shown in SEQ ID No.8, specifically:

(3) it is named as the fusion protein of ABP-L-DFE-3, N-terminal structural domain is staphylococcal protein A The a part of (Protein A), C-terminal structural domain are DNAse I, and described two structural domains pass through short rigid connection peptide Linker 1 connection.

The coding nucleotide sequence of a part of the Protein A as shown in SEQ ID No.9, specifically:

The amino acid sequence of a part of the Protein A as shown in SEQ ID No.10, specifically:

The coding nucleotide sequence of the DNAse I is the same as SEQ ID No.3.

The amino acid sequence of the DNAse I is the same as SEQ ID No.4.

The coding nucleotide sequence of the short rigid connection peptide Linker 1 is the same as SEQ ID No.5.

The amino acid sequence of the short rigid connection peptide Linker 1 is the same as SEQ ID No.6.

(4) it is named as the fusion protein of ABP-L-DFE-4, N-terminal structural domain is a part of Protein A, C-terminal structure Domain is DNAse I, and described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The coding nucleotide sequence of a part of the Protein A is the same as SEQ ID No.9.

The amino acid sequence of a part of the Protein A is the same as SEQ ID No.10.

The coding nucleotide sequence of the DNAse I is the same as SEQ ID No.3.

The amino acid sequence of the DNAse I is the same as SEQ ID No.4.

The coding nucleotide sequence of the rigid connection peptide Linker 2 of the length is the same as SEQ ID No.7.

The amino acid sequence of the rigid connection peptide Linker 2 of the length is the same as SEQ ID No.8.

(5) it is named as the fusion protein of ABP-L-DFE-5, N-terminal structural domain is a part of Protein G, C-terminal structure Domain is MNase, and described two structural domains pass through the short connection of rigid connection peptide Linker 1.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the MNase is as shown in SEQ ID No.11.

The amino acid sequence of the MNase is as shown in SEQ ID No.12.

(6) it is named as the fusion protein of ABP-L-DFE-6, N-terminal structural domain is a part of Protein G, C-terminal structure Domain is MNase, and described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the MNase is the same as SEQ ID No.11.

The amino acid sequence of the MNase is the same as SEQ ID No.12.

(7) it is named as the fusion protein of ABP-L-DFE-7, N-terminal structural domain is a part of Protein A, C-terminal structure Domain is MNase, and described two structural domains pass through the short connection of rigid connection peptide Linker 1.

The amino acid sequence of a part of the Protein A is the same as SEQ ID No.10.

The coding nucleotide sequence of the MNase is the same as SEQ ID No.11.

The amino acid sequence of the MNase is the same as SEQ ID No.12.

(8) it is named as the fusion protein of ABP-L-DFE-8, N-terminal structural domain is a part of Protein G, C-terminal structure Domain is MNase, and described two structural domains pass through the connection of rigid connection peptide Linker 3 of moderate-length.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the MNase is the same as SEQ ID No.11.

The amino acid sequence of the MNase is the same as SEQ ID No.12.

The coding nucleotide sequence of the rigid connection peptide Linker 3 of the moderate-length is as shown in SEQ ID No.13.

The amino acid sequence of the rigid connection peptide Linker 3 of the moderate-length is as shown in SEQ ID No.14.

(9) it is named as the fusion protein of ABP-L-DFE-9, N-terminal structural domain is a part of Protein A, C-terminal structure Domain is MNase, and described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The amino acid sequence of a part of the Protein A is the same as SEQ ID No.10.

The coding nucleotide sequence of the MNase is the same as SEQ ID No.11.

The amino acid sequence of the MNase is the same as SEQ ID No.12.

(10) it is named as the fusion protein of ABP-L-DFE-10, N-terminal structural domain is a part of Protein G, C-terminal knot Structure domain is Tn5m, and described two structural domains pass through the short connection of rigid connection peptide Linker 1.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the Tn5m is as shown in SEQ ID No.15.

The amino acid sequence of the Tn5m is as shown in SEQ ID No.16.

(11) it is named as the fusion protein of ABP-L-DFE-11, N-terminal structural domain is a part of Protein G, C-terminal knot Structure domain is Tn5m, and described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

(12) it is named as the fusion protein of ABP-L-DFE-12, N-terminal structural domain is a part of Protein A, C-terminal knot Structure domain is Tn5m, and described two structural domains pass through the short connection of rigid connection peptide Linker 1.

The amino acid sequence of a part of the Protein A is the same as SEQ ID No.10.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

(13) it is named as the fusion protein of ABP-L-DFE-13, N-terminal structural domain is a part of Protein G, C-terminal knot Structure domain is Tn5m, and described two structural domains pass through the connection of rigid connection peptide Linker 3 of moderate-length.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

The coding nucleotide sequence of the rigid connection peptide Linker 3 of the moderate-length is the same as SEQ ID No.13.

The amino acid sequence of the rigid connection peptide Linker 3 of the moderate-length is the same as SEQ ID No.14.

(14) it is named as the fusion protein of ABP-L-DFE-14, N-terminal structural domain is a part of Protein A, C-terminal knot Structure domain is Tn5m, and described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The amino acid sequence of a part of the Protein A is the same as SEQ ID No.10.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

(15) it is named as the fusion protein of ABP-L-DFE-15, N-terminal structural domain is a part of Protein G, C-terminal knot Structure domain is Tn5m, and described two structural domains pass through the long connection of flexible peptide linker Linker 4.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

The coding nucleotide sequence of the flexible peptide linker Linker 4 of the length is as shown in SEQ ID No.17.

The amino acid sequence of the flexible peptide linker Linker 4 of the length is as shown in SEQ ID No.18.

(16) it is named as the fusion protein of DFE-L-ABP-1, N-terminal structural domain is Tn5m, and C-terminal structural domain is Protein A part of A, described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

The amino acid sequence of a part of the Protein A is the same as SEQ ID No.10.

(17) it is named as the fusion protein of DFE-L-ABP-2, N-terminal structural domain is Tn5m, and C-terminal structural domain is ProteinG A part, described two structural domains pass through the long connection of rigid connection peptide Linker 2.

The coding nucleotide sequence of the Tn5m is the same as SEQ ID No.15.

The amino acid sequence of the Tn5m is the same as SEQ ID No.16.

The amino acid sequence of a part of the Protein G is the same as SEQ ID No.2.

