WO2021155712A1 - Application of substance for detecting mecp2 mutation in detecting whether mecp2 mutation is pathogenic mutation and selecting drug - Google Patents

Abstract

Provided is an application of a substance for detecting an MeCP2 mutation in detecting whether the MeCP2 mutation is a pathogenic mutation and selecting a drug. It is found that MeCP2 can be bound with nucleosome beads to generate a phase change, and both an MeCP2 pathogenic truncation mutation and a point mutation cause an abnormal phase change between MeCP2 and the nucleosome beads; an intracellular experimental result shows that only a pathogenic point mutation can cause the abnormal phase change, but a non-pathogenic point mutation has no obvious influence on the phase change, and it is further proved that the abnormal phase change is closely related to a disease caused by the MeCP2 mutation. Therefore, the reversion of the abnormal phase change is likely to become a new idea and direction for treating a related disease, a regulation substance capable of reversing the abnormal phase change can be obtained by means of high throughput screening and be applied to the development of a potential targeted drug, and the strategy can also provide reference for treating other diseases related to the abnormal phase change.

Description

Application of substances detecting MeCP2 mutations in detecting whether MeCP2 mutations are pathogenic mutations and screening drugs Technical field

The invention relates to the application of a substance for detecting a MeCP2 mutation in the field of biotechnology in detecting whether the MeCP2 mutation is a pathogenic mutation and screening drugs.

Background technique

"Phase transition" as a property of matter has long been well known in the physical world and daily life. In recent years, scientists have gradually discovered that the mechanism of phase transition (or phase separation) is also widely present in biological cells, and in the time and space of the cell life cycle. Regulation and other aspects exercise important biological functions.

Current studies have found that when the multivalent macromolecules in the solution interact with their multivalent ligands, larger complexes are easily produced, and the solubility of the latter generally decreases, thereby separating from the ordinary solution to form a complex It is an independent liquid phase enriched by substances. This transformation process is called "liquid-liquid phase change" (referred to as "phase change") (LLPS, liquid-liquid phase separation), and the resulting liquid phase is the phase change part Or phase change phase, the rest are non-phase change part or ordinary phase. Among them, the multivalent valence refers to the number of binding regions contained in a macromolecule or its ligand that can interact with each other. For protein interactions, multivalent proteins and their multivalent ligands will also undergo a "liquid-liquid separation phase transition" phenomenon in vitro, that is, a normal solution phase and a protein-rich viscous liquid can be produced Mutually. Under the microscope, it can be seen that the protein-enriched liquid phase contains a large number of small droplets (ie, phase change droplets), and the diameter of the droplets can reach micrometers or even larger.

Methyl CpG binding protein 2 (MeCP2) can bind methylated and unmethylated DNA, and has a stronger affinity for methylated DNA. MeCP2 is an important transcriptional regulatory factor. Abnormal expression or mutations (including point mutations and duplication or deletion mutations) will cause changes in the expression levels of genes regulated by MeCP2, causing abnormal development of neurons, axons, and dendrites. Causes serious neurological diseases. MeCP2 R106W mutation, R111G mutation, Y120D mutation, R133C mutation, P152R mutation, F157I mutation, T158M mutation, P225R mutation, R306C mutation, and early termination at position 168 (R168X), early termination at position 255 (R255X), and position 270 Early termination (R270X) and 294 premature termination (R294X) can lead to angel syndrome, patients with mental retardation, rigid limb movement, delayed speech development, and autism, among which the 106th, 111th, 120th, 157th, and 158th positions The mutations of R168X, R255X, and R270X truncation mutations cause more severe disease. The point mutations at positions 133 and 306, and the R294X truncation mutation have milder symptoms than the former.

Invention Disclosure

The technical problem to be solved by the present invention is how to detect whether the MeCP2 mutation is a pathogenic mutation, and how to screen drugs for treating and/or preventing diseases caused by the MeCP2 mutation.

To solve the above technical problems, the present invention first provides any of the following applications:

1. The application of the substance for detecting the MeCP2 mutation in the preparation, detection or auxiliary detection of whether the MeCP2 mutation is a pathogenic mutation product;

2. The application of the substance detecting the MeCP2 mutation in detecting or assisting the detection of whether the MeCP2 mutation is a pathogenic mutation;

3. Application of detecting MeCP2 mutations in screening or auxiliary screening of drugs for treatment and/or prevention of diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation;

4. The application of the substance detecting the MeCP2 mutation in the preparation, screening or auxiliary screening of drug products for the treatment and/or prevention of diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation;

5. The application of a substance for detecting MeCP2 mutations in the preparation of products for testing or assisting in detecting whether the MeCP2 mutations are pathogenic;

6. The application of the substance for detecting the MeCP2 mutation in the detection or auxiliary detection of whether the MeCP2 mutation is pathogenic;

7. The application of substances detecting MeCP2 mutations in the preparation of products for diagnosis or auxiliary diagnosis of diseases caused by the MeCP2 mutations;

8. The application of substances detecting MeCP2 mutations in the diagnosis or auxiliary diagnosis of diseases caused by the MeCP2 mutations.

In the above application, the substance for detecting the MeCP2 mutation may include the MeCP2 mutant protein in which the MeCP2 mutation occurs and the following a1) or a2) or a3) or a4):

a1) DNA fragments;

a2) Nucleosome beads;

a3) Substances used to prepare nucleosome beads;

a4) Cells containing beads of nucleosomes.

The nucleosome bead is a structure with a diameter of 11 nm formed by a DNA winding histone octamer.

In the above application, the DNA fragment in a1) can be a11) or a12) or a13):

a11) Nucleosome localization DNA;

a12) A DNA fragment containing the nucleosome positioning DNA;

a13) A DNA fragment formed by repeating n times with a12) as a repeating unit.

In the above application, the nucleosome localization DNA of a11) may specifically be a "601" sequence. The “601” sequence may be the 31st-177th position of the sequence 21 or the 24-170th position of the sequence 22.

a12) The DNA fragment may be positions 16-192 of sequence 21, or positions 9-185 or 186-362 or positions 1956-2132 of sequence 22.

In a13), n is any natural number greater than or equal to 1. Specifically, n can be 4-12. a13) The DNA fragment may specifically be the DNA fragment shown in sequence 21 or sequence 22 in the sequence listing.

The DNA fragments can be methylated or unmethylated DNA fragments.

In an embodiment of the present invention, the nucleosome bead is formed by a DNA fragment shown in sequence 21 or sequence 22 and a histone octamer.

In the above application, the substance mentioned in a3) can be a31) or a32):

a31) histone octamer and said DNA fragment;

a32) H2A, H2B, H3 and H4 and the DNA fragments.

In the above application, the substance for detecting MeCP2 mutation may also include MeCP2 unmutated protein.

The substance for detecting the mutation of MeCP2 may further include a reporter group, and the reporter group is used to label MeCP2 or its mutant protein.

The reporter group can be a dye (such as Alexa Fluor ^TM 568 NHS Ester (Succinimidyl Ester)) or a fluorescent protein (such as mCherry).

When the reporter group is the fluorescent protein, the fluorescent protein can be connected to the MeCP2 mutein or MeCP2 unmutated protein directly or through a connecting peptide.

The substance for detecting the MeCP2 mutation may be composed of the MeCP2 mutein and the above a1), may also be composed of the MeCP2 mutein and the above a2), may also be composed of the MeCP2 mutein and the above a3), or may be composed of the MeCP2 mutein and the above a3). The mutein is composed of the above a4), or may be composed of the MeCP2 mutein, the above a1) and the MeCP2 unmutated protein, or may be composed of the MeCP2 mutein, the above a2) and the MeCP2 unmutated protein, or may be composed of the MeCP2 mutant protein. Protein, the above a3) and MeCP2 unmutated protein, or the MeCP2 mutein, the above a4) and MeCP2 unmutated protein, and the MeCP2 mutein, the above a1), MeCP2 unmutated protein and the report The group composition can also be composed of the MeCP2 mutein, the above a2), the MeCP2 unmutated protein and the reporter group, or the MeCP2 mutein, the above a3), the MeCP2 unmutated protein, and the reporter group. The composition can also be composed of the MeCP2 mutein, the above a4), the MeCP2 unmutated protein and the reporter group.

Specifically, the method for detecting whether the MeCP2 mutation is a pathogenic mutation using the substance for detecting the MeCP2 mutation includes: mixing the MeCP2 mutant protein with the MeCP2 mutation and the nucleosome beads to obtain the system to be tested; mixing; The MeCP2 unmutated protein is beaded with the nucleosome to obtain a control system; compare the phase transition of the test system and the control system to determine whether the MeCP2 mutation is a pathogenic mutation: as the phase change of the test system The change ability is less than the phase change ability of the control system, and the MeCP2 mutation is or candidate is a pathogenic mutation; if the phase change ability of the test system is not less than the phase change ability of the control system, the MeCP2 mutation is Or the candidate is a non-pathogenic mutation.

A method for screening drugs for the treatment and/or prevention of diseases caused by the MeCP2 mutation by using the substance for detecting the MeCP2 mutation, wherein the MeCP2 mutation is a MeCP2 pathogenic mutation, and the method includes: mixing the drug to be tested and the occurrence of the MeCP2 mutation The MeCP2 mutant protein and the nucleosome bead beaded to obtain a test system; the MeCP2 mutant protein and the nucleosome bead beaded to obtain a control system; compare the phase transitions of the test system and the control system Determine whether the test drug is a drug that can treat and/or prevent the disease caused by the MeCP2 mutation: if the phase change ability of the test system is greater than the phase change ability of the control system, the test drug is or The candidate is a drug that can treat and/or prevent the disease caused by the MeCP2 mutation; if the phase change ability of the test system is not greater than the phase change ability of the control system, the test drug is not or the candidate is not Drugs for treating and/or preventing diseases caused by the MeCP2 mutation.

