SA-MBD recombinant protein and expression method and application

Abstract

The present invention discloses a SA-MBD recombinant protein, an expression method and an application; the SA-MBD recombinant protein is recombined from streptavidin (SA) and a methylated DNA binding domain (MBD) and is used for the detection of methylated DNA. The expression method of the SA-MBD recombinant protein is to artificially synthesize DNA encoding SA-MBD, clone it into the prokaryotic expression vector pET-30a(+), transform Escherichia coli BL21 (DE3), induce expression with IPTG at 37°C, and purify it with nickel column affinity. The SA-MBD recombinant protein of the present invention improves the affinity of the methylation binding domain MBD with methylated DNA and simplifies the fluorescent labeling process of the MBD protein. It can be used for in situ imaging analysis of methylated DNA in cells.

Description

Technical Field

[0001] The present invention relates to the fields of biology and biotechnology, and in particular to a SA-MBD recombinant protein and an expression method and application thereof. Background Art

[0002] DNA methylation is a type of epigenetic modification, and CpG island cytosine methylation is considered to be the main epigenetic modification in mammals. Gene methylation in the promoter region can silence genes, and abnormal DNA methylation at the genome-wide level leads to deregulation of gene expression and causes a variety of human diseases.

[0003] The methyl-CpG-binding domain (MBD) protein family is a key member that determines the transcriptional state of the epigenome, and MBD proteins are key regions that bind to methylated DNA. Through abnormal expression and post-translational modification, MBD proteins disrupt the interaction and localization with protein binding partners, constituting a potential mechanism for the occurrence of human diseases. Streptavidin (SA) consists of four subunits, each of which has a biotin binding site and has a very strong affinity for biotin.

[0004] The search has not yet found any reports on the recombinant SA and MBD to prepare SA-MBD recombinant protein. Summary of the invention

[0005] In view of the deficiencies of the prior art, the first object of the present invention is to provide a SA-MBD recombinant protein and an expression method and application;

[0006] The second object of the present invention is to provide a method for expressing SA-MBD recombinant protein.

[0007] The third object of the present invention is to provide the application of the aforementioned SA-MBD recombinant protein.

[0008] The present invention has the following technical solutions:

[0009] A SA-MBD recombinant protein, whose amino acid sequence is shown in SEQ ID NO: 1.

[0010] Streptavidin SA is a natural tetramer, so the SA-MBD recombinant protein will also form a tetramer. After testing, the SA-MBD recombinant protein monomer is composed of 299 amino acids, with a molecular weight of 31.56 KDa and an isoelectric point of 10.2.

[0011] The present invention also protects the gene encoding SA-MBD recombinant protein.

[0012] The nucleotide sequence of the aforementioned gene is shown in SEQ ID NO:2.

[0013] The present invention also protects expression cassettes, recombinant vectors, transgenic cell lines or recombinant engineering bacteria containing the above-mentioned genes.

[0014] The present invention also protects a method for expressing a SA-MBD recombinant protein, which includes artificially synthesizing DNA encoding SA-MBD, cloning it into a prokaryotic expression vector pET-30a(+), transforming Escherichia coli BL21(DE3), inducing expression with IPTG at 37°C, and affinity purification with a nickel column to obtain a SA-MBD recombinant protein with high affinity for methylated DNA.

[0015] As a preferred technical solution of the present application, the main steps of the expression method are as follows:

[0016] (1) Artificially synthesize the coding gene of the target protein SA-MBD;

[0017] (2) constructing the coding gene of the SA-MBD protein synthesized in step (1) into an Escherichia coli expression vector to obtain a recombinant expression vector;

[0018] (3) transforming Escherichia coli host cells with the recombinant expression vector obtained in step (2) to obtain recombinant genetically engineered bacteria;

[0019] (4) using the recombinant genetically engineered bacteria in step (3) as the production strain, adding IPTG, and inducing expression at 37° C. for 8 h;

[0020] (5) Ultrasonicate the cells in step (4), centrifuge, load the supernatant onto a nickel column, affinity purify the target protein, and remove small molecules such as imidazole through a gravity desalting column.