2, the expression and purifying of fusion protein

By the correct 17 kinds of recombinant plasmid transformeds of sequence to the Efficiency Competent E. coli expression strains BL21 of preparation (DE3) in, in the LB culture medium of 1mL, 37 DEG C of culture 45min are coated on the LB solid containing screening antibiotic kanamycins On plate, 37 DEG C are incubated overnight 16h, select monoclonal bacterium in being inoculated in the 3mL liquid containing final concentration of 50 μ g/mL kanamycins In body LB culture medium, 37 DEG C are incubated overnight 16h.It is added in the culture bottle of 1 250mL within second day and contains final concentration of 50 μ g/ The 50mL LB liquid medium of mL kanamycins accesses the bacterium solution being incubated overnight according to the ratio of 1:100,37 DEG C of culture 4- 6h.The 1L LB liquid medium for containing final concentration of 50ug/mL kanamycins is added in the culture bottle of 1 3L, 250mL is trained The bacterium solution supported in bottle is respectively connected to according to 1:100 ratio, 37 DEG C of cultures, and when OD600 reaches 0.6 or so, IPTG induction is added Agent is to final concentration 1mM, 37 DEG C of inducing expression 4h.Fermentation obtains the genetic engineering bacterium bacterium solution of 4L express express target protein, uses high speed Centrifuge (Hunan instrument) centrifugation, 12800g 4 DEG C, 5min, abandon supernatant, collect thallus.With 200mL buffer (50mM Tris-HCl, 500mM NaCl, 2mM EDTA, pH 7.5) thallus is resuspended, high-pressure homogeneous crusher machine thallus (condition) is centrifuged, 12800g again, 4 DEG C, 60min, supernatant is collected, abandons precipitating.Supernatant is crossed with 0.22 μm of filter and filters out granule foreign, and cationic chromatographic purifying bacterium is used Body cracks supernatant, obtains the destination protein of purity is high.

Fig. 3 is the SDS-PAGE electrophoresis of 17 kinds of fusion proteins, and 1-15 respectively corresponds ABP-L-DFE-1-ABP-L-DFE- 15,16 be DFE-L-ABP-1, and 17 be DFE-L-ABP-2, and M is molecular weight reference substance (Marker), and each fusion protein molecule amount is just Really, consistent with expection.

The verifying of 2 engineered protein antibody binding activity of embodiment

Activity is combined using the antibody (Ig) of ELISA (enzyme linked immunosorbent assay (ELISA)) principle detection fusion albumen, with coating Fusion enzyme to be checked is diluted to 1-10 μ g/mL by buffer (50mM carbonate buffer solution, pH 9.6), difference fusion enzymes it is mole dense Degree is consistent, mixes well, addition ELISA Plate, 100 holes μ L/, three multiple holes of each fusion protein, without containing fusion in negative control Albumen, 2-8 DEG C of overnight incubation；It is washed 3 times with washing lotion (1*PBST)；It is added confining liquid (1*PBS, 5%BSA), 200 holes μ L/, 37 DEG C incubator is incubated for 2-3h；Enzyme labelled antibody (goat-anti rabbit or sheep anti mouse) is diluted to 5 μ g/ with dilution (1*PBST, 5%BSA) ML is added in each hole, 200 holes μ L/, after 37 DEG C of incubators are incubated for 1h, outwells reaction solution；It is washed 4 times with washing lotion (1*PBST)；Often TMB developing solution is added in hole, and 100 holes μ L/, after 37 DEG C of incubators are incubated for 10min, terminate liquid (2M H2SO4) is added in every hole, 50 μ L/ OD450 is detected with microplate reader immediately after termination in hole.By Fig. 4 and Fig. 5 it is found that the fusion protein of all transformations all have it is good (1-15 respectively corresponds ABP-L-DFE-1-ABP-L-DFE-15, and 16 be DFE-L-ABP-1, and 17 are for rabbit and mouse antibody binding activity DFE-L-ABP-2,18, No. 19 are negative control), the results showed that the fusion protein of all transformations all has good rabbit antibody knot Activity is closed, the rabbit-anti of different fusion proteins combines activity difference, and activity height is different, without evident regularity.

The DNA fragmentationization of embodiment 3ABP-L-DFE-1 (GsLDNase I) and ABP-L-DFE-3 (AsLDNase I) are living Property compares

The testing protein and 500ng BL21 genomic DNA for taking 5-10ng equimolar concentration are in Buffer A (20mM Tris-HCl, 10mM Ca2Cl, pH 8.0) in be uniformly mixed, 37 DEG C of incubations 10min, immediately loading race 1% Ago-Gel Electrophoresis, validating DNA fragmentation effect.It will be appreciated from fig. 6 that the DNA fragmentationization activity for merging the same DFE of different ABP is suitable, specifically To have merged the DNase I of Protein part A and the part Protein G respectively under equimolar concentration, DNA fragmentationization activity Quite.

Embodiment 4ABP-L-DFE-5 (GsLMNase), ABP-L-DFE-6 (GlLMNase) and ABP-L-DFE-8 (GmLMNase) DNA fragmentation expression activitiy

Take testing protein ABP-L-DFE-5 and ABP-L-DFE-6 and 500ng the BL21 gene of 5-10ng equimolar concentration Group DNA is uniformly mixed in Buffer A (20mM Tris-HCl, 10mM Ca2Cl, pH 8.0), 37 DEG C of incubation 10min, finally Pass through agarose gel electrophoresis validating DNA fragmentation effect.As shown in Figure 7, DNA fragmentation activity ABP-L-DFE-5 > ABP- The fragmentation activity of L-DFE-8 > ABP-L-DFE-6, ABP-L-DFE-5 are most strong, and ABP-L-DFE-6 is most weak, equimolar concentration Under, short Linker fusion protein activity is higher than long Linker fusion protein.

Embodiment 5ABP-L-DFE-11 (GlLTn5m), ABP-L-DFE-14 (AlLTn5m) and DFE-L-ABP-1 (Tn5m- LZ33), the DNA fragmentation expression activitiy of DFE-L-ABP-2 (Tn5m-LG)

Use the DNA fragmentationization activity of the TD501 kit verifying fusion protein of Vazyme.Adapter is prepared first Mix, by 1nmol Prime A (5'-phos-CTGTCTCTTATACACATCT-NH3-3') respectively with 1nmol PrimerB (5′-TAATACGACTCACTATAGGAGATGTGTATAAGAGACAG-3′)、1nmol PrimerC(PrimerE:5′-CCAA GGGGTTATGCTAGAGATGTGTATAAGAGACAG-3 ') it is mixed into AB and AC, it anneals, then mixes AB and AC equal respectively It is even, as Adapter Mix.By the fusion protein of equimolar concentration and 30 DEG C of incubation 1h of Adapter Mix, then with 600ng E. coli bl21 genomic DNA is uniformly mixed in Buffer A (20mM Tris-HCl, 10mM Ca2Cl, pH 8.0), and 55 As a result DEG C being incubated for 10min finally by agarose gel electrophoresis validating DNA fragmentation effect sees Fig. 8.