The method for detecting whether the MeCP2 mutation is pathogenic using the substance for detecting the MeCP2 mutation includes: mixing the MeCP2 mutant protein with the MeCP2 mutation and the nucleosome beads to obtain the system to be tested; mixing the MeCP2 unmutated protein with The nucleosomes are beaded to obtain a control system; compare the phase change of the test system and the control system to determine whether the MeCP2 mutation is pathogenic: if the phase change ability of the test system is less than that of the control system If the phase change ability of the test system is not less than the phase change ability of the control system, the MeCP2 mutation does not cause disease or the candidate does not cause disease.

In the above, the test system and the control system can both be an intracellular environment or a reaction buffer environment. The reaction buffer can be composed of a solute and a solvent. The solvent is water, and the solute and its content in the reaction buffer The concentrations in are 20mM HEPES and 100mM NaCl, pH 7.4.

The phase change ability may be reflected in whether a phase change occurs, the proportion of the phase change phase in the field of view in the field of view, the clarity of the phase separation boundary, and/or the signal strength of the reporter group in the phase change phase.

In the above application, the MeCP2 mutation may be a truncation mutation or a point mutation of MeCP2.

In an embodiment of the present invention, the truncation mutation of MeCP2 is an early termination at position 168, 255, 270 or 294 of MeCP2.

The point mutation of MeCP2 may be a mutation at positions 106, 111, 120, 133, 152, 157, 158, 225 or/and 306 of MeCP2. Specifically, the point mutation of MeCP2 may be R106W mutation, R111G mutation, Y120D mutation, R133C mutation, P152R mutation, F157I mutation, T158M mutation, P225R mutation or/and R306C mutation of MeCP2.

The disease caused by the MeCP2 mutation may be a dysplasia of the nervous system. Specifically, the disease caused by the MeCP2 mutation may be a dysplasia of the nervous system caused by a mutation of MeCP2, or a dysplasia of the nervous system containing a MeCP2 mutation. Furthermore, the disease caused by the MeCP2 mutation may be Angel syndrome.

The present invention also provides a method for detecting or assisting in detecting whether the MeCP2 mutation is a pathogenic mutation, and the method is completed by using the substance for detecting the MeCP2 mutation.

The present invention also provides a method for screening or assisting in screening drugs for the treatment and/or prevention of diseases caused by the MeCP2 mutation. The MeCP2 mutation is a MeCP2 pathogenic mutation; the method is completed by using the substance for detecting the MeCP2 mutation.

The present invention also provides a method for detecting or assisting in detecting whether the MeCP2 mutation is pathogenic, and the method is completed by using the substance for detecting the MeCP2 mutation.

The present invention also provides a method for diagnosing or assisting in diagnosing the diseases caused by the MeCP2 mutation, the disease caused by the MeCP2 mutation is a dysplasia of the nervous system, and the method is completed by using the substance for detecting the MeCP2 mutation.

The present invention also provides a product with any of the following functions, and the product is the substance for detecting MeCP2 mutation:

X1) Preparation and detection or auxiliary detection of whether the MeCP2 mutation is a pathogenic mutation product;

X2) Detect or assist in detecting whether the MeCP2 mutation is a pathogenic mutation;

X3) Screen or assist in screening drugs for treatment and/or prevention of diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation;

X4) Preparation of drug products for screening or auxiliary screening to treat and/or prevent diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation;

X5) Preparation and detection or auxiliary detection of whether the MeCP2 mutation is a pathogenic product;

X6) Detect or assist in detecting whether the MeCP2 mutation is pathogenic;

X7) Preparation of products for diagnosing or assisting in the diagnosis of diseases caused by the MeCP2 mutation;

X8) Diagnose or assist in the diagnosis of diseases caused by the MeCP2 mutation.

Description of the drawings

Figure 1 shows the phase transition analysis of MeCP2 and nucleosome beads. The upper left picture in A is control 1, the upper right picture is control 2, and the lower three pictures are the experimental group; the upper left picture in B picture is control 1, the upper right picture is control 3, and the lower three pictures are control 4. AI568 stands for Alexa Fluor ^TM 568 NHS Ester (Succinimidyl Ester).

Figure 2 shows the phase transition analysis of truncated MeCP2 and nucleosome beads. A is a schematic diagram of the structure of truncated MeCP2. B is the phase transition image analysis of different concentrations of full-length/truncated MeCP2 and 4×nucleosome beads. C is the quantitative analysis of the phase transition in Figure B. Area Occupied represents the percentage of the area occupied by the phase transition phase or precipitation in the field of view, and No LLPS represents the percentage of the area occupied by the precipitation with fluorescent signal formed by the phase transition in the field of view, and the value is relatively small; LLPS represents the percentage of the area occupied by the phase change phase in the field of view. In this figure, R168×, R255×, R270×, R294× and MeCP2 represent R168×-His, R255×-His, R270×-His, R294×-His and MeCP2-His, respectively, and 4×NA means 4×non Methylated nucleosomes beaded.

Figure 3 shows the phase transition analysis of pathogenic point mutation MeCP2 and methylated/unmethylated nucleosome beads. 12x Native Nucleosome Aray (DNA) means 12 x non-methylated nucleosome beads, and 12x Nudeosome Aray (5me-DNA) means 12 x methylated nucleosome beads. MeCP2 wt means MeCP2-His, MeCP2 R106W, MeCP2 R111G, MeCP2 Y120D, MeCP2 R133C, MeCP2 F157I, MeCP2 T158M, MeCP2 P225R, MeCP2 R306C respectively means MeCP2_R106W-His, MeCP2_R106W-His, MeCP2_R106W-His, MeCP2_R106W-His, MeCP2_R106W-His-HiCP1 His, MeCP2_T158M-His, MeCP2_P225R-His and MeCP2_R306C-His.

Figure 4 shows the effect of different point mutations of MeCP2 on intracellular phase transition. A is a schematic diagram of the site distribution of pathogenic point mutation MeCP2 (top) and non-pathogenic point mutation MeCP2 (bottom). B is the imaging analysis of cells transfected with pathogenic point mutation MeCP2 (left column) and non-pathogenic point mutation MeCP2 (right column). mCh-MeCP2, mCh-MeCP2_R106W, mCh-MeCP2_R111G, mCh-MeCP2_Y120D, mCh-MeCP2_R133C, mCh-MeCP2_P152R, mCh-MeCP2_F157I, mCh-MeCP2_K144R, mCh-MeCP2_MeCh2_MeCh-MeCP2_MeCh-MeCP2 MeCP2_R294P means the transfection of pmCherry-C1-MeCP2, pmCherry-C1-MeCP2_R106W, pmCherry-C1-MeCP2_R111G, pmCherry-C1-MeCP2_Y120D, pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_P152R, PMCherry-C1 -MeCP2_K144R, pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_T197M, pmCherry-C1-MeCP2_A201V, pmCherry-C1-MeCP2_R250H, pmCherry-C1-MeCP2_R294P cells; DAPI indicates the results of DAPI staining detection; Merger is the two images on the left; The merger.

Figure 5 shows the statistical analysis of the partition coefficient of mCherry fusion protein in the phase transition phase and the normal phase of the transfected cell line. The distribution coefficient shown in the figure is the ratio of the intensity of the mCherry fluorescence signal in the DAPI dark-stained area and the DAPI non-stained area of the transfected cell line. MeCP2, R106W, R111G, Y120D, R133C, P152R, F157I, K144R, P176R, T197M, A201V, R250H, R294P respectively represent the transfection of pmCherry-C1-MeCP2, pmCherry-C1-MeCP2_R106W, pmCherry-C1-Mepm2_R111G, Cherry-C1 -MeCP2_Y120D, pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_F157I, pmCherry-C1-MeCP2_K144R, pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_T201CV1-MeCP2Cherry-C1 , PmCherry-C1-MeCP2_R294P cells.

The best way to implement the invention

The present invention will be further described in detail below in conjunction with specific embodiments, and the examples given are only to illustrate the present invention, not to limit the scope of the present invention. The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence list is the 5'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3'terminal nucleus of the corresponding DNA/RNA Glycidic acid.

Example 1, MeCP2 mutation affects the phase transition state

1. Preparation of recombinant vector

1. Construction of the recombinant vector of the fusion protein of full-length MeCP2 and His tag

Synthesize the DNA molecules shown in positions 1-1466 in sequence 2 in the sequence table, and replace the DNA fragments (including the recognition sequences of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (Invitrogen) For the DNA molecule shown in position 1-1466 in sequence 2 in the sequence table, the recombinant vector pET-28a(+)-MeCP2-His was obtained, and pET-28a(+)-MeCP2-His contained the DNA fragment shown in sequence 2. , Can express the protein shown in sequence 1 (MeCP2 fused with His protein tag, denoted as MeCP2-His).

Among them, positions 1-6 and 1461-1466 of sequence 2 are the recognition sequences of NcoI and XhoI, respectively. The DNA molecule coding sequence shown in positions 3-1484 of sequence 2 is MeCP2-His shown in sequence 1, sequence 1 Positions 1-486 are the amino acid sequence of MeCP2, and positions 489-494 of Sequence 1 are the amino acid sequence of His-tag.

