CN117003850B - Methylation enriched protein and encoding gene, preparation method and application thereof - Google Patents

Abstract

The invention discloses a methylation enrichment protein, and belongs to the field of molecular biology. The amino acid sequence of the methylation enrichment protein is shown as SEQ ID No.3 or SEQ ID No. 4. The invention further discloses a gene of the methylation enrichment protein, a vector and a host cell containing the gene, a preparation method of the methylation enrichment protein and application of the methylation enrichment protein in preparation of a kit for enriching methylated DNA. The methylation enrichment protein has high purification efficiency, high enrichment efficiency on methylated DNA, strong specificity and high sensitivity, and has very important clinical application value.

Description

Methylation enriched protein and encoding gene, preparation method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a methylation enrichment protein, and a coding gene, a preparation method and application thereof.

Background

DNA methylation (DNA methylation) is an apparent regulatory modification of DNA that can alter genetic manifestations without altering the DNA sequence. By DNA methylation is meant covalent bonding of a methyl group at the cytosine carbon number 5 of a genomic CpG dinucleotide under the action of a DNA methyltransferase. Studies have shown that DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression.

MBD (methyl-CpG-binding domain) family proteins are the earliest discovered methylated DNA binding proteins, and mainly comprise MeCEP2 and several major members of MBD1, MBD2, MBD3, and MBD 4. In humans, MBD family proteins each contain MBD domains and other functional domains that inhibit expression of a target gene by recruiting different co-inhibitory complexes to the methylation site of the target gene. Meanwhile, by utilizing the characteristic that MBD family proteins can specifically bind methylated DNA, methylated DNA fragments are pulled down or captured in a genome DNA sample, so that the enrichment or purification effect is achieved, and the methylation level of a sample area or whole is detected by combining fluorescent quantitative PCR detection (qPCR) and Next Generation Sequencing (NGS) technology.

The current method for DNA methylation enrichment by using MBD family proteins is not generally used because of low protein purification expression efficiency, low yield and high cost. Meanwhile, compared with other methylation enrichment modes, the protein expressed by the existing protein sequence design has low enrichment efficiency, poor specificity and low sensitivity, and cannot be used in clinical detection kits.

Disclosure of Invention

In order to solve at least one of the technical problems, the invention adopts the following technical scheme:

the first aspect of the invention provides a methylation-enriched protein, the amino acid sequence of which is shown as SEQ ID No.3 or SEQ ID No. 4.

In the invention, SEQ ID No.3 is obtained by optimizing and modifying on the basis of SEQ ID No.2, and the sequence shown in SEQ ID No.2 is an MBD domain sequence in MBD2 protein. Specifically, the SEQ ID No.3 is obtained by respectively extending 5-6 amino acids before and after the SEQ ID No.2, the corresponding protein structure is closer to the natural state, the structure between peptide chains is more compact, and simultaneously, the methylation functional region can be better combined with CpG islands in methylated DNA, so that the methylation enrichment efficiency can be better improved.

Furthermore, SEQ ID No.4 is obtained by further optimizing on the basis of SEQ ID No.3, specifically, a specific amino acid sequence is added at the N end and the C end of SEQ ID No.3, and the main functions of the two end sequences are to stabilize the methylation binding protein function and improve the expression and purification efficiency thereof, and also provide a certain flexibility for the protein structure, so that the protein structure can be better added with subsequent purification and magnetic bead labels.

In a second aspect, the invention provides a gene encoding a methylation enrichment protein according to the first aspect of the invention, comprising a nucleotide sequence shown as SEQ ID No.6 or SEQ ID No.7, wherein the nucleotide sequence shown as SEQ ID No.6 encodes an amino acid sequence shown as SEQ ID No.3 and the nucleotide sequence shown as SEQ ID No.7 encodes an amino acid sequence shown as SEQ ID No. 4.

In some embodiments of the invention, the methylation-enriched protein further comprises a functional tag, and correspondingly, the gene further comprises a sequence for expressing the functional tag.