As shown in Figure 8, DNA fragmentation activity ABP-L-DFE-14 > ABP-L-DFE-11 > DFE-L-ABP-1 > DFE- The DNA fragmentation activity of L-ABP-2, i.e. ABP-L-DFE are better than DFE-L-ABP, and N-terminal activity of the ABP fusion in DFE exists than fusion C-terminal is some higher.Analysis reason may be activity to be played after dimerization because of Tn5m, and its polymerization site is in C-terminal, C-terminal fusion Albumen dimerization may be affected after albumen, to influence activity.

Embodiment 6ABP-L-DFE-10 (GsLTn5m), ABP-L-DFE-11 (GlLTn5m), ABP-L-DFE-12 (AsLTn5m) and the DNA fragmentation expression activitiy of ABP-L-DFE-14 (AlLTn5m)

Use the DNA fragmentationization activity of the TD501 kit verifying fusion protein of Vazyme.Adapter is prepared first Mix, by 1nmol Prime A (5'-phos-CTGTCTCTTATACACATCT-NH3-3') respectively with 1nmol PrimerB (5′-TAATACGACTCACTATAGGAGATGTGTATAAGAGACAG-3′)、1nmol PrimerC(PrimerE:5′-CCAA GGGGTTATGCTAGAGATGTGTATAAGAGACAG-3 ') it is mixed into AB and AC, it anneals, then mixes AB and AC equal respectively It is even, as Adapter Mix.By the fusion protein of equimolar concentration and 30 DEG C of incubation 1h of Adapter Mix, then with 600ng E. coli bl21 genomic DNA is uniformly mixed in Buffer A (20mM Tris-HCl, 10mM Ca2Cl, pH 8.0), and 55 As a result DEG C being incubated for 10min finally by agarose gel electrophoresis validating DNA fragmentation effect sees Fig. 9.

As shown in Figure 9, DNA fragmentation activity ABP-L-DFE-12 > ABP-L-DFE-10 > ABP-L-DFE-14 > ABP- The fusion protein activity of L-DFE-11, i.e., short Linker are higher than the fusion protein of long Linker, the result of the result and embodiment 4 Unanimously, Linker is shorter, and activity is higher.

The DNA fragmentation activity ratio of embodiment 7ABP-L-DFE-13 (GmLTn5m) and ABP-L-DFE-15 (GG4STn5m) Compared with

Use the DNA fragmentationization activity of the TD501 kit verifying fusion protein of Vazyme.Adapter is prepared first Mix, by 1nmol Prime A (5'-phos-CTGTCTCTTATACACATCT-NH3-3') respectively with 1nmol PrimerB (5′-TAATACGACTCACTATAGGAGATGTGTATAAGAGACAG-3′)、1nmol PrimerC(PrimerE:5′-CCAA GGGGTTATGCTAGAGATGTGTATAAGAGACAG-3 ') it is mixed into AB and AC, it anneals, then mixes AB and AC equal respectively It is even, as Adapter Mix.By the fusion protein of equimolar concentration and 30 DEG C of incubation 1h of Adapter Mix, then with 600ng E. coli bl21 genomic DNA is uniformly mixed in Buffer A (20mM Tris-HCl, 10mM Ca2Cl, pH 8.0), and 55 DEG C be incubated for 10min, finally by agarose gel electrophoresis validating DNA fragmentation effect, the result is shown in Figure 10.

As shown in Figure 10, DNA fragmentation activity ABP-L-DFE-13 > ABP-L-DFE-15, i.e. connection ABP and DFE are used Rigid Linker activity is better than flexibility Linker.

Application of the embodiment 8DNase I fusion protein in ADEACS (ABP-L-DFE assistant ChIP-seq)

By the 3T3 cell (mouse embryonic fibroblasts) of culture according to 100, the 1000 cell packing of every pipe, 1ml is used Tris buffer is resuspended.10ul concanavalin A magnetic bead (Concanavalin A-coated beads) is added into every pipe Adherent cell.It is washed cell 2 times with Tris buffer, removes cleaning solution.50ul anti-H3K27me3 (Cell is added Signaling Technology) (antibody is diluted with Tris buffer according to 1:100 antibody-solutions, and addition concentration is 0.05% Digoxin and 2mM EDTA) cell is resuspended, be added same amount of non-specific rabbit igg in negative control, mixed at room temperature 10 minutes Or 4 DEG C overnight.

After antibody incubation, liquid is removed on magnetic frame.With 1ml buffer solution A, (+0.05% ground of Tris buffer is high Pungent+2mM EDTA) washing cell is resuspended, liquid is removed on magnetic frame.

Every solencyte is separately added into the ABP-L-DFE-1 (GsLDNase I) and ABP-L-DFE-3 (AsLDNase of 50ul I) (buffer solution A is diluted to 0.1ug/ml) covers cell, gently flicks cell, mixes 10 minutes on vertical blending instrument, is centrifuged And with removing liquid on magnetic frame.150ul Tris buffer is added, cell is resuspended, 3ul 100mM calcium chloride solution is added, mixes It closes uniformly, activates the fixed point cleavage activity of DNase I in fusion protein, cut 20 minutes on ice, 6ul 250mM is added EDTA terminates reaction.37 DEG C of 10 minutes released dna compounds.

High speed centrifugation 5 minutes, centrifuge tube is placed on magnetic frame, target DNA is in centrifugation supernatant.Reagent is extracted with DNA The methods of box or phenol chloroform recycle the DNA in supernatant.With the ND606 kit DNA amplification of Vazyme and library is built, is used Illumina sequencing.Sequencing result is shown in Figure 11.

As shown in Figure 11, on 100 cellular levels, ABP-L-DFE-1 (GsLDNase I) and ABP-L-DFE-3 The cutting spectrogram and ENCODE (Encyclopedia of DNA Elements, DNA of (AsLDNase I) in the site H3K27me3 Element encyclopedia) it is closest referring to map, and also background is lower.And (ABP-L-DFE-1-Igg is added non-negative control Specific antibody) then there is no specificity to cut.