2. Construction of a recombinant vector of truncated MeCP2 and His tag fusion protein

1) Construction of a recombinant vector that terminates prematurely at position 168 of MeCP2

Artificially synthesized the DNA molecules shown in positions 1-509 in sequence 4 in the sequence listing, and replaced the DNA fragments between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with the sequence listing The DNA molecule shown in position 1-509 in sequence 4, to obtain the recombinant vector pET-28a(+)-R168×-His, pET-28a(+)-R168×-His contains the DNA fragment shown in sequence 4. It can express the protein shown in sequence 3 (the His protein tag is fused at positions 1-167 of MeCP2, denoted as R168×-His).

Among them, positions 1-6 and 504-509 of sequence 4 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-527 of sequence 4 are the R168×-His sequence shown in DNA molecule coding sequence 3. Position 1-167 of sequence 3 is position 1-167 of MeCP2, and position 170-175 of sequence 3 is the amino acid sequence of His-tag.

2) Construction of a recombinant vector that terminates prematurely at position 255 of MeCP2

Synthesize the DNA molecules shown in positions 1-770 in sequence 6 in the sequence list, and replace the DNA fragments between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with the sequence list In the DNA molecule shown at positions 1-770 in the sequence 6, the recombinant vector pET-28a(+)-R255×-His is obtained, and pET-28a(+)-R255×-His contains the DNA fragment shown in the sequence 6, It can express the protein shown in sequence 5 (the His protein tag is fused at positions 1-254 of MeCP2, denoted as R255×-His).

Among them, positions 1-6 and 765-770 of sequence 6 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-788 of sequence 6 are the R255×-His sequence shown in the coding sequence 5 of the DNA molecule, The 1-254th position of sequence 5 is the 1-254th position of MeCP2, and the 257-262th position of sequence 5 is the amino acid sequence of His-tag.

3) Construction of a recombinant vector that terminates prematurely at position 270 of MeCP2

Synthesize the DNA molecules shown in positions 1-815 in sequence 8 in the sequence listing, and replace the DNA fragments between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with the sequence listing In the DNA molecule shown in position 1-815 in sequence 8, the recombinant vector pET-28a(+)-R270×-His, pET-28a(+)-R270×-His contains the DNA fragment shown in sequence 8, It can express the protein shown in sequence 7 (the His protein tag is fused at positions 1-269 of MeCP2, denoted as R270×-His).

Among them, positions 1-6 and 810-815 of sequence 8 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-833 of sequence 8 are the R270×-His sequence shown in the coding sequence 7 of the DNA molecule, Position 1-269 of Sequence 7 is position 1-269 of MeCP2, and position 272-277 of Sequence 7 is the amino acid sequence of His-tag.

4) Construction of a recombinant vector that terminates prematurely at position 294 of MeCP2

Synthesize the DNA molecules shown in positions 1-887 in sequence 10 in the sequence listing, and replace the DNA fragments between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with the sequence listing In the DNA molecule shown at positions 1-887 in sequence 10, the recombinant vector pET-28a(+)-R294×-His was obtained, and pET-28a(+)-R294×-His contained the DNA fragment shown in sequence 10. The protein shown in sequence 9 can be expressed (the His protein tag is fused at positions 1-293 of MeCP2, denoted as R294×-His).

Among them, positions 1-6 and 882-887 of sequence 10 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-905 of sequence 10 are the R294×-His sequence shown in the coding sequence 9 of the DNA molecule, Position 1-293 of sequence 9 is position 1-293 of MeCP2, and position 296-301 of sequence 9 is the amino acid sequence of His-tag.

3. Construction of the recombinant vector of the fusion protein of point mutation MeCP2 and His tag

1) Recombinant vector expressing MeCP2_R106W-His

The 318th position of the DNA fragment shown in sequence 2 was replaced by a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 1). The sequence of the protein encoded by this DNA fragment is the 106th position of sequence 1 The amino acid sequence obtained by replacing the amino acid residue R with the tryptophan residue W, and the protein is designated as MeCP2_R106W-His.

Artificially synthesize DNA fragment 1, replace the DNA fragment between the NcoI and XhoI recognition sequences of pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 1, to obtain the recombinant vector pET-28a(+)-MeCP2_R106W -His, pET-28a(+)-MeCP2_R106W-His can express MeCP2_R106W-His.

2) Recombinant vector expressing MeCP2_R111G-His

The 333rd position of the DNA fragment shown in sequence 2 was replaced with G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 2). The sequence of the protein encoded by this DNA fragment is that the 111th position of sequence 1 is replaced by fine The amino acid sequence obtained by replacing the amino acid residue R with the glycine residue G, and the protein is designated as MeCP2_R111G-His.

Artificially synthesize DNA fragment 2, replace the DNA fragment between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 2, to obtain the recombinant vector pET-28a(+)-MeCP2_R111G -His, pET-28a(+)-MeCP2_R111G-His can express MeCP2_R111G-His.

3) Recombinant vector expressing MeCP2_Y120D-His

The 360th position of the DNA fragment shown in sequence 2 was replaced by T with G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 3). The sequence of the protein encoded by this DNA fragment is the 120th position of sequence 1 from The amino acid sequence obtained by replacing the amino acid residue Y with the aspartic acid residue D, and the protein is designated as MeCP2_Y120D-His.

Artificially synthesize DNA fragment 3, replace the DNA fragment between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 3 to obtain the recombinant vector pET-28a(+)-MeCP2_Y120D -His, pET-28a(+)-MeCP2_Y120D-His can express MeCP2_Y120D-His.

4) Recombinant vector expressing MeCP2_R133C-His

The 399th position of the DNA fragment described in sequence 2 was replaced by a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 4). The sequence of the protein encoded by this DNA fragment was changed from the 133rd position of sequence 1 to fine The amino acid sequence obtained by replacing the amino acid residue R with the cysteine residue C, and the protein is designated as MeCP2_R133C-His.

Artificially synthesized DNA fragment 4, and replaced the DNA fragment between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 4 to obtain the recombinant vector pET-28a(+)-MeCP2_R133C -His, pET-28a(+)-MeCP2_R133C-His can express MeCP2_R133C-His.

5) Recombinant vector expressing MeCP2_F157I-His

The 471th position of the DNA fragment shown in sequence 2 was replaced with A from T to obtain the point mutation MeCP2 DNA fragment (denoted as DNA fragment 5). The amino acid sequence obtained by replacing the alanine residue F with the isoleucine residue I, the protein is designated as MeCP2_F157I-His.

Artificially synthesized DNA fragment 5, and replaced the DNA fragment between the NcoI and XhoI recognition sequences of pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 5 to obtain the recombinant vector pET-28a(+)-MeCP2_F157I -His, pET-28a(+)-MeCP2_F157I-His can express MeCP2_F157I-His.

6) Recombinant vector expressing MeCP2_T158M-His

The 475th position of the DNA fragment shown in sequence 2 was replaced with T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 6). The sequence of the protein encoded by this DNA fragment is that the 158th position of The amino acid sequence obtained by replacing the amino acid residue T with the methionine residue M, and the protein is designated as MeCP2_T158M-His.

Artificially synthesize DNA fragment 6, replace the DNA fragment between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 6, to obtain the recombinant vector pET-28a(+)-MeCP2_T158M -His, pET-28a(+)-MeCP2_T158M-His can express MeCP2_T158M-His.

7) Recombinant vector expressing MeCP2_P225R-His

The 676th position of the DNA fragment shown in sequence 2 was replaced by C to G, and the DNA fragment of point mutation MeCP2 (denoted as DNA fragment 7) was obtained. The amino acid sequence obtained by replacing the amino acid residue P with the arginine residue R, and the protein is designated as MeCP2_P225R-His.

Artificially synthesized DNA fragment 7, and replaced the DNA fragment between the NcoI and XhoI recognition sequences of the pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 7, to obtain the recombinant vector pET-28a(+)-MeCP2_T158M -His, pET-28a(+)-MeCP2_T158M-His can express MeCP2_T158M-His.

8) Recombinant vector expressing MeCP2_R306C-His

The 918th position of the DNA fragment shown in sequence 2 was replaced with a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 8). The sequence of the protein encoded by this DNA fragment is that the 306th position of sequence 1 is replaced by fine The amino acid sequence obtained by replacing the amino acid residue R with the cysteine residue C, and the protein is designated as MeCP2_R306C-His.

Artificially synthesize DNA fragment 8, replace the DNA fragment between the NcoI and XhoI recognition sequences of pET-28a(+) vector (including the recognition sequences of NcoI and XhoI) with DNA fragment 8, to obtain the recombinant vector pET-28a(+)-MeCP2_R306C -His, pET-28a(+)-MeCP2_R306C-His can express MeCP2_R306C-His.

4. Construct a recombinant vector of mCherry and MeCP2 fusion protein

Synthesize the DNA molecule shown in sequence 12 in the sequence table, and replace the DNA fragment (including the recognition sequence of BspEI and HindIII) between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (Shenzhen Weitong Biotechnology Co., Ltd., article number zt333) The recombinant vector pmCherry-C1-MeCP2 is obtained for the DNA molecule shown in sequence 12 in the sequence table. pmCherry-C1-MeCP2 can express the protein shown in sequence 11 (mCherry fusion MeCP2, denoted as mCherry-MeCP2).