In some embodiments of the invention, the functional tag is a tag for purification and/or a tag for detection. In some preferred embodiments of the invention, the tag for purification is selected from at least one of the group consisting of NH tag and His tag; the functional tag is selected from at least one of the group consisting of Avi tag and Fc tag. In some more preferred embodiments of the invention, the functional tag consists of both a tag for detection and a tag for purification. Wherein the label for detection is enriched for the target substance using affinity, hybridization or any other specific recognition property.

In some preferred embodiments of the invention, the NH tag has an amino acid sequence of HNHNHNHNHNHN.

In some preferred embodiments of the invention, the amino acid sequence of the His-tag is hhhhhhhh.

In some preferred embodiments of the invention, the Avi tag has an amino acid sequence of GLNDIFEAQKIEWHEPGAAGHNHNHNHNHNHN.

In some preferred embodiments of the invention, the Fc tag has amino acid sequences MKWVTFLLLLFVSDSAFS and PGAAGAGSDQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, respectively, added to the N-and C-terminus of the methylation-enriched protein.

In a third aspect the invention provides an expression vector comprising the sequence of the gene according to the second aspect of the invention.

In some embodiments of the invention, the expression vector is a prokaryotic expression vector, such as a pET-28a (+) vector.

In a fourth aspect, the invention provides a host cell comprising an expression vector according to the third aspect of the invention.

In some embodiments of the invention, the host cell is a prokaryotic cell, such as E.coli.

In a fifth aspect, the invention provides a method of producing a methylation-enriched protein according to the first aspect of the invention, comprising the step of inducing expression in a host cell according to the fourth aspect of the invention.

In some embodiments of the invention, the expression vector is pET-28a and the host cell is E.coli.

In some embodiments of the invention, the step of inducing the host cell to perform protein expression is:

s1, culturing the escherichia coli at 37 ℃ by using an LB medium containing 50 mug/mL kanamycin,

s2, when the OD600 of the escherichia coli culture solution is 0.5-0.7, the induction expression is carried out by using IPTG with the final concentration of 1mM, wherein the induction conditions are as follows: 16 ℃,220rpm, overnight;

s3, centrifuging the culture solution at 6000rpm for 5min, and collecting thalli;

s4, protein purification is carried out after the bacterial cells are crushed.

In a sixth aspect, the invention provides the use of a methylation-rich protein according to the first aspect of the invention for the preparation of a kit for enriching methylated DNA.

In a seventh aspect, the invention provides a kit for enriching methylated DNA comprising a methylation-enriched protein according to the first aspect of the invention.

The beneficial effects of the invention are that

Compared with the prior art, the invention has the following technical effects:

the methylation enrichment protein has high purification efficiency, high enrichment efficiency on methylated DNA, strong specificity and high sensitivity, and has very important clinical application value.

The methylation enrichment protein is arranged on His tag-Ni/Co magnetic beads; NH label-Ni/Co magnetic beads; biotin tag-streptavidin magnetic beads; the Fc label-protein A/G magnetic beads and other different coordination effects can have good enrichment effect.

Drawings

FIG. 1 shows a schematic structural diagram of MBD2 protein.

FIG. 2 shows a schematic representation of the MBD-1 prediction structure in an embodiment of the invention.

FIG. 3 shows a schematic representation of the MBD-2 prediction structure in an embodiment of the invention.

FIG. 4 shows the effect of expressing each of MBD-4 to MBD-6 proteins in the examples of the present invention.

FIG. 5 shows the amplification results of the reference enriched product.

FIG. 6 shows the results of MBD-4 to MBD-6 enriched product amplification.

FIG. 7 shows the TIC profile of MBD-6 protein.

FIG. 8 shows a mass spectrum detection profile of MBD-6 protein.

FIG. 9 shows a convolved molecular weight map of MBD-6 protein.

FIG. 10 shows the amplification results of MBD-6 to MBD-9 enriched products.