The Mnase fusion protein of 9 different length link peptide of embodiment answering in ADEACS (methylation histone site) With

By the MEF cell (mouse embryonic fibroblasts) of culture according to 100, the 1000 cell packing of every pipe, 1ml is used Tris buffer is resuspended.10ul concanavalin A magnetic bead adherent cell is added into every pipe.Cell is washed with Tris buffer 2 times, remove cleaning solution.50ul anti-H3K27me3 (Cell Signaling Technology) antibody-solutions (antibody is added Diluted with Tris buffer according to 1:100, the digoxin and 2mM EDTA that addition concentration is 0.05%) cell is resuspended, room temperature is mixed Close 10 minutes or 4 DEG C overnight.

After antibody incubation, liquid is removed on magnetic frame.With 1ml buffer solution A, (+0.05% ground of Tris buffer is high Pungent+2mM EDTA) washing cell is resuspended, it is then centrifuged again and removes liquid on magnetic frame.

It is separately added into the ABP-L-DFE-5 (GsLMnase), ABP-L-DFE-6 (GlLMnase) and ABP-L-DFE- of 50ul 8 (GmLMnase) (buffer solution A is diluted to 1ug/ml) cover cell, gently flick cell, and 10 points are mixed on vertical blending instrument Clock, with removing liquid on magnetic frame.150ul Tris buffer is added, cell is resuspended, 3ul 100mM magnesium chloride solution is added, mixes It closes uniformly, activation is bound to the DNA cleavage activity of Mnase in the fusion protein of target site, cuts 30 minutes on ice, is added 6ul 250mM EDTA terminates reaction.37 DEG C of 10 minutes released dna compounds.

High speed centrifugation 5 minutes, centrifuge tube is placed on magnetic frame, target DNA is in centrifugation supernatant.Reagent is extracted with DNA The methods of box or phenol chloroform recycle the DNA in supernatant.With the ND606 kit DNA amplification of Vazyme and library is built, is used Illumina sequencing.Sequencing result is shown in Figure 12.

As shown in Figure 12, on 100 cellular levels, ABP-L-DFE-5 (GsLMnase) cutting in the site H3K27me3 It is closest referring to map with ENCODE to cut spectrogram, and background is very low, and ABP-L-DFE-6 (GlLMnase) and ABP-L- DFE-8 (GmLMnase) is then incapable of efficiently cutting, and illustrates that the link peptide (L) among fusion protein cuts the fixed point of fusion protein It cuts and has a major impact, it may be possible to which, because the distance between histone and DNA of methylation are shorter, the fusion protein of long link peptide is logical When crossing antibody in conjunction with so far methylating on histone, since link peptide is too long, cause DNA fragmentation enzyme that cannot effectively connect with DNA Touching is cut from without causing specificity, so, for the DNA binding protein such as histone methylated site and CTCF, Need just to can be carried out fixed point specificity cutting with the fusion protein of shorter link peptide.

The Mnase fusion protein of 10 different length link peptide of embodiment is in ADEACS (CTCF, CCCTC binding factor) Using

By the MEF cell (mouse embryonic fibroblasts) of culture according to 500, the 5000 cell packing of every pipe, 1ml is used Tris buffer is resuspended.10ul concanavalin A magnetic bead adherent cell is added into every pipe.Cell is washed with Tris buffer 2 times, remove cleaning solution.Addition 50ul anti-CTCF (Millipore) antibody-solutions (antibody is with Tris buffer according to 1: 100 dilutions, the digoxin and 2mM EDTA that addition concentration is 0.05%) cell, mixed at room temperature 10 minutes or 4 DEG C of mistakes are resuspended Night.

It is separately added into the ABP-L-DFE-5 (GsLMnase), ABP-L-DFE-6 (GlLMnase) and ABP-L-DFE- of 50ul 8 (GmLMnase) (buffer solution A is diluted to 1ug/ml) cover cell, gently flick cell, and 10 points are mixed on vertical blending instrument Clock is centrifuged and with removing liquid on magnetic frame.3ul 100mM magnesium chloride solution is added, is uniformly mixed, activation is bound to target site Fusion protein in Mnase DNA cleavage activity, on ice cut 30 minutes, be added 6ul 250mM EDTA terminate reaction.37 DEG C 10 minutes released dna compounds.

High speed centrifugation 5 minutes, centrifuge tube is placed on magnetic frame, target DNA is in centrifugation supernatant.Reagent is extracted with DNA The methods of box or phenol chloroform recycle the DNA in supernatant.With the ND606 kit DNA amplification of Vazyme and library is built, is used Illumina sequencing.Sequencing result is shown in Figure 13.

As shown in Figure 13, on 500 cellular levels, ABP-L-DFE-5 (GsLMnase) cutting in CTCF binding site It is closest referring to map with ENCODE to cut spectrogram, and ABP-L-DFE-6 (GlLMnase) and ABP-L-DFE-8 (GmLMnase) Then be incapable of efficiently cutting it is similar with the site H3K27me3 in example 10, between this chromosome knob hop protein of CTCF and DNA Distance is smaller, needs the fusion enzyme of shorter link peptide that could effectively cut.

The Mnase fusion protein of 11 different length link peptide of embodiment is in ADEACS (pol II transcription factor complex) Application

By the MEF cell (mouse embryonic fibroblasts) of culture according to 500, the 5000 cell packing of every pipe, use 1mlTris buffer is resuspended.10ul concanavalin A magnetic bead adherent cell is added into every pipe.It is washed with Tris buffer Cell 2 times, remove cleaning solution.Addition 50ul anti-pol II (Abcam) antibody-solutions (antibody is with Tris buffer according to 1: 100 dilutions, the digoxin and 2mM EDTA that addition concentration is 0.05%) cell, mixed at room temperature 10 minutes or 4 DEG C of mistakes are resuspended Night.

It is separately added into the ABP-L-DFE-5 (GsLMnase), ABP-L-DFE-6 (GlLMnase) and ABP-L-DFE- of 50ul 8 (GmLMnase) (buffer solution A is diluted to 1ug/ml) cover cell, gently flick cell, and 10 points are mixed on vertical blending instrument Clock is centrifuged and with removing liquid on magnetic frame.150ul Tris buffer is added, cell is resuspended, 3ul 100mM magnesium chloride is added Solution is uniformly mixed, and activation is bound to the DNA cleavage activity of Mnase in the fusion protein of target site, cuts 30 points on ice Clock is added 6ul 250mM EDTA and terminates reaction.37 DEG C of 10 minutes released dna compounds.

High speed centrifugation 5 minutes, centrifuge tube is placed on magnetic frame, target DNA is in centrifugation supernatant.Reagent is extracted with DNA The methods of box or phenol chloroform recycle the DNA in supernatant.With the ND606 kit DNA amplification of Vazyme and library is built, is used Illumina sequencing.Sequencing result is shown in Figure 14.