Among them, positions 1-6 of sequence 12 are the recognition sequence of BspEI, positions 1465-1470 are the recognition sequence of HindIII, positions 7-1464 are the coding gene sequence of MeCP2, and positions 1-236 of sequence 11 are mCherry's Amino acid sequence, the 239-724th position of sequence 11 is the amino acid sequence of MeCP2.

5. Construction of a recombinant vector of the fusion protein of mCherry and point mutation MeCP2

1) Recombinant vector expressing mCherry-MeCP2_R106W

The 316th position of the MeCP2 coding sequence in the sequence 12 (ie the 322nd position in the sequence 12) was replaced with a T to obtain a DNA fragment of the point mutation MeCP2 (denoted as DNA fragment 9). Artificially synthesized DNA fragment 9, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 9, to obtain the recombinant vector pmCherry-C1-MeCP2_R106W, pmCherry-C1-MeCP2_R106W It can express the fusion protein obtained by replacing the arginine residue R with the tryptophan residue W in the 106th position of MeCP2 in the mCherry-MeCP2 of step 4 (ie the 344th position of the sequence 11), and this protein is denoted as mCherry -MeCP2_R106W.

2) Recombinant vector expressing mCherry-MeCP2_R111G

The 331st position of the MeCP2 coding sequence in sequence 12 was replaced by A to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 10). Artificially synthesized DNA fragment 10, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 10 to obtain the recombinant vector pmCherry-C1-MeCP2_R111G, pmCherry-C1-MeCP2_R111G The fusion protein obtained by replacing the arginine residue R with the glycine residue G at position 111 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is denoted as mCherry-MeCP2_R111G.

3) Recombinant vector expressing mCherry-MeCP2_Y120D

The 358th position of the MeCP2 coding sequence in sequence 12 was replaced by T to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 11). Artificially synthesized DNA fragment 11, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 11 to obtain the recombinant vector pmCherry-C1-MeCP2_Y120D, pmCherry-C1-MeCP2_Y120D It can express the fusion protein obtained by replacing the tyrosine residue Y with the aspartic acid residue D at position 120 of MeCP2 in the mCherry-MeCP2 of step 4, and this protein is denoted as mCherry-MeCP2_Y120D.

4) Recombinant vector expressing mCherry-MeCP2_R133C

The 397th position of the MeCP2 coding sequence in sequence 12 was replaced with a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 12). Artificially synthesized DNA fragment 12, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 12 to obtain the recombinant vector pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_R133C The fusion protein obtained by replacing the arginine residue R with the cysteine residue C at position 133 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_R133C.

5) Recombinant vector expressing mCherry-MeCP2_P152R

The 455th position of the MeCP2 coding sequence in sequence 12 was replaced by C to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 13). Artificially synthesize DNA fragment 13, replace the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 13, to obtain the recombinant vector pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_P152R The fusion protein obtained by replacing the proline residue P with the arginine residue R at position 152 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_P152R.

6) Recombinant vector expressing mCherry-MeCP2_F157I

The 469th position of the MeCP2 coding sequence in sequence 12 was replaced with A from T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 14). Artificially synthesized DNA fragment 14, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 14, to obtain the recombinant vector pmCherry-C1-MeCP2_F157I, pmCherry-C1-MeCP2_F157I The fusion protein obtained by replacing the phenylalanine residue F with the isoleucine residue I at position 157 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and the protein is designated as mCherry-MeCP2_F157I.

7) Recombinant vector expressing mCherry-MeCP2_K144R

The 431st position of the MeCP2 coding sequence in sequence 12 was replaced by A to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 15). Artificially synthesized DNA fragment 15, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 15, to obtain the recombinant vector pmCherry-C1-MeCP2_K144R, pmCherry-C1-MeCP2_K144R The fusion protein obtained by replacing the lysine residue K with the arginine residue R at position 144 of MeCP2 in pmCherry-C1-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_K144R.

8) Recombinant vector expressing mCherry-MeCP2_P176R

The 527th position of the MeCP2 coding sequence in Sequence 12 was replaced by C to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 16). Artificially synthesized DNA fragment 16, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 16, to obtain the recombinant vector pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_P176R The fusion protein obtained by replacing the proline residue P with the arginine residue R at position 176 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_P176R.

9) Recombinant vector expressing mCherry-MeCP2_T197M

The 590th position of the MeCP2 coding sequence in sequence 12 was replaced with T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 17). Artificially synthesized DNA fragment 17, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 17, to obtain the recombinant vector pmCherry-C1-MeCP2_T197M, pmCherry-C1-MeCP2_T197M It can express the fusion protein obtained by replacing the threonine residue T with the methionine residue M at position 197 of MeCP2 in the mCherry-MeCP2 of step 4, and this protein is denoted as mCherry-MeCP2_T197M.

10) Recombinant vector expressing mCherry-MeCP2_A201V

The 602th position of the MeCP2 coding sequence in the sequence 12 was replaced with a T to obtain a DNA fragment of the point mutation MeCP2 (denoted as DNA fragment 18). Artificially synthesized DNA fragment 18, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 18 to obtain the recombinant vector pmCherry-C1-MeCP2_A201V, pmCherry-C1-MeCP2_A201V The fusion protein obtained by replacing the alanine residue A with the valine residue V at position 201 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_A201V.

11) Recombinant vector expressing mCherry-MeCP2_R250H

The 749th position of the MeCP2 coding sequence in sequence 12 was replaced by G to A, and a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 19) was obtained. Artificially synthesized DNA fragment 19, and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 19 to obtain the recombinant vector pmCherry-C1-MeCP2_R250H, pmCherry-C1-MeCP2_R250H It can express a fusion protein obtained by replacing the arginine residue R with histidine residue H at position 250 of MeCP2 in the mCherry-MeCP2 of step 4, and this protein is denoted as mCherry-MeCP2_R250H.

12) Recombinant vector expressing mCherry-MeCP2_R294P

The 881th position of the MeCP2 coding sequence in sequence 12 was replaced by G to C to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 20). The DNA fragment 20 was artificially synthesized, and the DNA fragment between the BspEI and HindIII recognition sequences of the pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) was replaced with DNA fragment 20 to obtain the recombinant vector pmCherry-C1-MeCP2_R294P, pmCherry-C1-MeCP2_R294P The fusion protein obtained by replacing the arginine residue R with the proline residue P at position 294 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_R294P.

6. Construction of histone recombinant vector

1) Recombinant vector expressing H2A

The DNA molecule shown in sequence 14 in the sequence listing was artificially synthesized, and the DNA fragment between the NdeI and BamHI recognition sequences of the pET-3a vector (Invitrogen) (including the recognition sequences of NdeI and BamHI) was replaced with the sequence shown in sequence 14 in the sequence listing DNA molecules, the recombinant vector pET-3a-H2A is obtained, and pET-3a-H2A can express the H2A protein shown in sequence 13.

Among them, positions 1-6 and 399-404 of sequence 14 are the recognition sequences of NdeI and BamHI, respectively, positions 3-396 of sequence 14 are the H2A sequence of Xenopus laevis, and sequence 13 is the H2A sequence Amino acid sequence.

2) Recombinant vector expressing H2B

The DNA molecule shown in sequence 16 in the sequence listing was artificially synthesized, and the DNA fragment between the XbaI and BamHI recognition sequences of the pET-3a vector (including the recognition sequences of XbaI and BamHI) was replaced with the DNA molecule shown in sequence 16 in the sequence listing, The recombinant vector pET-3a-H2B is obtained, and pET-3a-H2B can express the H2B protein shown in sequence 15.

Among them, positions 1-6 and 423-428 of sequence 16 are the recognition sequences of XbaI and BamHI, respectively, positions 40-408 of sequence 16 are the H2B sequence of Xenopus laevis, and sequence 15 is the amino acid sequence of H2B.

3) Recombinant vector expressing H3

The DNA molecule shown in sequence 18 in the sequence listing was artificially synthesized, and the DNA fragment between the XbaI and BlpI recognition sequences of the pET-3a vector (including the recognition sequences of XbaI and BlpI) was replaced with the DNA molecule shown in sequence 18 in the sequence listing, The recombinant vector pET-3a-H3 is obtained, and pET-3a-H3 can express the H3 protein shown in sequence 17.

Among them, positions 1-6 and 503-509 of sequence 18 are the recognition sequences of XbaI and BlpI, respectively, positions 42-452 are the H3 sequence of Xenopus laevis, and sequence 17 is the amino acid sequence of H3.

4) Recombinant vector expressing H4

The DNA molecule shown in sequence 20 in the sequence listing was artificially synthesized, and the DNA fragment between the recognition sequences of NdeI and BlpI of the pET-3a vector (including the recognition sequences of NdeI and BlpI) was replaced with the DNA molecule shown in sequence 20 in the sequence listing. The recombinant vector pET-3a-H4 is obtained, and pET-3a-H4 can express the H4 protein shown in sequence 19.

Among them, positions 1-6 and 368-374 of sequence 20 are the recognition sequences of NdeI and BlpI, respectively, positions 4-315 of sequence 20 are the H4 sequence of Xenopus laevis, and sequence 19 is the amino acid sequence of H4.

7. Construction of 177-4 recombinant vector

The DNA molecule shown in sequence 21 in the sequence listing was artificially synthesized, and the DNA fragment between the PstI and SalI recognition sequences of the EZ-T vector (Invitrogen) (including the recognition sequences of PstI and SalI) was replaced with the sequence shown in sequence 21 in the sequence listing DNA molecules to obtain the recombinant vector EZ-T-177-4.