Detailed Description

Unless otherwise indicated, implied from the context, or common denominator in the art, all parts and percentages in the present application are based on weight and the test and characterization methods used are synchronized with the filing date of the present application. Where applicable, the disclosure of any patent, patent application, or publication referred to in this application is incorporated by reference in its entirety, and the equivalent patents to those cited are incorporated by reference, particularly as they relate to the definitions of terms in the art. If the definition of a particular term disclosed in the prior art does not conform to any definition provided in this application, the definition of that term provided in this application controls.

Numerical ranges in this application are approximations, so that it may include the numerical values outside of the range unless otherwise indicated. The numerical range includes all values from the lower value to the upper value that increase by 1 unit, provided that there is a spacing of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1,1.5, etc.), then 1 unit is suitably considered to be 0.0001,0.001,0.01, or 0.1. For a range containing units of less than 10 (e.g., 1 to 5), 1 unit is generally considered to be 0.1. These are merely specific examples of what is intended to be provided, and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

The terms "comprises," "comprising," "including," and their derivatives do not exclude the presence of any other component, step or procedure, and are not related to whether or not such other component, step or procedure is disclosed in the present application. For the avoidance of any doubt, all use of the terms "comprising," "including," or "having" herein, unless expressly stated otherwise, may include any additional additive, adjuvant, or compound. Rather, the term "consisting essentially of … …" excludes any other component, step or process from the scope of any of the terms recited below, as those out of necessity for operability. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. The term "or" refers to the listed individual members or any combination thereof unless explicitly stated otherwise.

In order to make the technical problems, technical schemes and beneficial effects solved by the invention more clear, the invention is further described in detail below with reference to the embodiments.

Examples

The following examples are presented herein to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

The molecular biology experiments described in the following examples, which are not specifically described, were performed according to the specific methods listed in the "guidelines for molecular cloning experiments" (fourth edition) (j. Sambrook, m.r. Green, 2017) or according to the kit and product specifications. Other experimental methods, unless otherwise specified, are all conventional. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1MBD Domain

According to NCBI database information (accession number: NP-003918.1), the original sequence of the MBD2 protein is:

MRAHPGGGRCCPEQEEGESAAGGSGAGGDSAIEQGGQGSALAPSPVSGVRREGARGGGRGRGRWKQAGRGGGVCGRGRGRGRGRGRGRGRGRGRGRPPSGGSGLGGDGGGCGGGGSGGGGAPRREPVPFPSGSAGPGPRGPRATESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKNKQRLRNDPLNQNKGKPDLNTTLPIRQTASIFKQPVTKVTNHPSNKVKSDPQRMNEQPRQLFWEKRLQGLSASDVTEQIIKTMELPKGLQGVGPGSNDETLLSAVASALHTSSAPITGQVSAAVEKNPAVWLNTSQPLCKAFIVTDEDIRKQEERVQQVRKKLEEALMADILSRAADTEEMDIEMDSGDEA(SEQ ID No.1)

as shown in FIG. 1, MBD2 proteins mainly contain G/R (Glycine/arginine rich domain ); MBD (methylated CpG binding domain, methyl-CoG binding domain); TRD (transcription repression domain ); CC (Coiled-coil domain) four main domains. Because MBD2 protein is larger, purification difficulty is high, and enrichment effect is poor. Thus, the inventors selected MBD domains therein for optimization.

The MBD domain is a main functional structural part of methylation CpG binding, and is marked as MBD-1, and the amino acid sequence is as follows:

MDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYLGNTVD LSSFDFRTGKMMPSKLQK(SEQ ID No.2)。

the structure of MBD-1 was calculated using the alpha Fold based Swiss-Model as shown in FIG. 2.

When the sequence structure is used for artificial synthesis and expression purification, two main problems are found:

a. the expression effect of the synthesized protein in the strain is unstable, the purification efficiency is low, the experimental repeatability is poor, and the standardized production cannot be realized;

b. synthetic proteins are inefficient to enrich in enrichment efficiency tests, which are not specific for methylation standards.