As shown in Figure 14, on 500 cellular levels, ABP-L-DFE-6 (GlLMnase) is in pol II transcription complex The cutting spectrogram in site is closest referring to map with ENCODE, and ABP-L-DFE-5 (GsLMnase) and ABP-L-DFE-8 (GmLMnase) it is then incapable of efficiently cutting, pol different with the histone and the chromosome knobs such as CTCF hop protein of methylation II and multiple transcription factors form macromolecular transcription complex, it may be possible to which pol II transcription complex volume is larger, with pol II The fusion protein that compound and its antibody combine needs the fusion protein of sufficiently long link peptide that could contact with chromosomal DNA And pinpoint cutting.

Application of the 12 difference Tn5m fusion protein of embodiment in ADEACS (pol II transcription factor complex)

By the mES cell (mouse embryo stem cell) of culture according to the 200 cell packing of every pipe, with 1ml Tris buffer It is resuspended.10ul concanavalin A magnetic bead adherent cell is added into every pipe.It is washed cell 2 times with Tris buffer, removal is washed Wash liquid.50ul anti-pol II (Abcam) antibody-solutions are added, and (antibody is diluted with Tris buffer according to 1:100, is added dense Digoxin and 2mM EDTA of the degree for 0.05%) cell is resuspended, mixed at room temperature 10 minutes or 4 DEG C are overnight.

It is separately added into the ABP-L-DFE-10 (GsLTn5m) that 50ul has wrapped connector, ABP-L-DFE-11 (GlLTn5m),ABP-L-DFE-13(GmLTn5m),ABP-L-DFE-15(GG4STn5m),DEF-L-ABP-2(Tn5m-LG) (buffer solution A is diluted to 1ug/ml) covers cell, gently flicks cell, mixes 10 minutes on vertical blending instrument, and centrifugation is used in combination Liquid is removed on magnetic frame.Addition 150ul Tris buffer resuspension cell, addition 3ul 100mM magnesium chloride solution (or 40ul 5 × TTBL (Vazyme TD501)), it is uniformly mixed, the DNA cutting that activation is bound to Tn5m in the fusion protein of target site is lived Property, 37 DEG C are cut 30 minutes, and 6ul 250mM EDTA is added and terminates reaction.With the TD501 kit DNA amplification of Vazyme and build Library is sequenced and is analyzed with illumina.Sequencing result is shown in Figure 15.

As shown in Figure 15, on 200 cellular levels, ABP-L-DFE-11 (GlLTn5m) is in pol II transcription complex The cutting spectrogram in site and ENCODE are closest referring to map, and background is very clean, ABP-L-DFE-15 (GG4STn5m) its It is secondary, and then effect is poor by ABP-L-DFE-10 (GsLTn5m) and ABP-L-DFE-13 (GmLTn5m), this result and embodiment The cutting that Mnase fusion protein (ABP-L-DFE-6, ABP-L-DFE-5, ABP-L-DFE-8) mediates in 11 is similar, only longer Link peptide could effectively mediated fusion albumen pinpoint cutting on pol II transcription factor complex, short link peptide then effect ratio It is poor, wherein ABP-L-DFE-11 (GlLTn5m) and this rigid connection being of convenient length of ABP-L-DFE-14 (AlLTn5m) The effect of peptide fusion protein is best.DEF-L-ABP-2 (Tn5m-LG) does not generate special map, and signal is suitable with background, explanation Relative ranks between ABP and DFE have a significant impact the cutting performance of fusion protein (especially Tn5m fusion protein), ABP It is smaller (consistent with result in embodiment 5) to the activity interference of DFE in the N-terminal of fusion protein.

Compared with Dnase I and Mnase merge enzyme, Tn5m fusion protein is not required to due to that can add connector simultaneously in cutting DNA extraction and jointing are carried out, the loss of DNA is less, can carry out on 200 even lower quantitative cells special Property cutting, sensitivity is lower, using more convenient.

Application of the 13 difference Tn5m fusion protein of embodiment in ADEACS (methylation histone)

By the mES cell (mouse embryo stem cell) of culture according to the 100 cell packing of every pipe, with 1ml Tris buffer It is resuspended.10ul concanavalin A magnetic bead adherent cell is added into every pipe.It is washed cell 2 times with Tris buffer, removal is washed Wash liquid.Addition 50ul anti-H3K4me3 (Millipore) antibody-solutions (antibody is diluted with Tris buffer according to 1:100, Add digoxin and the 2mM EDTA that concentration is 0.05%) cell is resuspended, mixed at room temperature 10 minutes or 4 DEG C are overnight.

It is separately added into the ABP-L-DFE-10 (GsLTn5m) that 50ul has wrapped connector, ABP-L-DFE-11 (GlLTn5m), ABP-L-DFE-12 (AsLTn5m), ABP-L-DFE-13 (GmLTn5m), (buffer solution A is dilute by ABP-L-DFE-15 It releases to 1ug/ml) and covers cell, gently flick cell, mixed 10 minutes on vertical blending instrument, be centrifuged and got on magnetic frame Except liquid.Addition 150ul Tris buffer resuspension cell, addition 3ul 100mM magnesium chloride solution (or 5 × TTBL of 40ul (Vazyme TD501)), it is uniformly mixed, activation is bound to the DNA cleavage activity of Tn5m in the fusion protein of target site, and 37 DEG C are cut It cuts 30 minutes, 6ul 250mM EDTA is added and terminates reaction.With the TD501 kit DNA amplification of Vazyme and library is built, is used Illumina is sequenced and analyzes.Sequencing result is shown in Figure 16.

As shown in Figure 16, on 100 cellular levels, ABP-L-DFE-10 (GsLTn5m), ABP-L-DFE-12 (AsLTn5m) the cutting spectrogram and ENCODE in the site H3K4me3 are closest referring to map, and ABP-L-DFE-11 (GlLTn5m), the longer fusion enzyme of the link peptides such as ABP-L-DFE-13 (GmLTn5m), ABP-L-DFE-15 (GG4STn5m) is then The phenomenon that cannot effectively cutting with embodiment 9, is similar, illustrates that the fusion protein of long link peptide is not suitable for the histone etc. that methylates The specificity cutting in more close albumen (or transcription factor) site in conjunction with DNA.