Among them, positions 1-6 and 801-806 of sequence 21 are the recognition sequences of PstI and SalI, respectively, positions 7-12 and 727-732 are the recognition sequences of EcoRV, respectively, and positions 16-192 of sequence 21 are Position, position 193-369, position 370-546 and position 547-723 are 4 177bp DNA sequences respectively.

Among them, each 177bp sequence contains a 147bp "601" sequence, which can bind to histone octamer with high affinity. There are 15bp linker DNA at each end of the "601" sequence, making every two nuclei The interval between the bodies is 30 bp. Among them, the "601" sequence is also called the nucleosome positioning DNA sequence, which is a region where nucleosomes are more easily formed than other sequences.

8. Construction of 177-12 recombinant vector

The DNA molecule shown in sequence 22 in the sequence listing was artificially synthesized, and the DNA fragment between the PstI and ClaI recognition sequences of the pWM530 vector (Invitrogen) (including the recognition sequence of PstI and ClaI) was replaced with the DNA molecule shown in sequence 22 in the sequence listing , The recombinant vector pWM530-177-12 was obtained.

Among them, positions 1-6 and 2133-2138 of sequence 22 are the recognition sequences of PstI and ClaI, respectively, positions 6-11 and 2130-2135 are the recognition sequences of EcoRV, respectively, and positions 9-185 of sequence 22 are No. 186-362, No. 363-539, No. 540-716, No. 717-893, No. 894-1070, No. 1071-1247, No. 1248-1424, No. 1425-1601, The 1602-1778th, 1779-1955th, and 1956-2132th positions are 12 177bp DNA sequences, respectively.

Among them, each 177bp sequence contains a 147bp "601" sequence, which can bind to histone octamer with high affinity. There are 15bp linker DNA at each end of the "601" sequence, making every two nuclei The interval between the bodies is 30 bp. MeCP2 binds to the import and export ends of the linker DNA and has no sequence specificity.

2. Expression and purification of recombinant protein

1. Expression and purification of wild-type MeCP2 with His tag, truncated MeCP2 and point mutation MeCP2

Introduce the recombinant vectors from steps 1-3 in step one into E. coli BL21 (DE3) (Tiangen Biochemical Technology (Beijing) Co., Ltd.) to obtain recombinant bacteria containing different recombinant vectors, and express the recombinant bacteria according to the following steps. Purification of the target protein:

1) Inoculate the recombinant bacteria into 100 mL of LB liquid medium, culture at 37°C, 220 rpm, for 12 hours to obtain seed liquid.

2) After the completion of step 1), inoculate the seed solution into 6 750mL LB liquid mediums at a ratio of 1:50 and expand the culture (37℃, 220rpm). When the culture reaches OD=0.6, add IPTG (final concentration is 0.5mM) induce 4h.

3) After step 2) is completed, centrifuge the obtained culture solution, discard the supernatant, resuspend it in two 50ml tubes with 100ml PBS and centrifuge (4°C, 4000rpm, 30min), discard the supernatant.

4) After step 3) is completed, the obtained bacterial pellet is resuspended in a beaker with 80 mL of lysis buffer, and lysozyme (sigma, catalog number 10837059001) is added, with a final concentration of 0.2 mg/mL) placed at 4°C for 10 minutes.

5) After step 4 is completed, ultrasonically crush the obtained product, and then perform high-speed centrifugation on the crushed product (4° C., 18000 RPM, centrifugation for 30 minutes), and collect the supernatant.

6) After step 5) is completed, incubate the supernatant with 2mL His-beads at 4°C for 3h, transfer to the bio-rad empty column tube and collect the flow-through fluid.

7) After step 6) is completed, wash the beads with 100ml wash buffer.

8) After step 7) is completed, the protein is eluted with elution buffers of different imidazole concentrations, and the eluate is collected.

9) After step 8) is completed, SDS-PAGE gel electrophoresis is used to detect the purification result in the eluate, and the eluate containing the target protein obtained in step 8 is purified by Hitrap SP HP column.

10) After step 9) is completed, use low-salt buffer A and high-salt buffer B to elute the protein bound to the Hitrap SP HP column, collect the eluate, and use SDS-PAGE gel electrophoresis again to detect the purification results. The results showed that high purity target protein was obtained.

11) Dialysis the eluate containing the target protein obtained in step 10) (the dialysate is a reaction buffer). After the dialysis is completed, freeze the protein sample at -80°C for later use. The obtained protein samples are MeCP2-His solution, R168× -His solution, R255×-His solution, R270×-His solution, R294×-His solution, MeCP2_R106W-His solution, MeCP2_R111G-His solution, MeCP2_Y120D-His solution, MeCP2_R133C-His solution, MeCP2_F157I-His solution, MeCP2_T158M-His Solution, MeCP2_P225R-His solution and MeCP2_R306C-His solution.

Among them, the specific reagents are as follows:

Analysis buffer: 20mM Tris, 500mM NaCl, 20mM imidazole, 1mM PMSF, pH 7.4, the balance is water.

Wash buffer: 20mM Tris, 300mM NaCl, 20mM imidazole, pH 7.4, the balance is water.

Elution buffer: 20mM Tris, 300mM NaCl, 100mM/300mM/500mM/1M imidazole, pH 7.4, the balance is water.

Buffer A: 20mM Tris, 300mM NaCl, pH 7.4, the balance is water.

Buffer B: 20mM Tris, 1M NaCl, pH 7.4, the balance is water.

reaction buffer: 20mM HEPES, 100mM NaCl, pH 7.4, the balance is water.

2. Histone expression and purification

The recombinant vectors pET-3a-H2A, pET-3a-H2B, pET-3a-H3 and pET-3a-H4 obtained in step 1 were respectively introduced into E. coli BL21(DE3) Plys (Tiangen Biochemical Technology (Beijing) Co., Ltd.), Obtain recombinant strains BL21(DE3)Plys-pET-3a-H2A, BL21(DE3)Plys-pET-3a-H2B, BL21(DE3)Plys-pET-3a-H3 and BL21(DE3)Plys-pET-3a-H4 .

The proteins expressed by the above recombinant strains were expressed and purified according to the following methods:

1) Inoculate the above recombinant strains into liquid LB medium, and after overnight culture (37°C, 200rpm), transfer the obtained bacterial solution to 50mL liquid LB medium according to the volume ratio of 1:500-1:1000, and again Incubate overnight (37°C, 200 rpm).

2) After step 1), transfer the obtained bacterial solution to 750mL liquid LB medium at a volume ratio of 1:50 and cultivate until the OD600 reaches 0.5-0.6, then add IPTG to a final concentration of 0.5mM, and incubate at 37°C for 3hrs. Protein.

3) After step 2), centrifuge the obtained bacterial solution and collect the precipitate. Use 100ml 1×wash buffer for every 6 bottles of bacteria (50mM Tris, 100mM NaCl, 1mM EDTA, 5mM B-ME, PH8.0, the balance is water) The bacteria were resuspended and sonicated (5s/5s, 400W, 2-3 rounds of sonication, 99 times per round), then the sonicated product was ultracentrifuged (23000g for 20min), the supernatant was discarded, and the precipitate was collected.

4) After step 3), resuspend the obtained precipitate in 100ml 1×wash buffer containing 1%(v/v) TritonX-100 and then ultrasonically break it again (5s/5s, 400W, two rounds of ultrasound, 99 per round Times), then ultracentrifuge the sonicated product (20000g for 10min), discard the supernatant, and collect the precipitate.

5) Repeat step 4) once (or more times).

6) Resuspend the pellet obtained in step 5) with 100ml 1×wash buffer and centrifuge (centrifuge at 20000g for 10min), discard the supernatant, and collect the pellet.

7) Repeat step 6) once (or multiple times).

8) Use 30ml unfolding buffer (20mM Tris(PH8.0), 7M Guanidine hydrochloride, 5mM B-ME) to resuspend the precipitate obtained in step 7 (stir and dissolve at room temperature for 1hr), ultracentrifuge (23000g for 20min), collect the supernatant, Obtain a protein solution.

9) Gel electrophoresis to detect the purity and concentration of the protein in the protein solution obtained in step 8).

The above purification steps are carried out on ice unless otherwise specified.

After step 9), high purity H2A solution, H2B solution, H3 solution and H4 solution containing the target protein are obtained respectively.

3. Histone octamer assembly and purification

1. Assemble the octamer:

Take H2A solution, H2B solution, H3 solution and H4 solution into the dialysis bag. The amount of H2A, H2B, H3 and H4 are 4mg each, and then put in the dialysate—refolding buffer (2M NaCl, 10mM Tris, 1mM EDTA, pH 8.0, The remainder is water). After stirring for 12 hours at 4°C, the dialysate is changed again and dialyzed at 4°C for 24 hours.

2. Purified octamer:

After step 1, centrifuge the obtained protein sample (4°C, 21000g for 5 min), collect the supernatant and concentrate to 500μl, purify the histone octamer by gel filtration chromatography, the purification steps are as follows:

Balance the superdex 200 column: balance the superdex 200 column with refolding buffer (2M Nacl, 10mM Tris, 1mM EDTA, pH 8.0);

Loading: Load 500μl of sample into the column through the loading loop;

Separate octamers: use refolding buffer to send the sample to the column for separation, collect different effluents, and identify histone octamers by SDS-PAGE.