Example 2 optimization of MBD Domains

The inventors have tried improvements and optimizations in three directions in an attempt to solve the two problems described above.

Firstly, aiming at the MBD-1 sequence, the inventor lengthens each 5-6 amino acids from front to back to enable the structure of the synthesized protein (MBD-2) to be closer to the natural state, so that the structure between peptide chains is more compact, and simultaneously, a methylation functional region can be better combined with CpG islands in methylated DNA, so that the methylation enrichment efficiency can be better improved. The amino acid sequence of MBD-2 is as follows:

ESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKPQLARYL GNTVDLSSFDFRTGKMMPSKLQKNKQRLRND(SEQ ID No.3)

secondly, considering the expression purification efficiency and the subsequent connection with a magnetic bead tag, the inventor adds a section of specific amino acid sequence at the N end and the C end of MBD-2, the main functions of the two end sequences are to stabilize the methylation binding protein function and improve the expression purification efficiency, and also provide a certain flexibility for the protein structure, so that the subsequent purification and the magnetic bead tag can be better added, and the MBD-3 protein is obtained, wherein the amino acid sequence is as follows:

GEKMDESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKP QLARYLGNTVDLSSFDFRTGKMMPSKLQKNKQRLRNDVYYGAAG(SEQ ID No.4)

the structure of MBD-3 was calculated using the alpha Fold based Swiss-Model as shown in FIG. 3.

It was found by predicting the structure that the structure of MBD-3 did not undergo a large change in folding compared to the original MBD-1 domain structure, indicating that MBD-3 is able to achieve methylation-specific enrichment of the retained structure.

Example 2 test of expression Effect of methylation-enriched proteins

To verify that the optimization of the above structure can improve protein yield, the inventors expressed and purified the three protein structures using a conventional 6×nh tag.

1. Design of Gene sequences

In order to express MBD-1, MBD-2 and MBD-3 proteins, the inventors designed the corresponding gene sequences for the amino acid sequences as follows:

the coding gene sequence corresponding to the MBD-1 protein is as follows:

ATGGATTGCCCGGCGCTGCCGCCGGGCTGGAAAAAAGAAGAAGTGATTCGCAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGCCCGAGCGGCAAAAAATTTCGCAGCAAACCGCAGCTGGCGCGCTATCTGGGCAACACCGTGGATCTGAGCAGCTTTGATTTTCGCACCGGCAAAATGATGCCGAGCAAACTGCAGAAA(SEQ ID No.5)

the coding gene sequence corresponding to the MBD-2 protein is as follows:

GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGCTGGAAAAAAGAAGAAGTGATTCGCAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGCCCGAGCGGCAAAAAATTTCGCAGCAAACCGCAGCTGGCGCGCTATCTGGGCAACACCGTGGATCTGAGCAGCTTTGATTTTCGCACCGGCAAAATGATGCCGAGCAAACTGCAGAAAAACAAACAGCGCCTGCGCAACGAT(SEQ ID No.6)

the coding gene sequence corresponding to the MBD-3 protein is as follows:

GGCGAAAAAATGGATGAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGCTGGAAAAAAGAAGAAGTGATTCGCAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGCCCGAGCGGCAAAAAATTTCGCAGCAAACCGCAGCTGGCGCGCTATCTGGGCAACACCGTGGATCTGAGCAGCTTTGATTTTCGCACCGGCAAAATGATGCCGAGCAAACTGCAGAAAAACAAACAGCGCCTGCGCAACGATGTGTATTATGGCGCGGCGGGC(SEQ ID No.7)

for convenient purification, the inventors respectively add 6 XNH tags to MBD-1, MBD-2 and MBD-3 proteins to obtain MBD-4, MBD-5 and MBD-6. The coding sequences of the genes with the 6 XNH tag (namely HNHNHNHNHNHN) are CATAACCATAACCATAACCATAACCATAACCATAAC, MBD-4, MBD-5 and MBD-6 respectively as follows:

the coding gene sequence corresponding to the MBD-4 protein is as follows:

ATGGATTGCCCGGCGCTGCCGCCGGGCTGGAAAAAAGAAGAAGTGATTCGCAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGCCCGAGCGGCAAAAAATTTCGCAGCAAACCGCAGCTGGCGCGCTATCTGGGCAACACCGTGGATCTGAGCAGCTTTGATTTTCGCACCGGCAAAATGATGCCGAGCAAACTGCAGAAACATAACCATAACCATAACCATAACCATAACCATAAC(SEQ ID No.8)

the coding gene sequence corresponding to the MBD-5 protein is as follows:

GAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGCTGGAAAAAAGAAGAAGTGATTCGCAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGCCCGAGCGGCAAAAAATTTCGCAGCAAACCGCAGCTGGCGCGCTATCTGGGCAACACCGTGGATCTGAGCAGCTTTGATTTTCGCACCGGCAAAATGATGCCGAGCAAACTGCAGAAAAACAAACAGCGCCTGCGCAACGATCATAACCATAACCATAACCATAACCATAACCATAAC(SEQ ID No.9)

the coding gene sequence corresponding to the MBD-6 protein is as follows:

GGCGAAAAAATGGATGAAAGCGGCAAACGCATGGATTGCCCGGCGCTGCCGCCGGGCTGGAAAAAAGAAGAAGTGATTCGCAAAAGCGGCCTGAGCGCGGGCAAAAGCGATGTGTATTATTTTAGCCCGAGCGGCAAAAAATTTCGCAGCAAACCGCAGCTGGCGCGCTATCTGGGCAACACCGTGGATCTGAGCAGCTTTGATTTTCGCACCGGCAAAATGATGCCGAGCAAACTGCAGAAAAACAAACAGCGCCTGCGCAACGATGTGTATTATGGCGCGGCGGGCCATAACCATAACCATAACCATAACCATAACCATAAC(SEQ ID No.10)

2. vector construction

Each of the above gene fragments was cloned into pET-28a (+) vector, and NcoI and XhoI were selected as cloning sites, to obtain plasmids capable of expressing each protein.

3. Protein expression

(1) 1. Mu.L of plasmid was added to 100. Mu.L of BL21 (DE 3) competent bacteria and placed on ice for 20min;

(2) Heat-shock at 42 ℃ for 90sec, rapidly placing in ice for 3min, and adding 500 mu L of LB culture solution;

(3) Shaking culture at 37deg.C and 220rpm for 2 hr, spreading 200 μl of the bacterial liquid on LB plate containing 50 μg/mL Kanamycin (Kanamycin), and culturing at 37deg.C under inversion overnight;

(4) The following morning, the single clone on the plate was picked up and inoculated into a test tube containing 50. Mu.g/mL Kanamycin in 4mL LB medium, and shake-cultured at 37℃and 220rpm until the OD was about 0.6;

(5) Respectively inoculating into 1L LB culture solution (3L culture solution) of 100 mug/mL Kan according to the ratio of 1:250, and shaking at 37 ℃ and 220rpm until the cell OD600 is 0.5-0.6 (about 4 h);

(6) Adding an inducer IPTG into each 1L of fermentation medium to a final concentration of 0.1mM, and culturing at 220rpm and 16 ℃ overnight;

(7) Centrifuging at 6000rpm for 5min, collecting the supernatant, and preserving at-20deg.C for crushing and purifying.

4. Protein purification

In this example, protein purification was performed by affinity chromatography using 20mM Tris,500mM NaCl (ph=7.4) as an equilibration buffer with the target protein in 500mM and 1M imidazole eluates, and after electrophoresis to detect purity and concentration, the protein was replaced in a dialysis bag with 20mM Tris,200mM NaCl (ph=7.4) protein buffer.

5. Protein validation

After protein purification, purity yield was verified by coomassie brilliant blue staining and a micro-spectrophotometer instrument (Nanodrop), as shown in fig. 4 and table 1.