[0021] As the preferred technical solution of the present application, the expression vector is pET30a(+).

[0022] As a preferred technical solution of the present application, in the SA-MBD recombinant protein:

[0023] The SA sequence includes 160 amino acids, and the sequence is shown in SEQ ID NO: 3;

[0024] The MBD sequence includes 112 amino acids, and the sequence is shown in SEQ ID NO: 4;

[0025] The linker sequence includes 20 amino acids, and the sequence is shown in SEQ ID NO: 5;

[0026] The separation and purification tag sequence tag comprises 6 histidines, and the sequence is shown in SEQ ID NO:6.

[0027] The present invention also protects the aforementioned SA-MBD recombinant protein, and / or, the gene mentioned above, and / or, the use of an expression cassette, a recombinant vector, a transgenic cell line or a recombinant engineered bacterium containing the gene mentioned above in the in situ detection of methylated DNA.

[0028] The MBD part of the recombinant protein binds to the methylated DNA, and then the biotin part of biotin-FITC binds to the SA part of the recombinant protein, and the methylated DNA can be imaged using a fluorescence microscope.

[0029] As a preferred technical solution of this application, the application includes the following specific steps:

[0030] (1) Culture A549 cells for 15 h;

[0031] (2) Wash the cells twice with PBS solution;

[0032] (3) Digest the cells with 2 mL of trypsin for 10 min, centrifuge at 1000 rpm for 5 min, and resuspend the A549 cells in 1 mL of 1% Triton X-100 in 75 mM KCl solution;

[0033] (4) Spread 200 μL of the resuspension onto a glass slide and air-dry;

[0034] (5) Place 20 μL of 1 μM SA-MBD recombinant protein on a glass slide and incubate for 3 h;

[0035] (6) After rinsing with PBS for 1 min, 20 μL of 1.5 μM Biotin-FITC was placed on the slide and incubated for 2 h;

[0036] (7) After rinsing with PBS for 1 min, 20 μL of DAPI solution was placed on the slide for counterstaining;

[0037] (8) Imaging was performed using an inverted fluorescence microscope.

[0038] Beneficial Effects

[0039] The present invention has the following advantages and effects:

[0040] (1) The present invention utilizes the characteristic of streptavidin to naturally form tetramers, and fuses streptavidin and a methylation binding domain (MBD) for expression, thereby increasing the affinity of MBD for methylated DNA.

[0041] (2) The present invention utilizes a streptavidin and biotin system to simplify the fluorescent labeling process of recombinant proteins and can be used for in situ detection of methylated DNA in cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is an SDS-PAGE electrophoresis diagram of the induced expression of the SA-MBD recombinant protein provided in an embodiment of the present invention;

[0043] Figure 2 The figure is an SDS-PAGE electrophoresis diagram of the purified SA-MBD recombinant protein provided according to an embodiment of the present invention; wherein, 1-Marker; 2-uninduced; 3-supernatant; 4-1×binding buffer eluent; 5-30mM imidazole eluent; 6-60mM imidazole eluent; 7-100mM imidazole eluent; 8-150mM imidazole eluent; 9-200mM imidazole eluent; 10-300mM imidazole eluent;

[0044] Figure 3 It is a schematic diagram of Western blot identification of SA-MBD recombinant protein according to an embodiment of the present invention;

[0045] Figure 4 is an agarose gel electrophoresis diagram of DNA containing a methylation site binding to SA-MBD recombinant protein or MBD protein provided in an embodiment of the present invention;

[0046] Figure 5 and Figure 6 is an SPR affinity diagram of DNA containing three methylation sites and SA-MBD recombinant protein or MBD protein provided in an embodiment of the present invention;

[0047] Figure 7 This is an imaging diagram of the in situ detection of methylated DNA by the SA-MBD recombinant protein provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0048] The embodiments of the present invention are described in detail below. It should be noted that the described embodiments are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0049] In order to facilitate the understanding of those skilled in the art, some terms in this document are explained and illustrated. These explanations and illustrations are only used to facilitate understanding and should not be regarded as limiting the scope of protection of the present invention.