Fourth, prepare 177-4 and 177-12 DNA

Use the recombinant vectors EZ-T-177-4 and pWM530-177-12 of step one to prepare 177-4 and 177-12 DNA. Firstly, the two recombinant vectors are introduced into E. coli DH5α (Tiangen Biochemical Technology (Beijing) Co., Ltd. In ), two recombinant bacteria are obtained, and then the two recombinant bacteria are operated separately according to the following steps:

1. Vaccination

The recombinant bacteria were inoculated in 50ml of liquid LB medium and cultured overnight at 37°C to obtain a culture solution.

2. Expansion culture

The culture solution obtained in step 1 was transferred to 800ml liquid LB medium containing 100ug/ml ampicillin, and after culturing at 37°C for 4-5 hours, the culture temperature was increased to 42°C and the culture was continued for about 12-13 hours to obtain a culture solution.

3. Collect bacteria

Centrifuge the culture broth obtained in step 2 and collect the bacteria (4000rpm, 30min).

4. Broken bacteria

① Add 30ml of ice-cold S1 (25mM Tris, 50mM glucose, 10mM EDTA, pH8.0, the balance is water) to the bacteria collected in step 3, and shake the centrifuge tube to make a uniform bacterial suspension.

②Add 120ml of freshly prepared S2 (0.2M NaOH, 1% (mass ratio) SDS, water, room temperature) to the bacterial suspension of ①, gently invert to mix well, and place at room temperature for 5 minutes. At this time, the solution should be Clear and transparent, with high viscosity.

③After step ② is completed, carefully add 210ml of ice-cold S3 (3M potassium acetate, adjust the pH to 5.2 with acetic acid, the balance is water) to the obtained liquid, and shake the centrifuge tube in one direction so that the liquid phase does not separate. , The impurities are in the shape of egg flowers, put on ice for 10 minutes.

④ After step ③ is completed, centrifuge the obtained liquid at 4°C at 4000 rpm for 30 minutes, carefully aspirate the supernatant, filter through 4 layers of gauze to remove suspended impurities, and transfer to a 4L beaker to obtain solution 1.

5. Plasmid DNA recovery

① Add 0.52 times the volume of isopropanol to solution 1 obtained in step 4, mix well, and place at room temperature for ≥ 15 minutes.

②After step ① is completed, centrifuge the obtained liquid (15000g 15min), and recover the nucleic acid precipitate with the supernatant.

③After step ② is completed, rinse the sediment on the wall and bottom of the centrifuge tube with 70% (v/v) ethanol aqueous solution, centrifuge (15000g 15min, 4℃), discard the supernatant, and make the remaining ethanol evaporate clean (no alcohol smell and precipitation) It is damp and opaque) to obtain nucleic acid precipitation.

6.Remove RNA

① Use 100ml TE10/50 (10mM Tris, 50mM EDTA, pH8.0, the balance is water) to dissolve the nucleic acid precipitate obtained in step 5 into a 250ml centrifuge tube, add RNase (final concentration 100ug/ml) for digestion overnight.

② After step ① is completed, add 1/5 volume of 4M NaCl aqueous solution and 2/5 volume of 40% (mass ratio) PEG 6000 aqueous solution to the obtained product, mix well, incubate at 37°C for 5 min, and place on ice for 30 min.

③After step ② is completed, centrifuge the obtained liquid at 4°C (20000g×15min), discard the supernatant, wash once with 70% (v/v) ethanol aqueous solution, and dissolve the obtained precipitate in 50ml TE10/0.1(10mM Tris, 0.1 mM EDTA, pH 8.0, the balance is water) to obtain solution 2.

7. Remove protein pollution and PEG6000

① Add 1/5 volume of phenol:chloroform (V/V=1:1) of solution 2 to solution 2 obtained in step 6, shake and mix well, centrifuge at room temperature (20000g×10min), carefully aspirate the upper aqueous phase, repeat this Step twice.

②After step ① is completed, mix the obtained aqueous phase, and add 1/5 volume of phenol:chloroform:isoamyl alcohol (V/V=25:24:1) to the total aqueous phase, shake and mix, and centrifuge at room temperature (20000g× 10min), carefully suck out the upper aqueous phase.

③After step ② is completed, add 1/10 volume of 3M NaAc (PH5.2) and 2.5 times volume of absolute ethanol to the water phase, and place it at -20°C for at least 1 hour.

④Centrifuge at 4°C (20000g×15min) to collect the precipitate.

⑤ Wash the precipitate with a 70% (v/v) ethanol aqueous solution, and dissolve the precipitate in TE10/0.1 after the ethanol evaporates to obtain a solution 3 containing a carrier.

8. Enzyme digestion and isolation of target DNA

① Use restriction endonuclease EcoRV to digest the plasmid in solution 3 obtained in step 7.

②Use PEG6000 to separate the vector backbone from the target DNA to obtain the target DNA.

③Phenol-chloroform extraction of the obtained target DNA (remove PEG6000), ethanol precipitation, and TE (10mM Tris, 1mM EDTA, pH 8.0, the balance is water) to dissolve the DNA to obtain 177-4DNA solution and 177-12DNA respectively The solution should be stored at -20°C for later use.

5. Preparation of methylated 177-12DNA

1. The 177-12 DNA obtained in step 4 is subjected to methylation reaction (using NEB methyltransferase M.SssI to complete), the system is as follows

NEBuffer2(10x), S-adenosylmethionine and Methyltransferase are all NEB products.

The resulting system was incubated at 37°C for 8h.

2. Use dot hybridization to detect methylation results

1) Take 3μl of the reaction mixture in step 1, add 7μl of water, add 10μl of 0.2M NaOH aqueous solution after mixing, then incubate at 95°C for 10min;

2) After step 1) is completed, add 20μl 1M ammonium acetate to the system to neutralize the reaction;

3) After step 2) is completed, take 0.1μl and 0.3μl of the neutralized samples respectively and drop them on the N+ membrane (GE Healthcare) to dry;

4) After step 3) is completed, place the membrane at 80°C and incubate for 30 minutes;

5) After step 4) is completed, seal the N+ membrane with 5% (mass ratio) milk at room temperature, and wash 3 times with TBST for 1 hour;

6) After step 5) is completed, bind the membrane with an anti-5mC primary antibody (active motif), and carry out the binding at 4°C for 8 hours; wash with TBST three times;

7) After step 6) is completed, place the membrane in a goat anti-mouse secondary antibody (Zhongshan Jinqiao) and incubate at room temperature for 1 hour;

8) After step 7) is completed, add a luminescent substrate (invitrogen) to the film for 3 minutes at room temperature for development.

3. Purify the 177-12 DNA that has undergone methylation modification. The purification method is the same as Step 4 and Step 7 to obtain the 177-12 methylation modified DNA solution.

6. In vitro assembly of 4× and 12× methylated/unmethylated nucleosome beads

The 177-4 DNA and 177-12 DNA obtained in step four, the 177-12 methylated modified DNA obtained in step five and the histone octamer obtained in step three were used to assemble nucleosome beading. The assembly system is as follows:

Among them, 1×TE: 10mM Tris, 1mM EDTA, pH 8.0, and the balance is water.

The samples were mixed according to the above assembly system and put into the refolding buffer for gradient dialysis. The initial buffer was 450ml refolding buffer (the solution with NaCl concentration of 2M obtained by adding NaCl to 1×TE), and the 1050ml 1×TE pump was used by the peristaltic pump. In the refolding buffer, gradually reduce the salt ion concentration, and after at least 16 hours of dialysis, the salt ion concentration of the assembled system is reduced to 0.6M NaCl. Then it is further dialyzed into a low-salt reaction buffer to obtain nucleosome beads.

The nucleosome beads obtained by 177-4DNA, 177-12DNA, and 177-12 methylation modified DNA were recorded as 4×unmethylated nucleosome beads, 12×unmethylated nucleosome beads, and 12 ×Methylated nucleosomes beaded.

Seven, the phase transition between MeCP2 and nucleosome beads

Take 10 mg of MeCP2-His obtained in step 2 at a molar ratio of 1:1 with the dye Alexa Fluor ^TM 568 NHS Ester (Succinimidyl Ester) (ThermoFisher, catalog number A20003), mix and incubate (placed on a rotating shaker, room temperature for 1h) to achieve Fluorescent labeling of proteins. Then use reaction buffer to perform gel filtration chromatography on the above sample (the method is the same as step 3) to remove the remaining fluorescent dye to obtain dye-labeled MeCP2-His, which is frozen at -80°C for use.

The obtained dye-labeled MeCP2-His and unlabeled MeCP2-His are mixed to obtain a protein mixture, so that the molar concentration of the labeled protein accounts for 5%. Mix the obtained protein mixture with the 4×non-methylated nucleosome beads (4×NA or 4×601NA, NA is nucleosome array) obtained in step 6 to obtain the reaction system of the experimental group, 4×non- The concentration of methylated nucleosome beads in the reaction system was 112.5 nM, and the concentration of the total protein in the protein mixture in the reaction system was 10 μM. And set up the following control system:

Control 1 reaction system: only add the above protein mixture, and use the reaction buffer to dilute to a total protein concentration of 20μM.

Control 2 reaction system: only add 4 x unmethylated nucleosome beads, and use reaction buffer to dilute to a concentration of 225 nM.

Control 3 reaction system: only add the 177-4 DNA obtained in step 4, and use the reaction buffer to dilute to a concentration of 37.5 nM.

Control 4 reaction system: only add the 177-4DNA obtained in step 4 and the above-mentioned protein mixture, the concentration of 177-4DNA and MeCP2-His total protein are 37.5nM and 10μM, respectively.