TABLE 1 Effect of protein expression

As can be seen from FIG. 4 and Table 1, the expression effect of MBD-4 was not good, and the expression effects of MBD-5 and MBD-6 were improved after the sequence modification. As can be seen from the coomassie blue stained bands, the expression of MBD-4, MBD-5 and MBD-6 were all expected, with MBD-6 being the most effective band than MBD-4 and MBD-5. From the expression yield point of view, the expression yield of MBD-4 is the lowest, and after sequence modification, the expression yields of MBD-5 and MBD-6 are improved, wherein the yield of MBD-6 is the highest. The results prove that the technical effect of improving the expression and the yield of the protein is achieved after the two-step optimization.

Example 3 enrichment efficiency test of methylation-enriched protein

In this example, the inventors used the following procedure to test the methylation enrichment efficiency of the corresponding purified proteins MBD-4, MBD-5 and MBD-6 based on MBD-1, MBD-2 and MBD-3, respectively, in example 2. The method comprises the following steps:

1. preparing experimental materials:

protein buffer: 20mM Tris (MACHLIN, T823912), 200mM NaCl (MACHLIN, T823912), pH=7.4;

DNA eluent: 20mM Tris,2000mM NaCl,pH =7.4;

methylation binding proteins (MBD-4, MBD-5, MBD-6): 10 μg protein (concentration: 1 mg/mL).

Protein-binding magnetic beads: 100. Mu.L of Ni magnetic beads (Thermo, 88831).

Simulation sample DNA: positive and negative references 10ng (Thermo, ME 10025).

Primer liquid: 10. Mu.L of methylated primer and unmethylated primer (Thermo, ME 10025).

Pre-solution for chromogenic real-time quantitative PCR amplification: fluorescent dye 1×SYBR Green I, 10. Mu.L (Yiasen, 11184ES08, the next holy organism).

2. Experimental reaction steps:

(1) For each methylation enrichment reaction, 100 μl of protein-binding magnetic beads were added to a 1.5mL centrifuge tube;

(2) Adding 100 mu L of protein buffer solution, cleaning, standing on a magnetic rack for 1min, and removing supernatant;

(3) Adding 10 mug of methylation binding protein and 90 mug of protein buffer solution, and shaking and mixing uniformly;

(4) Adding 10ng of simulated sample DNA and shaking and uniformly mixing for about 20 minutes;

(5) Placing the centrifuge tube on a magnetic rack for standing for 1min, and removing supernatant;

(6) Adding 20 mu L of DNA eluent, shaking and uniformly mixing for 5 minutes, then performing instantaneous centrifugation, placing the centrifuge tube on a magnetic rack, standing for 1 minute, removing supernatant and transferring to a new centrifuge tube.

The collected enriched samples were formulated into the reaction system according to table 2:

TABLE 2 reaction system

Component (A)	Volume (mu L)
		Enriching a sample	1
ddH2O	8.2
		Primer liquid	0.8
Reaction liquid	10
		Total volume of	20

Detection was performed using a fluorescent quantitative PCR instrument (example: yaRui MA-6000) based on SYBR Green fluorescence, the reaction procedure was as follows:

first part step 1: setting the temperature to 98 ℃ and the time to 30s

Second part step 1: setting the temperature to 95 ℃ and the time to 3s;

step 2: setting the temperature to be 60 ℃ and the time to be 60s;

repeating step 2 and step 3 for 40 cycles

Third partial step 4: setting the temperature to 94 ℃ and the time to 30s;

step 5: setting the temperature to 60 ℃ and the time to 90s;

step 6: setting the temperature to 94 ℃ and the time to 10s;

the results are shown in FIG. 5, FIG. 6, table 3, and Table 4.

TABLE 3 methylation enrichment test reference results

TABLE 4 methylation enrichment test protein detection results

As can be seen from fig. 5 and table 3, for the reference, the reference average CT values of the unmethylated and methylated regions amplified with the specific primers, respectively, were 19.89 and 17.64, respectively, the differences of which were mainly caused by the sample itself, and were used as reference for calculation of subsequent experiments.