[0050] Explanation of the terms in this invention:

[0051] Blocking DNA sequence: 5′-GATCTC GTCTGT CGCCTA GCGAGC ATCGGA CTGCGG ACTG-3′.

[0052] Reagent preparation:

[0053] Buffer 1: 1× Binding Buffer, composed of 1 M NaCl, 40 mM Tris, and 10 mM imidazole;

[0054] Buffer 2: 1× Charge Buffer, composed of 50 mM NiSO4;

[0055] Buffer 3: contains 30 mM imidazole, composed of 0.5 M NaCl, 20 mM Tris, 30 mM imidazole;

[0056] Buffer 4: contains 60 mM imidazole, composed of 0.5 M NaCl, 20 mM Tris, 60 mM imidazole;

[0057] Buffer 5: contains 100 mM imidazole, composed of 0.5 M NaCl, 20 mM Tris, 100 mM imidazole;

[0058] Buffer 6: contains 300 mM imidazole, composed of 0.5 M NaCl, 20 mM Tris, 300 mM imidazole;

[0059] Buffer 7: 1× Strip Buffer, composed of 0.5 M NaCl, 20 mM Tris, 100 mM EDTA;

[0060] Buffer 8: composed of 20 mM HEPES, 3 mM MgCl2, 1 mM DTT, 100 mM KCl, 0.1% Triton X-100.

[0061] 1. Preparation of SA-MBD recombinant protein by prokaryotic expression system

[0062] 1. Synthesis of the nucleic acid sequence of SA-MBD recombinant protein, as shown in SEQ ID NO: 2.

[0063] 2. Inducible expression of SA-MBD recombinant protein

[0064] Pick the positive clones and inoculate them in a test tube containing 5μL kanamycin (30mg / mL) + 5μL chloramphenicol (34mg / mL) + 5mL LB medium, and shake culture at 37℃ for 12h. Transfer the bacterial solution after 12h culture at a ratio of 1:100 to fresh LB medium containing kanamycin and chloramphenicol, shake culture at 37℃ until OD reaches 0.6, add IPTG to a final concentration of 0.1mM, and shake and induce expression at 37℃ for 8h.

[0065] 3. Purification of SA-MBD recombinant protein

[0066] Centrifuge at 8000×g for 3 min at 4°C and discard the supernatant; wash the bacteria with buffer 1 and resuspend them with 30 mL of buffer 1; add 1‰ volume of lysozyme (10 mg / mL) and let it stand at room temperature for 40 min; ultrasonically break the bacteria (place it in an ice bath) using a power of 400 watts, 10 mL per batch, ultrasonicate for 10 s, stop for 10 s, and the total ultrasonic time for each batch is 10 min; after ultrasonication, centrifuge at 12000×g for 30 min and collect the supernatant.

[0067] Adjust the nucleic acid protein instrument to 280nm, 0.1A, take out the nickel column, rinse the resin with sterile water, adjust the flow rate to one drop / 3s; wash with 5 volumes of buffer 2; balance with 3 volumes of buffer 1, and zero the instrument; load the collected sample supernatant onto the column; after the sample is loaded, wash the column with 10 volumes of buffer 1; elute with 6 volumes of buffer 3; elute with 6 volumes of buffer 4; elute with 6 volumes of buffer 5; elute with 6 volumes of buffer 6, and collect all the parts flowing out from the place where the signal increases; wash the column with 5 volumes of buffer 7.

[0068] The eluted protein was ultrafiltered using a 10KDa ultrafiltration tube, replaced with buffer 8 three times, and centrifuged at 12,000×g for 4 min at 4°C.

[0069] 2. SDS-PAGE electrophoresis and Western blot identification of SA-MBD expression products

[0070] 1. Determination of the molecular weight of SA-MBD recombinant protein by SDS-PAGE

[0071] SDS-PAGE uses a separation gel concentration of 12% and a concentrated gel concentration of 5%; the sample is boiled for 10 minutes and then 10 μL is taken out for electrophoresis, and a standard molecular weight is added as a control; the initial voltage is 60V for 30 minutes, then the voltage is adjusted to 110V for 90 minutes, and the gel is stripped and stained with Coomassie Brilliant Blue; after electrophoresis, the molecular weight of the protein to be tested is obtained based on the molecular weight of the standard protein.