The reaction systems obtained were allowed to stand overnight at 4°C, and imaged and analyzed by a laser confocal scanning microscope.

The results showed that the reaction system of the experimental group had a phase change, the control system 4 had a phase change, and the control systems 1-3 did not have a phase change, indicating that MeCP2 can interact with the nucleosome positioning sequence and the nucleosome containing the nucleosome positioning sequence. The body string undergoes a phase change (Figure 1).

8. Phase transition analysis of truncated MeCP2 and nucleosome beads

Many missense mutations and nonsense mutations that occur on MeCP2 can cause ReTT syndrome. Clinical studies have found that among the nonsense mutations that cause ReTT syndrome, nonsense mutations at positions 168, 255, 270, and 294 of the MeCP2 amino acid sequence account for more than 90%. The following examines whether these nonsense mutations affect the phase transition mediated by MeCP2.

Take 10 mg of MeCP2-His and R168×-His, R255×-His, R270×-His, and R294×-His obtained in step two with the dye Alexa Fluor ^TM 568 NHS Ester (Succinimidyl Ester) at a molar ratio of 1:1. (The brightness and light stability of the conjugate prepared by using the amino-reactive Alexa Fluor 488 carboxylic acid and succinimide ester is significantly better than that of fluorescein. Alexa Fluor 488 dye can emit bright green fluorescence, and the spectrum is similar to that of fluorescein. Stable between pH 4 and pH 10.) (ThermoFisher, catalog number A20003), mix and incubate (placed on a rotating shaker, room temperature for 1 hour) to achieve fluorescent labeling of the corresponding protein. Subsequently, the above samples were subjected to gel filtration chromatography with reaction buffer (the method is the same as step 3), and the remaining fluorescent dyes were removed to obtain dye-labeled MeCP2-His, dye-labeled R168×-His, dye-labeled R255×-His, Dye-labeled R270×-His and dye-labeled R294×-His, the resulting protein samples were frozen at -80°C for later use.

The obtained five dye-labeled proteins are respectively mixed with the respective unlabeled proteins to obtain a protein mixture, so that the molar concentration of the labeled protein is 5%. The five protein mixtures obtained were mixed with the 4× unmethylated nucleosome beads (4×NA) obtained in step 6 according to the final concentration shown in Figure 2 to obtain different reaction systems, 4× unmethylated nucleosome The concentration of body beads in the reaction system was set to 14.06, 28.13, 56.25, 112.5 nM, and the concentration of total protein in the protein mixture in the reaction system was set to 1.25, 2.5, 5, and 10 μM, respectively. The obtained reaction systems were allowed to stand overnight at 4°C, and imaged and analyzed by a laser confocal scanning microscope.

The results showed that, compared with the full-length MeCP2, the ability of all truncated mutations to undergo phase transition with nucleosome beads was weakened to varying degrees, and the more the C-terminal truncation, the weaker the phase transition ability, as shown in Figure 2 and Table 1. Previous studies have found that the more severe the C-terminal truncation, the more severe the disease. It can be seen that the ability of MeCP2 to undergo phase transition with nucleosome beads is negatively correlated with its pathogenicity, that is, the weaker the phase transition ability caused by the mutation, the more serious the corresponding disease.

Table 1. Test results of the phase change ability of each reaction system

9. Phase transition analysis of point mutation MeCP2 and nucleosome beads

The wild-type MeCP2-His obtained in step two and the MeCP2-His of each point mutation were labeled according to the operation process of step eight to obtain dye-labeled MeCP2-His, dye-labeled MeCP2_R106W-His, dye-labeled MeCP2_R111G-His, Dye-labeled MeCP2_Y120D-His, dye-labeled MeCP2_R133C-His, dye-labeled MeCP2_F157I-His, dye-labeled MeCP2_T158M-His, dye-labeled MeCP2_P225R-His and dye-labeled MeCP2_R306C-His, frozen at -80°C for later use.

The obtained nine dye-labeled proteins are respectively mixed with the respective unlabeled proteins to obtain a protein mixture, so that the molar concentration of the labeled protein is all 5%. Mix the resulting protein mixture with the 12× unmethylated nucleosome beads or 12× methylated nucleosome beads obtained in step 6 according to the final concentration shown in Figure 3 to obtain different reaction systems. The concentration settings of nucleosome beads and 12×methylated nucleosome beads in the reaction system are 4.6875, 9.375, 18.75, 37.5nM, and the concentration of total protein in the protein mixture in the reaction system except MeCP2_R111G-His Set to 1.25, 2.5, 5, 10μM, and the concentration of MeCP2_R111G-His to 0.63, 1.25, 2.5, 5μM. The resulting reaction system was allowed to stand overnight at 4°C, and imaged and analyzed by a laser confocal scanning microscope.

It was found that wild-type MeCP2-His can undergo phase transition with unmethylated and methylated nucleosome beads, and has a stronger phase transition ability with the latter. Previous studies have found that MeCP2 protein can bind methylated and unmethylated DNA, and its affinity with methylated DNA is stronger, which is consistent with the experimental results of the present invention. Figure 3 also shows that, compared with wild-type MeCP2-His, the point mutation MeCP2 (the point mutations shown in Figure 3 are pathogenic) and the ability of methylated and unmethylated nucleosome beads to undergo phase transition Different degrees of weakening, and the degree of this abnormal phase transition is basically positively correlated with the severity of the disease caused by the MeCP2 point mutation.

10. The effect of different point mutations of MeCP2 on intracellular phase transition

Transfection of NIH 3T3 cells: The recombinant vector expressing mCherry fusion protein obtained in steps 4 and 5 in step 1 (pmCherry-C1-MeCP2, pmCherry-C1-MeCP2_R106W, pmCherry-C1-MeCP2_R111G, pmCherry-C1-MeCP2_Y120D, pmCherry-C1 -MeCP2_R133C, pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_F157I, pmCherry-C1-MeCP2_K144R, pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_T197M, pmCherry-C1-MeCP2_R201V, CP2Cherry-294P ) Were transfected into NIH 3T3 cells (adherent culture), the transfection reagent used was VigoFect (Viglas Biotechnology (Beijing) Co., Ltd.), and the cells were transfected according to the reagent instructions. DAPI staining was performed 36 hours after transfection. DAPI staining steps:

1. Discard the cell culture medium and wash the cells 3 times with PBS;

2. Fix the cells with 4% paraformaldehyde for 15 minutes;

3. Discard the paraformaldehyde and wash with PBS three times;

4. DAPI(sigma) staining for 5min;

5. Discard DAPI and wash twice with PBS;

6. Add anti-quenching agent (SlowFade ^TM Diamond Antifade Mountant with DAPI invitrogen), and mount the slide.

The obtained DAPI stained cells were analyzed by laser confocal scanning microscopy.

The results (Figure 4) found that the phase separation boundary produced by transfection of wild-type MeCP2 was clear, and the red fluorescence signal (the fluorescence signal produced by mCherry) was highly concentrated in the phase change phase, which was significantly different from the red fluorescence signal intensity of the non-phase change part. . Compared with the phase separation produced by transfection of wild-type MeCP2, the pathogenic point mutation MeCP2 (the left column of Figure 4) either does not undergo phase separation, or the phase separation boundary produced is blurred, and the red fluorescence signal is The degree of aggregation in the concentrated phase decreases, and the difference in intensity of the red fluorescence signal from the non-phase-change part decreases. However, the phase separation morphology, the distribution of red fluorescent signal and the degree of aggregation in the phase transition phase produced by transfection of non-pathogenic point mutation MeCP2 (right column in Figure 4) are not significantly different from those of transfection of wild-type MeCP2. Calculate the partition coefficient, see Figure 5. The partition coefficient is the ratio of the mCherry fluorescence signal intensity in the DAPI darkly stained area of the transfected cell line and the DAPI non-stained area. Transfection pmCherry-C1-MeCP2, pmCherry-C1-MeCP2_R106W, pmCherry-C1-MeCP2_R111G, pmCherry-C1-MeCP2_Y120D, pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_K-C144I, pmCherry-C1-MeCP2_F157I, The distribution coefficients of pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_T197M, pmCherry-C1-MeCP2_A201V, pmCherry-C1-MeCP2_R250H, pmCherry-C1-MeCP2_R294P are 2.2877±1.733792, 1.620161±1.110083, 1.065981±0.177234, 1.065981±0.177234, 1.065981±0.1772, respectively 1.900664, 3.288191±2.781871, 3.01643±1.964515, 1.01914±0.724986, 5.641106±0.431235, 5.235031±0.375481, 4.113438±2.736971, 2.861968±1.881579, 2.900806±3.608265, 3.964675±4.663274. The experimental data in the cell further confirmed that the MeCP2-mediated phase transition abnormality is closely related to the disease.

In vitro experiments of the present invention show that MeCP2 can combine with nucleosome beads to undergo phase change, and that MeCP2 pathogenic truncation mutations and point mutations both cause abnormal phase transitions between MeCP2 and nucleosome beads; and the intracellular experimental results show that , Only pathogenic point mutations can cause abnormal phase transitions, and non-pathogenic point mutations have no significant effect on phase transitions, further confirming that abnormal phase transitions are closely related to the occurrence of diseases caused by MeCP2 mutations. Therefore, reversing the abnormal phase transition is likely to become a new idea and direction for the treatment of related diseases. High-throughput screening can be used to obtain reversible abnormal phase transition regulators and be applied to the development of potential targeted drugs. This strategy can also be used to treat other diseases. Provide reference for diseases related to abnormal phase transition.