As can be seen from fig. 6 and table 4, when the sample is subjected to different protein enrichment processes, the reference average CT values of the unmethylated and methylated regions amplified by the specific primers are changed, and the enrichment efficiency of one reference is calculated by:

Δct= (non-methylated region test value-non-methylated region reference value) - (methylated region test value-methylated region reference value)

The enrichment efficiency that can be obtained according to the formula is shown in table 5:

TABLE 5 methylation enrichment test protein enrichment efficiency results

			MBD-4	2.51	82.44％
MBD-5	5.57	97.89％
			MBD-6	7.9	99.58％

As can be seen from Table 5, the enrichment efficiency of MBD-4 results is 82.44%, the enrichment efficiency of MBD-5 after optimization is 97.89%, and the enrichment efficiency of MBD-6 is 99.58%, wherein the enrichment effect of MBD-6 is the best, and the enrichment effect of protein after two-step optimization is improved.

Example 4 Mass spectrometric identification of MBD-6 proteins

1. Principal materials and reagents

FA (Fluka, 06450); acetonitrile (Sigma, 34851).

2. Main instrument and software

High resolution mass spectrometers (Waters, xex-G2-XS-Qtof);

ultra high performance liquid chromatography (Waters, acquisition UPLC H-Class);

c4 column (ACQUITY UPLC BEH 300C 4,5.0 μm 2.1100 mm).

3. Experimental procedure

Preparing a sample and a mobile phase; and taking a proper amount of MBD-6 protein in a sample injection bottle to be injected.

And (3) on-machine analysis: MBD-6 protein is separated by an ultra-high performance liquid chromatography system.

Mobile phase a (0.1% aqueous FA): weighing 900mL of ultrapure water, adding 1mL of formic acid, uniformly mixing, adding water to a constant volume of 1000mL, preserving at room temperature, and keeping the effective period for 1 month;

mobile phase B (0.1% FA acetonitrile solution): 900mL of acetonitrile is measured, 1mL of formic acid is added, acetonitrile is added to the mixture to reach 1000mL after uniform mixing, and the mixture is preserved at room temperature for 1 month in the effective period.

The mobile phase is replaced to start the gradient balance system until the baseline balance is reached, and sample injection is performed according to the sequence of Blank and test sample after the mass spectrum correction is completed, and the specific liquid phase method is shown in Table 6.

Table 6 liquid phase mass spectrometry method

Data analysis: deconvolution analysis is carried out on the original data to obtain accurate molecular weight values. TOF Resolution:10000; output range:5000-30000.

The TIC profile, mass spectrum detection profile and deconvolution molecular weight profile of MBD-6 protein are shown in FIGS. 7, 8 and 9, respectively.

Example 5 testing of enrichment efficiency under other protein tags

To further verify the effect of different purification tags on purification effect and enrichment efficiency, the inventors compared and tested the tags of four commonly used magnetic bead-protein binding modes on the basis of MBD-3: his tag-Ni/Co magnetic beads; NH label-Ni/Co magnetic beads; biotin tag-streptavidin magnetic beads; fc label-protein A/G magnetic beads prove that the optimized sequence structure has good enrichment efficiency under various label-magnetic bead combination modes.

And using a 6 XNH label to obtain the MBD-6.

Using the 6×His tag, MBD-7 was obtained with the amino acid sequence as follows:

GEKMDESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKP QLARYLGNTVDLSSFDFRTGKMMPSKLQKNKQRLRNDVYYGAAGHHHHHH(SEQ ID No.11)

MBD-8 was obtained using biotin Avi tag, the amino acid sequence was as follows:

GEKMDESGKRMDCPALPPGWKKEEVIRKSGLSAGKSDVYYFSPSGKKFRSKP QLARYLGNTVDLSSFDFRTGKMMPSKLQKNKQRLRNDVYYGAAGGLNDIFEAQKI EWHEPGAAGHNHNHNHNHNHN(SEQ ID No.12)

MBD-9 was obtained using Fc protein tags, and its amino acid sequence was as follows:

MKWVTFLLLLFVSDSAFSGEKMDESGKRMDCPALPPGWKKEEVIRKSGLSAG

KSDVYYFSPSGKKFRSKPQLARYLGNTVDLSSFDFRTGKMMPSKLQKNKQRLRND

VYYGAAGPGAAGAGSDQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH

QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT

CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV

FSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID No.13)

preparing experimental materials:

the materials were substantially identical to those of example 3 except for the magnetic bead portion.