[0072] like Figure 1 As shown: After IPTG induction, SA-MBD recombinant protein was expressed compared with that without induction.

[0073] like Figure 2 As shown: Nickel column affinity purification protein, 150mM, 200mM, 300mM imidazole elution all have the target band, finally 30mM, 60mM, 100mM imidazole are used to elute the impurities, and 300mM imidazole is used to elute the target protein.

[0074] 2. Western blot identification of SA-MBD recombinant protein

[0075] The recombinant protein MBD designed by ourselves and irrelevant proteins were selected as negative controls. After electrophoresis, the 25Kda-48KDa gel was cut under the indication of the marker and transferred to the membrane at 4°C and 300mA for 1.5h; the blocking solution was used for 10min; 8mL TBST-diluted HRP conjugated Biotin (1:500) was added and incubated at room temperature for 1h; TBST was washed 3 times, 10min each time; 100μL of developer was evenly dropped on the membrane surface and developed after 2min of light protection.

[0076] like Figure 3 As shown: After incubating the bands with biotin-linked horseradish peroxidase, only the SA-MBD recombinant protein has a specific band.

[0077] 3. High affinity of SA-MBD recombinant protein for methylated DNA

[0078] 1. Agarose gel electrophoresis verifies the high affinity of SA-MBD recombinant protein to DNA containing a methylation site

[0079] The concentration of blocked DNA is 30μM, the concentration of protein is 5μM, and the concentration of methylated DNA is 1μM. According to Table 1 below, dilute the protein with buffer 8, then add 2μL of blocked DNA and incubate at room temperature for 10min; then add methylated DNA and incubate at room temperature for 1h; take 10μL of the solution after the reaction and run agarose gel electrophoresis, the gel concentration used for electrophoresis is 1%, 100V, 20min.

[0080] Table 1 Addition volume of SA-MBD protein and buffer 8

[0081] SA-MBD / μL 0 1 2 4 6 8 10 Buffer 8 / μL 14 13 12 10 8 6 4

[0082] like Figure 4 As shown: After adding protein, DNA and protein bind to cause the molecular weight to increase and run slower. Compared with the original MBD protein, SA-MBD recombinant protein has a higher affinity for methylated DNA.

[0083] 2. SPR verification of the high affinity of SA-MBD recombinant protein to DNA containing three methylation sites

[0084] The affinity of recombinant protein to DNA was determined by Biacore T200. An appropriate amount of recombinant protein was fixed on a CM5 chip, and PBS-P (10mM NaH2PO4 / Na2HPO4+2.7mM KCl+137mM NaCl+0.05% P20) was used as the running buffer. DNA was introduced at a flow rate of 30μL / min for 60s, dissociated with the running buffer for 60s, and the concentration of DNA was diluted by 2 times.

[0085] like Figure 5 Shown: K of methylated DNA and SA-MBD D 6.333nM;

[0086] like Figure 6 Shown: K of methylated DNA and MBD D The SPR results further demonstrated that SA-MBD had a higher affinity for methylated DNA.

[0087] 4. Application of SA-MBD recombinant protein for in situ detection of methylated DNA

[0088] (1) Culture A549 cells for 15 h;

[0089] (2) Wash the cells twice with PBS solution;

[0090] (3) Digest the cells with 2 mL of trypsin for 10 min, centrifuge at 1000 rpm for 5 min, and resuspend the A549 cells in 1 mL of 1% Triton X-100 in 75 mM KCl solution;

[0091] (4) Spread 200 μL of the resuspension onto a glass slide and air-dry;

[0092] (5) Place 20 μL of 1 μM SA-MBD recombinant protein on a glass slide and incubate for 3 h;

[0093] (6) After rinsing with PBS for 1 min, 20 μL of 1.5 μM Biotin-FITC was placed on the slide and incubated for 2 h;

[0094] (7) After rinsing with PBS for 1 min, take 20 μL of DAPI solution and apply it to the slide for counterstaining.