In summary: the fusion protein (ABP-L-DFE-1,3,5,7,10,12) containing short link peptide is suitable for methylation The specific fragment in more close albumen (or transcription factor) site in conjunction with DNA such as histone；Contain melting for long link peptide Hop protein (ABP-L-DFE-2,4,6,9,11,14,15) is suitable for the fixed point fragmentation of the macromoleculars transcription complexes such as pol II. The fusion protein of different length link peptide is applied to different experiment purposes.These fusion proteins can be in less cell (100 A cell is less) on carry out In situcut, obtain high quality DNA piece segment information, clip size is suitable, is highly suitable for NGS analysis all obtains the sequencing result of high quality in the application of different cells, consistent or more referring to map with ENCODE It is excellent.The ABP-L-DFE and ADEACS (ABP-L-DFE assistant ChIP-seq) that we develop are in epigenetics Be widely used potentiality.

Sequence table

<110>Nanjing Vazyme Biotechnology Co., Ltd.

<120>recombination fusion protein of antibody target and its application in epigenetics

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 165

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

acctataaac tggtgatcaa cggcaaaacc ctgaaaggcg agaccaccac caaggcagtt 60

gatgcagaaa ccgccgagaa agccttcaag cagtatgcca acgataacgg cgtggatggt 120

gtgtggacct atgacgacgc caccaaaacc tttaccgtga ccgaa 165

<210> 2

<211> 55

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr

1 5 10 15

Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys Gln Tyr

20 25 30

Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp Asp Ala Thr

35 40 45

Lys Thr Phe Thr Val Thr Glu

50 55

<210> 3

<211> 780

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

ctgaaaattg ccgcctttaa catccgtacc ttcggcgaga ccaagatgag taacgcaacc 60

ctggccagct atattgtgcg catcgtgcgc cgctacgata tcgtgctgat tcaggaagtg 120

cgcgatagcc atctggtggc cgtgggcaag ctgctggact atctgaacca ggacgatccg 180

aacacctacc attacgtggt tagcgaaccg ctgggtcgca acagctacaa agagcgctac 240

ctgttcctgt tccgtcctaa caaagtgagc gtgctggaca cctatcagta tgacgatggc 300

tgcgaaagct gcggtaacga tagcttcagt cgcgaaccgg ccgtggtgaa attcagcagt 360

cacagcacca aggtgaagga attcgccatt gtggccctgc atagtgcccc gagcgatgcc 420

gtggccgaaa tcaatagcct gtacgacgtg tatctggacg tgcagcagaa gtggcactta 480

aacgacgtga tgctgatggg tgatttcaac gccgactgca gttacgtgac cagcagtcag 540

tggagcagca ttcgtctgcg cacaagcagc acctttcagt ggctgattcc ggatagcgcc 600

gataccaccg ccacaagcac caactgcgcc tatgatcgca ttgtggtggc cggtagtctg 660

ctgcagagta gtgtggtgcc gggcagcgca gcaccgtttg attttcaggc cgcttatggt 720

ctgagcaacg aaatggccct ggccattagc gatcattacc ctgtggaagt gaccctgacc 780

<210> 4

<211> 260

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 4

Leu Lys Ile Ala Ala Phe Asn Ile Arg Thr Phe Gly Glu Thr Lys Met

1 5 10 15

Ser Asn Ala Thr Leu Ala Ser Tyr Ile Val Arg Ile Val Arg Arg Tyr

20 25 30

Asp Ile Val Leu Ile Gln Glu Val Arg Asp Ser His Leu Val Ala Val

35 40 45

Gly Lys Leu Leu Asp Tyr Leu Asn Gln Asp Asp Pro Asn Thr Tyr His

50 55 60

Tyr Val Val Ser Glu Pro Leu Gly Arg Asn Ser Tyr Lys Glu Arg Tyr

65 70 75 80

Leu Phe Leu Phe Arg Pro Asn Lys Val Ser Val Leu Asp Thr Tyr Gln

85 90 95

Tyr Asp Asp Gly Cys Glu Ser Cys Gly Asn Asp Ser Phe Ser Arg Glu

100 105 110

Pro Ala Val Val Lys Phe Ser Ser His Ser Thr Lys Val Lys Glu Phe

115 120 125

Ala Ile Val Ala Leu His Ser Ala Pro Ser Asp Ala Val Ala Glu Ile

130 135 140

Asn Ser Leu Tyr Asp Val Tyr Leu Asp Val Gln Gln Lys Trp His Leu

145 150 155 160

Asn Asp Val Met Leu Met Gly Asp Phe Asn Ala Asp Cys Ser Tyr Val

165 170 175

Thr Ser Ser Gln Trp Ser Ser Ile Arg Leu Arg Thr Ser Ser Thr Phe

180 185 190

Gln Trp Leu Ile Pro Asp Ser Ala Asp Thr Thr Ala Thr Ser Thr Asn

195 200 205

Cys Ala Tyr Asp Arg Ile Val Val Ala Gly Ser Leu Leu Gln Ser Ser

210 215 220

Val Val Pro Gly Ser Ala Ala Pro Phe Asp Phe Gln Ala Ala Tyr Gly

225 230 235 240

Leu Ser Asn Glu Met Ala Leu Ala Ile Ser Asp His Tyr Pro Val Glu

245 250 255

Val Thr Leu Thr

260

<210> 5

<211> 15

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gaagctgccg ccaag 15

<210> 6

<211> 5

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 6

Glu Ala Ala Ala Lys

1 5

<210> 7

<211> 138

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gccgaggccg cagcaaaaga agctgcagca aaggaagccg ccgccaaaga agctgccgca 60

aaagcactgg aagcagaggc agccgccaaa gaggcagccg ccaaagaagc agcagcaaaa 120

gaggccgccg ccaaggcc 138

<210> 8

<211> 46

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala

20 25 30

Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

35 40 45

<210> 9

<211> 174

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gtggataata aatttaataa ggagcagcag aatgcctttt acgagatcct gcacctgccg 60

aacctgaatg aggagcagcg caacgcattc attcagagcc tgaaggatga cccgagtcag 120

agcgccaacc tgctggccga agcaaaaaag ctgaatgacg cccaggcacc gaaa 174

<210> 10

<211> 58

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 11

<211> 447

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

gcgaccagca ccaagaaact gcacaaggag ccggcgaccc tgatcaaagc gattgacggt 60

gataccgtga agctgatgta caaaggccag ccgatgacct tccgtctgct gctggttgac 120

accccggaga ccaagcaccc gaagaaaggt gttgagaaat atggcccgga agcgagcgcg 180

ttcaccaaga aaatggtgga aaacgcgaag aaaatcgagg ttgaatttga caagggtcag 240

cgtaccgata aatacggtcg tggcctggcg tacatttatg cggatggcaa gatggtgaac 300

gaagcgctgg ttcgtcaagg cctggcgaag gtggcgtacg tttataaacc gaacaacacc 360

cacgagcagc acctgcgtaa gagcgaggcg caagcgaaga aagaaaaact gaacatctgg 420

agcgaagaca acgcggatag cggccaa 447

<210> 12

<211> 149

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Ala Thr Ser Thr Lys Lys Leu His Lys Glu Pro Ala Thr Leu Ile Lys