Protein-binding magnetic beads for MBD-6: ni magnetic beads (Thermo, 88831); or Ni magnetic beads (Beaverbio, 70501); the method comprises the steps of carrying out a first treatment on the surface of the Or Co magnetic beads (Beaverbio, 70502)

Protein-binding magnetic beads for MBD-7: ni magnetic beads (Thermo, 88831); or Ni magnetic beads (Beaverbio, 70501); or Co magnetic beads (Beaverbio, 70502).

Protein-binding magnetic beads for MBD-8: streptavidin magnetic beads (Thermo, 88017); or streptavidin magnetic beads (Beaverbio, 22309).

Protein-binding magnetic beads for MBD-9: protein a/G magnetic beads (Thermo, 88802); or protein a magnetic beads (Thermo, 88845). The method comprises the steps of carrying out a first treatment on the surface of the Or protein G magnetic beads (Thermo, 88848).

According to the experimental procedure of example 2 and example 3 (reference may be made to the magnetic bead protocol for different combinations of magnetic beads), the corresponding enrichment test results are shown in fig. 10 and tables 7 and 8:

TABLE 7 methylation enrichment test protein detection results

TABLE 8 methylation enrichment test protein enrichment efficiency results

			MBD-6	8.6	99.74％
MBD-7	8.59	99.48％
			MBD-8	8.37	99.40％
MBD-9	7.81	99.12％

As can be seen from the results of FIG. 10, table 7 and Table 8, the delta CT values of MBD-6, MBD-7, MBD-8 and MBD-9 can reach about 8, and the enrichment efficiency can be higher than 99% in different tag designs. The results thus demonstrate that the protein based on MBD-3 structure, on His tag-Ni/Co magnetic beads; NH label-Ni/Co magnetic beads; biotin tag-streptavidin magnetic beads; the Fc label-protein A/G magnetic beads and other different coordination effects can have good enrichment effect.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (10)

1. A methylation enrichment protein is characterized in that the amino acid sequence of the methylation enrichment protein is shown in SEQ ID No. 4.

2. A gene encoding a methylation enriched protein according to claim 1, wherein the nucleotide sequence is shown in SEQ ID No. 7.

3. An expression vector comprising the sequence of the gene of claim 2.

4. The expression vector of claim 3, further comprising a sequence for expressing a functional tag selected from at least one of an NH tag, a His tag, an Avi tag, and an Fc tag.

5. A host cell comprising the expression vector of claim 3 or 4.

6. A method of making a methylation-enriched protein of claim 1, comprising the step of inducing expression of the host cell of claim 5.

7. The method of claim 6, wherein the expression vector is pET-28a and the host cell is e.

8. The method of claim 7, wherein the step of inducing the host cell to express the protein comprises:

s1, culturing the escherichia coli at 37 ℃ by using an LB medium containing 50 mug/mL kanamycin,

s2, when the OD600 of the escherichia coli culture solution is 0.5-0.7, the induction expression is carried out by using IPTG with the final concentration of 1mM, wherein the induction conditions are as follows: 16 ℃,220rpm, overnight;

s3, centrifuging the culture solution at 6000rpm for 5min, and collecting thalli;

s4, protein purification is carried out after the bacterial cells are crushed.

9. Use of a methylation-rich protein according to claim 1 for the preparation of a kit for enriching methylated DNA.

10. A kit for enriching methylated DNA comprising a methylation-enriched protein according to claim 1.