Industrial application

In vitro experiments of the present invention show that MeCP2 can combine with nucleosome beads to undergo phase change, and that MeCP2 pathogenic truncation mutations and point mutations both cause abnormal phase transitions between MeCP2 and nucleosome beads; and the intracellular experimental results show that , Only pathogenic point mutations can cause abnormal phase transitions, and non-pathogenic point mutations have no significant effect on phase transitions, further confirming that abnormal phase transitions are closely related to the occurrence of diseases caused by MeCP2 mutations. Therefore, reversing the abnormal phase transition is likely to become a new idea and direction for the treatment of related diseases. High-throughput screening can be used to obtain reversible abnormal phase transition regulators and be applied to the development of potential targeted drugs. This strategy can also be used to treat other diseases. Provide reference for diseases related to abnormal phase transition.

Claims (37)

1. The application of the substance for detecting the MeCP2 mutation in the preparation, detection or auxiliary detection of whether the MeCP2 mutation is a pathogenic mutation product.
2. The application according to claim 1, wherein the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein in which the MeCP2 mutation occurs and the following a1) or a2) or a3) or a4):

   a1) DNA fragments;

   a2) Nucleosome beads;

   a3) Substances used to prepare nucleosome beads;

   a4) Cells containing beads of nucleosomes.
3. The application according to claim 2, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

   a11) Nucleosome localization DNA;

   a12) A DNA fragment containing the nucleosome positioning DNA;

   a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

   a3) The substance is a31) or a32):

   a31) histone octamer and said DNA fragment;

   a32) H2A, H2B, H3 and H4 and the DNA fragments.
4. The use according to any one of claims 1-3, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
5. The application of a substance for detecting a MeCP2 mutation in detecting or assisting in detecting whether the MeCP2 mutation is a pathogenic mutation.
6. The application according to claim 5, wherein the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein in which the MeCP2 mutation occurs and the following a1) or a2) or a3) or a4):

   a1) DNA fragments;

   a2) Nucleosome beads;

   a3) Substances used to prepare nucleosome beads;

   a4) Cells containing beads of nucleosomes.
7. The application according to claim 6, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

   a11) Nucleosome localization DNA;

   a12) A DNA fragment containing the nucleosome positioning DNA;

   a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

   a3) The substance is a31) or a32):

   a31) histone octamer and said DNA fragment;

   a32) H2A, H2B, H3 and H4 and the DNA fragments.
8. The use according to any one of claims 5-7, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
9. Application of a substance for detecting MeCP2 mutations in screening or auxiliary screening of drugs for the treatment and/or prevention of diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation.
10. The application according to claim 9, characterized in that the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein in which the MeCP2 mutation occurs and the following a1) or a2) or a3) or a4):

    a1) DNA fragments;

    a2) Nucleosome beads;

    a3) Substances used to prepare nucleosome beads;

    a4) Cells containing beads of nucleosomes.
11. The application according to claim 10, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

    a11) Nucleosome localization DNA;

    a12) A DNA fragment containing the nucleosome positioning DNA;

    a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

    a3) The substance is a31) or a32):

    a31) histone octamer and said DNA fragment;

    a32) H2A, H2B, H3 and H4 and the DNA fragments.
12. The use according to any one of claims 9-11, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
13. Use of a substance for detecting MeCP2 mutations in preparing, screening or assisting screening of drug products for the treatment and/or prevention of diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation.
14. The application according to claim 13, wherein the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein in which the MeCP2 mutation occurs and the following a1) or a2) or a3) or a4):

    a1) DNA fragments;

    a2) Nucleosome beads;

    a3) Substances used to prepare nucleosome beads;

    a4) Cells containing beads of nucleosomes.
15. The application according to claim 14, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

    a11) Nucleosome localization DNA;

    a12) A DNA fragment containing the nucleosome positioning DNA;

    a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

    a3) The substance is a31) or a32):

    a31) histone octamer and said DNA fragment;

    a32) H2A, H2B, H3 and H4 and the DNA fragments.
16. The use according to any one of claims 13-15, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
17. The application of the substance for detecting the MeCP2 mutation in the preparation of a product for detecting or assisting in detecting whether the MeCP2 mutation is pathogenic.
18. The application according to claim 17, characterized in that the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein with the MeCP2 mutation and the following a1) or a2) or a3) or a4):

    a1) DNA fragments;

    a2) Nucleosome beads;

    a3) Substances used to prepare nucleosome beads;

    a4) Cells containing beads of nucleosomes.
19. The application according to claim 18, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

    a11) Nucleosome localization DNA;

    a12) A DNA fragment containing the nucleosome positioning DNA;

    a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

    a3) The substance is a31) or a32):

    a31) histone octamer and said DNA fragment;

    a32) H2A, H2B, H3 and H4 and the DNA fragments.
20. The use according to any one of claims 17-19, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
21. Use of a substance for detecting MeCP2 mutation in detecting or assisting in detecting whether the MeCP2 mutation is pathogenic.
22. The application according to claim 21, characterized in that the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein with the MeCP2 mutation and the following a1) or a2) or a3) or a4):

    a1) DNA fragments;

    a2) Nucleosome beads;

    a3) Substances used to prepare nucleosome beads;

    a4) Cells containing beads of nucleosomes.
23. The application according to claim 22, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

    a11) Nucleosome localization DNA;

    a12) A DNA fragment containing the nucleosome positioning DNA;

    a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

    a3) The substance is a31) or a32):

    a31) histone octamer and said DNA fragment;

    a32) H2A, H2B, H3 and H4 and the DNA fragments.
24. The use according to any one of claims 21-23, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
25. The use of a substance for detecting MeCP2 mutations in the preparation of products for diagnosing or assisting in the diagnosis of diseases caused by the MeCP2 mutations.
26. The application according to claim 25, characterized in that the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein with the MeCP2 mutation and the following a1) or a2) or a3) or a4):

    a1) DNA fragments;

    a2) Nucleosome beads;

    a3) Substances used to prepare nucleosome beads;

    a4) Cells containing beads of nucleosomes.
27. The application according to claim 26, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

    a11) Nucleosome localization DNA;

    a12) A DNA fragment containing the nucleosome positioning DNA;

    a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

    a3) The substance is a31) or a32):

    a31) histone octamer and said DNA fragment;

    a32) H2A, H2B, H3 and H4 and the DNA fragments.
28. The use according to any one of claims 25-27, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
29. The application of the substance detecting the MeCP2 mutation in the diagnosis or auxiliary diagnosis of the diseases caused by the MeCP2 mutation.
30. The application according to claim 29, characterized in that the substance for detecting the MeCP2 mutation includes the MeCP2 mutant protein with the MeCP2 mutation and the following a1) or a2) or a3) or a4):

    a1) DNA fragments;

    a2) Nucleosome beads;

    a3) Substances used to prepare nucleosome beads;

    a4) Cells containing beads of nucleosomes.
31. The application according to claim 30, characterized in that: a1) the DNA fragment is a11) or a12) or a13):

    a11) Nucleosome localization DNA;

    a12) A DNA fragment containing the nucleosome positioning DNA;

    a13) A DNA fragment formed by repeating n times with a12) as a repeating unit;

    a3) The substance is a31) or a32):

    a31) histone octamer and said DNA fragment;

    a32) H2A, H2B, H3 and H4 and the DNA fragments.
32. The use according to any one of claims 29-31, wherein the MeCP2 mutation is a truncation mutation or a point mutation of MeCP2.
33. The method for detecting or assisting in detecting whether the MeCP2 mutation is a pathogenic mutation is accomplished by using the substance for detecting MeCP2 mutation in any one of claims 1-4.
34. Screening or auxiliary screening methods for treating and/or preventing diseases caused by the MeCP2 mutation, the MeCP2 mutation is a MeCP2 pathogenic mutation; the method is completed by using the substance for detecting the MeCP2 mutation in any one of claims 1-4 .
35. The method for detecting or assisting in detecting whether the MeCP2 mutation is pathogenic is accomplished by using the substance for detecting the MeCP2 mutation in any one of claims 1-4.
36. A method for diagnosing or assisting in the diagnosis of the disease caused by the MeCP2 mutation, the disease caused by the MeCP2 mutation is a dysplasia of the nervous system, and the method is completed by using the substance for detecting the MeCP2 mutation in any one of claims 1-4.
37. A product with any of the following functions is the substance for detecting MeCP2 mutations according to any one of claims 1-4:

    X1) Preparation and detection or auxiliary detection of whether the MeCP2 mutation is a pathogenic mutation product;

    X2) Detect or assist in detecting whether the MeCP2 mutation is a pathogenic mutation;

    X3) Screen or assist in screening drugs for treatment and/or prevention of diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation;

    X4) Preparation of drug products for screening or auxiliary screening to treat and/or prevent diseases caused by the MeCP2 mutation; the MeCP2 mutation is a MeCP2 pathogenic mutation;

    X5) Preparation and detection or auxiliary detection of whether the MeCP2 mutation is a pathogenic product;

    X6) Detect or assist in detecting whether the MeCP2 mutation is pathogenic;

    X7) Preparation of products for diagnosing or assisting in the diagnosis of diseases caused by the MeCP2 mutation;

    X8) Diagnose or assist in the diagnosis of diseases caused by the MeCP2 mutation.

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