1 5 10 15

Ala Ile Asp Gly Asp Thr Val Lys Leu Met Tyr Lys Gly Gln Pro Met

20 25 30

Thr Phe Arg Leu Leu Leu Val Asp Thr Pro Glu Thr Lys His Pro Lys

35 40 45

Lys Gly Val Glu Lys Tyr Gly Pro Glu Ala Ser Ala Phe Thr Lys Lys

50 55 60

Met Val Glu Asn Ala Lys Lys Ile Glu Val Glu Phe Asp Lys Gly Gln

65 70 75 80

Arg Thr Asp Lys Tyr Gly Arg Gly Leu Ala Tyr Ile Tyr Ala Asp Gly

85 90 95

Lys Met Val Asn Glu Ala Leu Val Arg Gln Gly Leu Ala Lys Val Ala

100 105 110

Tyr Val Tyr Lys Pro Asn Asn Thr His Glu Gln His Leu Arg Lys Ser

115 120 125

Glu Ala Gln Ala Lys Lys Glu Lys Leu Asn Ile Trp Ser Glu Asp Asn

130 135 140

Ala Asp Ser Gly Gln

145

<210> 13

<211> 45

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

gaagctgccg ccaaggaagc cgccgccaag gaagcagccg ccaag 45

<210> 14

<211> 15

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

<210> 15

<211> 1428

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

atgataactt ctgctcttca tcgtgcggcc gactgggcta aatctgtgtt ctcttcggcg 60

gcgctgggtg atcctcgccg tactgcccgc ttggttaacg tcgccgccca attggcaaaa 120

tattctggta aatcaataac catctcatca gagggtagta aagccgcgca ggaaggcgct 180

taccgattta tccgcaatcc caacgtttct gccgaggcga tcagaaaggc tggcgccatg 240

caaacagtca agttggctca ggagtttccc gaactgctgg ccattgagga aaccacctct 300

ttgagttatc gccaccaggt cgccgaagag cttggcaagc tgggctctat tcaggataaa 360

tcccgcggat ggtgggttca ctccgttctc ttgctcgagg ccaccacatt ccgcaccgta 420

ggattactgc atcaggagtg gtggatgcgc ccggatgacc ctgccgatgc ggatgaaaag 480

gagagtggca aatggctggc agcggccgca actagccggt tacgcatggg cagcatgatg 540

agcaacgtga ttgcggtctg tgaacgcgaa gccgatattc atgcttatct gcaggacaaa 600

ctggcgcata acgagcgctt cgtggtgcgc tccaagcacc cacgcaagga cgtagagtct 660

gggttgtatc tgtacgacca tctgaagaac caaccggagt tgggtggcta tcagatcagc 720

attccgcaaa agggcgtggt ggataaacgc ggtaaacgta aaaatcgacc agcccgcaag 780

gcgagcttga gcctgcgcag tgggcgcatc acgctaaaac aggggaatat cacgctcaac 840

gcggtgctgg ccgaggagat taacccgccc aagggtgaga ccccgttgaa atggttgttg 900

ctgaccagcg aaccggtcga gtcgctagcc caagccttgc gcgtcatcga catttatacc 960

catcgctggc ggatcgacga gttccataag gcatggaaaa ccggagcagg agccgagagg 1020

caacgcatgg aggagccgga taatctggag cggatggtct cgatcctctc gtttgttgcg 1080

gtcaggctgt tacagctcag agaaagcttc acgccgccgc aagcactcag ggcgcaaggg 1140

ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat 1200

gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt 1260

agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga 1320

accggaattg ccagctgggg cgccctctgg gaaggttggg aagccctgca aagtaaactg 1380

gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatc 1428

<210> 16

<211> 476

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 16

Met Ile Thr Ser Ala Leu His Arg Ala Ala Asp Trp Ala Lys Ser Val

1 5 10 15

Phe Ser Ser Ala Ala Leu Gly Asp Pro Arg Arg Thr Ala Arg Leu Val

20 25 30

Asn Val Ala Ala Gln Leu Ala Lys Tyr Ser Gly Lys Ser Ile Thr Ile

35 40 45

Ser Ser Glu Gly Ser Lys Ala Ala Gln Glu Gly Ala Tyr Arg Phe Ile

50 55 60

Arg Asn Pro Asn Val Ser Ala Glu Ala Ile Arg Lys Ala Gly Ala Met

65 70 75 80

Gln Thr Val Lys Leu Ala Gln Glu Phe Pro Glu Leu Leu Ala Ile Glu

85 90 95

Glu Thr Thr Ser Leu Ser Tyr Arg His Gln Val Ala Glu Glu Leu Gly

100 105 110

Lys Leu Gly Ser Ile Gln Asp Lys Ser Arg Gly Trp Trp Val His Ser

115 120 125

Val Leu Leu Leu Glu Ala Thr Thr Phe Arg Thr Val Gly Leu Leu His

130 135 140

Gln Glu Trp Trp Met Arg Pro Asp Asp Pro Ala Asp Ala Asp Glu Lys

145 150 155 160

Glu Ser Gly Lys Trp Leu Ala Ala Ala Ala Thr Ser Arg Leu Arg Met

165 170 175

Gly Ser Met Met Ser Asn Val Ile Ala Val Cys Glu Arg Glu Ala Asp

180 185 190

Ile His Ala Tyr Leu Gln Asp Lys Leu Ala His Asn Glu Arg Phe Val

195 200 205

Val Arg Ser Lys His Pro Arg Lys Asp Val Glu Ser Gly Leu Tyr Leu

210 215 220

Tyr Asp His Leu Lys Asn Gln Pro Glu Leu Gly Gly Tyr Gln Ile Ser

225 230 235 240

Ile Pro Gln Lys Gly Val Val Asp Lys Arg Gly Lys Arg Lys Asn Arg

245 250 255

Pro Ala Arg Lys Ala Ser Leu Ser Leu Arg Ser Gly Arg Ile Thr Leu

260 265 270

Lys Gln Gly Asn Ile Thr Leu Asn Ala Val Leu Ala Glu Glu Ile Asn

275 280 285

Pro Pro Lys Gly Glu Thr Pro Leu Lys Trp Leu Leu Leu Thr Ser Glu

290 295 300

Pro Val Glu Ser Leu Ala Gln Ala Leu Arg Val Ile Asp Ile Tyr Thr

305 310 315 320

His Arg Trp Arg Ile Asp Glu Phe His Lys Ala Trp Lys Thr Gly Ala

325 330 335

Gly Ala Glu Arg Gln Arg Met Glu Glu Pro Asp Asn Leu Glu Arg Met

340 345 350

Val Ser Ile Leu Ser Phe Val Ala Val Arg Leu Leu Gln Leu Arg Glu

355 360 365

Ser Phe Thr Pro Pro Gln Ala Leu Arg Ala Gln Gly Leu Leu Lys Glu

370 375 380

Ala Glu His Val Glu Ser Gln Ser Ala Glu Thr Val Leu Thr Pro Asp

385 390 395 400

Glu Cys Gln Leu Leu Gly Tyr Leu Asp Lys Gly Lys Arg Lys Arg Lys

405 410 415

Glu Lys Ala Gly Ser Leu Gln Trp Ala Tyr Met Ala Ile Ala Arg Leu

420 425 430

Gly Gly Phe Met Asp Ser Lys Arg Thr Gly Ile Ala Ser Trp Gly Ala

435 440 445

Leu Trp Glu Gly Trp Glu Ala Leu Gln Ser Lys Leu Asp Gly Phe Leu

450 455 460

Ala Ala Lys Asp Leu Met Ala Gln Gly Ile Lys Ile

465 470 475

<210> 17

<211> 135