[0095] (8) Imaging was performed using an inverted fluorescence microscope.

[0096] like Figure 7 As shown: After Biotin-FITC was incubated with SA-MBD protein, FITC co-localized with DAPI dye under inverted fluorescence microscopy.

[0097] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims. Sequence Listing <110> China Pharmaceutical University <120> SA-MBD recombinant protein and expression method and application <160> 6 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 299 <212> PRT <213> Artificial Sequence <400> 1 Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser 1 5 10 15 Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp 20 25 30 Ser Pro Asp Leu Gly Thr Gly Ala Ala Glu Ala Gly Ile Thr Gly Thr 35 40 45 Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr Ala Gly Ala Asp 50 55 60 Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser 65 70 75 80 Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro Ala Thr Asp Gly 85 90 95 Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg 100 105 110 Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala 115 120 125 Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Glu 130 135 140 Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys 145 150 155 160 Val Lys Pro Ser Ala Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 165 170 175 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Glu Ser Trp 180 185 190 Gln Asp Cys Pro Ala Leu Gly Pro Gly Trp Lys Arg Arg Glu Ser Phe 195 200 205 Arg Lys Ser Gly Ala Ser Phe Gly Arg Ser Asp Ile Tyr Tyr Gln Ser 210 215 220 Pro Thr Gly Glu Lys Ile Arg Ser Lys Val Glu Leu Thr Arg Tyr Leu 225 230 235 240 Gly Pro Ala Cys Asp Leu Thr Leu Phe Asp Phe Arg Gln Gly Thr Leu 245 250 255 Cys His Pro Ile Pro Lys Thr His Pro Leu Ala Val Pro Ser Lys Lys 260 265 270 Lys Lys Lys Pro Ser Lys Pro Ala Lys Thr Lys Lys Gln Gln Val Gly 275 280 285 Leu Gln Arg Ser Glu Val Arg Arg Glu Thr Pro 290 295 <210> 2 <211> 786 <212> DNA <213> Artificial Sequence <400> 2 ggtgctgctg aagcaggtat caccggcacc tggtacaacc agctcggctc gaccttcatc 60 gtgaccgcgg gcgccgacgg cgccctgacc ggaacctacg agtcggccgt cggcaacgcc 120 gagagccgct acgtcctgac cggtcgttac gacagcgccc cggccaccga cggcagcggc 180 accgccctcg gttggacggt ggcctggaag aataactacc gcaacgccca ctccgcgacc 240 acgtggagcg gccagtacgt cggcggcgcc gaggcgagga tcaacaccca gtggctgctg 300 acctccggca caaccgaggc caacgcctgg aagtccacgc tggtcggcca cgacaccttc 360 accaaggtga agccgtccgc cgcctccgga ggaggaggaa gtggcggcgg cggcagcggt 420 ggtggtggtt ccggcggcgg cggcagtatg gctgagtcct ggcaggactg cccagccctg 480 ggccctggct ggaaacgccg agagtccttt cgaaagtcag gggcctcgtt tggacgctca 540 gacatctatt accagagccc cacaggagaa aagatccgaa gcaaagttga gctgactcgg 600 tacttgggcc ctgcatgtga tcttaccctg tttgacttca gacaaggcac cttatgccat 660 cccattccca agactcatcc cttggctgtc cccagcaaga agaaaaagaa gccttctaaa 720 ccagccaaga ctaagaaaca gcaggttggg ctgcagcgaa gtgaggtcag gagagagact 780 ccataa 786 <210> 3 <211> 160 <212> PRT <213> Artificial Sequence <400> 3 Ser Ser Gly Leu Val Pro Arg Gly Ser Gly Met Lys Glu Thr Ala Ala 1 5 10 15 Ala Lys Phe Glu Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly 20 25 30 Ala Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser 35 40 45 Thr Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr 50 55 60 Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg 65 70 75 80 Tyr Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp 85 90 95 Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr 100 105 110 Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln 115 120 125 Trp Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr 130 135 140 Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser 145 150 155 160 <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <400> 4 Met Ala Glu Ser Trp Gln Asp Cys Pro Ala Leu Gly Pro Gly Trp Lys 1 5 10 15 Arg Arg Glu Ser Phe Arg Lys Ser Gly Ala Ser Phe Gly Arg Ser Asp 20 25 30 Ile Tyr Tyr Gln Ser Pro Thr Gly Glu Lys Ile Arg Ser Lys Val Glu 35 40 45 Leu Thr Arg Tyr Leu Gly Pro Ala Cys Asp Leu Thr Leu Phe Asp Phe 50 55 60 Arg Gln Gly Thr Leu Cys His Pro Ile Pro Lys Thr His Pro Leu Ala 65 70 75 80 Val Pro Ser Lys Lys Lys Lys Lys Pro Ser Lys Pro Ala Lys Thr Lys 85 90 95 Lys Gln Gln Val Gly Leu Gln Arg Ser Glu Val Arg Arg Glu Thr Pro 100 105 110 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <400> 5 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <400> 6 Met His His His His His His 1 5

Claims

1. SA-MBD recombinant protein, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. A method for expressing SA-MBD recombinant protein, characterized in that: DNA encoding SA-MBD was artificially synthesized, cloned into the prokaryotic expression vector pET-30a(+), transformed into Escherichia coli BL21(DE3), induced for expression with IPTG at 37°C, and affinity purified by nickel column to obtain SA-MBD recombinant protein with high affinity for methylated DNA, wherein the amino acid sequence of the SA-MBD recombinant protein is shown in SEQ ID NO:

1.

3. The method for expressing the SA-MBD recombinant protein according to claim 2, characterized in that: The main steps are as follows: (1) Artificially synthesize the coding gene of the target protein SA-MBD; (2) constructing the coding gene of the SA-MBD protein synthesized in step (1) into an Escherichia coli expression vector to obtain a recombinant expression vector; (3) transforming Escherichia coli host cells with the recombinant expression vector obtained in step (2) to obtain recombinant genetically engineered bacteria; (4) Use the recombinant genetically engineered bacteria in step (3) as the production strain, add IPTG, and induce expression at 37°C for 8 hours; (5) Ultrasonicate the cells in step (4), centrifuge, load the supernatant onto a nickel column, affinity purify the target protein, and remove the imidazole small molecules through a gravity desalting column.

4. The method for expressing the SA-MBD recombinant protein according to claim 3, characterized in that: The expression vector was pET30a(+).

5. The method for expressing the SA-MBD recombinant protein according to claim 4, characterized in that: The SA sequence includes 160 amino acids, and the sequence is shown in SEQ ID NO: 3; The MBD sequence includes 112 amino acids, and the sequence is shown in SEQ ID NO: 4; The linker sequence includes 20 amino acids, and the sequence is shown in SEQ ID NO: 5; The separation and purification tag sequence tag contains 6 histidines, and the sequence is shown in SEQ ID NO:

6.

6. Use of the recombinant protein according to claim 1 in in situ detection of methylated DNA, wherein the use is for non-disease diagnosis and treatment purposes.

7. The use according to claim 6, characterized in that: The specific steps include: (1) Culture A549 cells for 15 h; (2) Wash the cells twice with PBS solution; (3) Digest the cells with 2 mL of trypsin for 10 min, centrifuge at 1000 rpm for 5 min, and resuspend the A549 cells in 1 mL of 1% Triton X-100 in 75 mM KCl solution; (4) Take 200 μL of the resuspension and spread it on a glass slide and air dry; (5) Place 20 μL of 1 μM SA-MBD recombinant protein on a glass slide and incubate for 3 h; (6) After rinsing with PBS for 1 min, apply 20 μL of 1.5 μM Biotin-FITC on the slide and incubate for 2 h; (7) After rinsing with PBS for 1 min, take 20 μL of DAPI solution and apply it to the slide for counterstaining; (8) Imaging was performed using an inverted fluorescence microscope.

Images