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

ggtggtggtg gcagtggtgg tggtggtagc ggtggtggtg gcagcggtgg tggtggcagt 60

ggtggtggtg gtagcggtgg tggtggcagc ggtggtggtg gcagtggtgg tggtggtagc 120

ggtggtggtg gcagc 135

<210> 18

<211> 45

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

35 40 45

Claims

1. a kind of fusion protein, the fusion protein includes the structure of ABP-L-DFE, and wherein ABP is located at the N-terminal of fusion protein, Indicate that the structural domain with binding antibody function, L indicate link peptide, DFE indicates the structural domain with DNA fragmentation function；L long Degree is 0-50 amino acid, further preferred 2-50 amino acid.

2. fusion protein according to claim 1, which is characterized in that the L be flexible peptide linker or rigid connection peptide, Preferably be rigidly connected peptide.

3. fusion protein according to claim 1, which is characterized in that the L be 1-6 amino acid short linker, The long linker of moderate-length linker or > 20 amino acid of 7-20 amino acid；Preferably, L sequence such as SEQ ID No.6, SEQ ID No.8, shown in SEQ ID No.14 or SEQ ID No.18.

4. fusion protein according to claim 1, which is characterized in that ABP is staphylococcal protein A, streptococcus G Albumen, streptococcus L albumen or other albumen or polypeptide analog with binding antibody function；Wherein staphylococcus aureus A albumen is overall length staphylococcal protein A, the partial function domain of staphylococcal protein A, staphylococcus aureus A protein mutant, the overall length staphylococcal protein A of tape label, tape label staphylococcal protein A part The staphylococcal protein A mutant of functional domain or tape label；The streptococcal protein G be overall length streptococcal protein G, The partial function domain of streptococcal protein G, streptococcal protein G mutant, the overall length streptococcal protein G of tape label, tape label chain The partial function domain of coccus G-protein or the streptococcal protein G mutant of tape label；Preferably, the amino acid sequence of ABP is selected from SEQ ID No.2 or SEQ ID No.10；The DFE is micrococcal nuclease, DNA enzymatic 1 or TnA swivel base enzyme family；It is described TnA transposase be preferably Tn5m.

5. fusion protein according to claim 1, which is characterized in that the amino acid sequence of the DFE such as SEQ ID Shown in No.4, SEQ ID No.12 or SEQ ID No.16.

6. a kind of fusion protein, selected from following any: ABP-L-DFE1, ABP-L-DFE2, ABP-L-DFE3, ABP-L-DFE4, ABP-L-DFE5、ABP-L-DFE6、ABP-L-DFE7、ABP-L-DFE8、ABP-L-DFE9、ABP-L-DFE10、ABP-L- DFE11、ABP-L-DFE12、ABP-L-DFE13、ABP-L-DFE14、ABP-L-DFE15。

7. a kind of for encoding the polynucleotides of claim 1~6 fusion protein.

8. polynucleotides according to claim 7, sequence is selected from following any:

The nucleotide sequence of the ABP of SEQ ID No.2 is encoded as shown in SEQ ID No.1；

The nucleotide sequence of the ABP of SEQ ID No.10 is encoded as shown in SEQ ID No.9；

The nucleotide sequence of the DFE of SEQ ID No.4 is encoded as shown in SEQ ID No.3；

The nucleotide sequence of the DFE of SEQ ID No.12 is encoded as shown in SEQ ID No.11；

The nucleotide sequence of the DFE of SEQ ID No.16 is encoded as shown in SEQ ID No.15；

The nucleotide sequence of the L of SEQ ID No.6 is encoded as shown in SEQ ID No.5；

The nucleotide sequence of the L of SEQ ID No.8 is encoded as shown in SEQ ID No.7；

The nucleotide sequence of the L of SEQ ID No.14 is encoded as shown in SEQ ID No.13；

The nucleotide sequence of the L of SEQ ID No.18 is encoded as shown in SEQ ID No.17.

9. a kind of expression vector contains polynucleotides as claimed in claim 7.

10. a kind of host cell is converted by expression vector as claimed in claim 9.

11. host cell described in expression vector described in claim 1~6 fusion protein, claim 9 or claim 10 Application in building sequencing library, epigenetics research or research albumen-chromatin interaction.

12. a kind of application of claim 1~6 fusion protein in chromatin immune co-precipitation, includes the following steps:

(1) it is burrowed on cell with detergent；

(3) fusion protein of the present invention is added in conjunction with antibody specificity, is formed ternary complex, is preferably cleaned removal again The fusion protein and antibody of non-specific binding, to reduce background；

(4) addition metal ion activates fusion protein, fragmentation chromosome；

13. application according to claim 12, which is characterized in that metal ion described in step (4) be magnesium ion or Calcium ion；The detergent of step (1) is triton or digoxin.

14. application according to claim 12, which is characterized in that when for the more close methylation group egg in conjunction with DNA When the specific fragment of Bai Weidian, step (3) selects the fusion protein for containing short linker；Turn when for pol II macromolecular When recording the fixed point fragmentation of compound, step (3) selects the fusion protein for containing long linker.