m6A-binding proteins and their applications

Abstract

This invention provides an m6A binding protein and its applications, relating to the field of biotechnology. The m6A binding protein provided by this invention has variable regions comprising complementarity-determining regions CDR1-VH, CDR2-VH, CDR3-VH, CDR1-VL, CDR2-VL, and CDR3-VL with specific amino acid sequences. This m6A binding protein can specifically recognize m6A, exhibits high affinity for m6A, and can be used in the preparation of m6A detection products.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an m6A binding protein and its applications. Background Technology

[0002] With the adoption of classic hybridoma technology, rabbit monoclonal antibodies have become more widely used in scientific research and pathological testing. Simultaneously, monoclonal antibodies are also widely used as highly specific diagnostic tools and as therapeutic agents for viral infections, tumors, autoimmune diseases, metabolic diseases, and neurological disorders.

[0003] However, due to limitations imposed by rabbit myeloma cells, very few companies currently utilize hybridoma technology to develop rabbit monoclonal antibodies. To circumvent patents and technological barriers associated with rabbit B-cell hybridoma technology, other antibody discovery technologies based on single B cells have been developed.

[0004] In vitro culture of single B cells and screening of single B cells using microfluidic technology are two commonly used single B cell sorting methods. In vitro culture of single B cells requires well-established protocols for B cell enrichment, feeder cell construction, and cytokine stimulation. The entire protocol necessitates optimization of the B cell enrichment protocol, feeder cell viability, and cytokine stimulation protocol, presenting significant technical challenges and requiring a long optimization cycle for reagent ratios. On the other hand, the development of monoclonal antibodies using microfluidic technology, primarily exemplified by the Beacon platform developed by Berkeley Lights in recent years, integrates single B cell PCR technology through microfluidic manipulation of single B cells for recombinant antibody development. However, its main drawbacks are the extremely high cost of the Beacon equipment and its low cell screening throughput, allowing only tens of thousands of cells to be screened at a time.

[0005] Based on the above, there is an urgent need for new antibody screening technologies that are convenient and have high throughput to meet the growing demand for antibody discovery. Flow cytometry, with its ability to perform high-throughput cell sorting using different fluorescent labels, can sort hundreds of thousands to tens of millions of cell populations in a short time. Target B cells selected through screening using B cell surface membrane-bound immunoglobulins and specific fluorescently labeled antigens can be separated and their single-cell RNA pre-amplified in vitro to obtain recombinant antibody sequence information, significantly increasing the speed at which specific antigen-antibody sequences can be obtained from immunized animals. Therefore, establishing antibody development protocols based on flow cytometry for the isolation of single B cells is essential.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a binding protein for m6A (N6-methyladenosine) that exhibits high specificity and strong affinity for m6A.

[0008] A second objective of this invention is to provide an application of the above-mentioned binding protein in the preparation of m6A detection products.

[0009] A third objective of this invention is to provide an ELISA kit for detecting m6A.

[0010] A fourth objective of the present invention is to provide a gene encoding the said binding protein.

[0011] The fifth objective of this invention is to provide a recombinant plasmid.

[0012] The sixth objective of this invention is to provide a cell line.

[0013] The seventh objective of this invention is to provide a method for preparing the above-mentioned binding protein.

[0014] In a first aspect, the present invention provides an m6A binding protein, the variable region of which comprises: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.6.

[0015] As a further technical solution, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0016] As a further technical solution, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO.8.

[0017] Secondly, the present invention provides the application of the above-mentioned binding protein in the preparation of m6A detection products.

[0018] As a further technical solution, the product includes immunochromatographic test strips and ELISA kits.

[0019] Thirdly, the present invention provides an ELISA kit for detecting m6A, wherein the kit uses the aforementioned binding protein as a capture protein.

[0020] Fourthly, the present invention provides a gene encoding the binding protein, the gene having nucleic acid sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0021] Fifthly, the present invention provides a recombinant plasmid comprising a vector and the gene.

[0022] In a sixth aspect, the present invention provides a cell line expressing the aforementioned binding protein.

[0023] In a seventh aspect, the present invention provides a method for preparing a binding protein, comprising: culturing the cell line in a culture medium, and then isolating the binding protein.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The m6A binding protein provided by this invention can specifically recognize m6A, has a high affinity for m6A, and can be used in the preparation of m6A detection products. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 It has the structural formula of N6-methyladenosine;

[0028] Figure 2 The SDS-PAGE detection results provided in Example 1;

[0029] Figure 3 The flowchart for antigen fluorescent labeling provided in Example 5;

[0030] Figure 4 The detection results provided in Example 6;

[0031] Figure 5 The competing ELISA detection results provided in Example 9;

[0032] Figure 6 The structural formula is for a small molecule analog of m6A;

[0033] Figure 7 The results are for the specific detection of the 1G1 antibody provided in Example 12. Detailed Implementation

[0034] The embodiments and examples of the present invention will be described in detail below. However, those skilled in the art will understand that the following embodiments and examples are for illustrative purposes only and should not be considered as limiting the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0035] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domain of the heavy chain can be referred to as "VH," and the variable domain of the light chain as "VL." These domains are typically the most variable parts of the antibody and contain antigen-binding sites. The variable region of the light or heavy chain consists of a framework region interrupted by three hypervariable regions called "complementarity-determining regions" or "CDRs." The framework region of the antibody, that is, the framework region of the combination of the light and heavy chains, plays a role in locating and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0036] The “framework” or “FR” region refers to the region outside of those defined as CDRs of the antibody variable domain. Each antibody variable domain framework can be further subdivided into adjacent regions separated by CDRs (FR1, FR2, FR3, and FR4).

[0037] Typically, the variable regions VL / VH of heavy and light chains can be obtained by connecting the following numbered CDRs with FRs in the following combination: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0038] In this invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three highly variable regions of the heavy chain variable region, and correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three highly variable regions of the light chain variable region.

[0039] In a first aspect, the present invention provides an m6A binding protein, the variable region of which comprises: a complementarity-determining region CDR1-VH having the amino acid sequence shown in SEQ ID NO.1, a complementarity-determining region CDR2-VH having the amino acid sequence shown in SEQ ID NO.2, a complementarity-determining region CDR3-VH having the amino acid sequence shown in SEQ ID NO.3, a complementarity-determining region CDR1-VL having the amino acid sequence shown in SEQ ID NO.4, a complementarity-determining region CDR2-VL having the amino acid sequence shown in SEQ ID NO.5, and a complementarity-determining region CDR3-VL having the amino acid sequence shown in SEQ ID NO.6.

[0040] The sequences of the variable regions mentioned above are shown in Table 1:

[0041] Table 1

[0042] CDR1-VH YYHMS SEQ ID NO.1 CDR2-VH ISSPGGTTYYASWAKG SEQ ID NO.2 CDR3-VH GWSSSSKFML SEQ ID NO.3 CDR1-VL QASQNVYNNLA SEQ ID NO.4 CDR2-VL DASALAS SEQ ID NO.5 CDR3-VL QGGYYYYNYIGG SEQ ID NO.6

[0043] The m6A binding protein provided by this invention can specifically recognize m6A, has a high affinity for m6A, and can be used for the detection of m6A.

[0044] In some preferred embodiments, the variable region includes a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.7.

[0045] The amino acid sequence of the heavy chain variable region VH in the variable region is as follows:

[0046] QSVEESGGRLVTPGTPLTLTCTASGFSTSYYHMSWVRQVPGKGLEW IGISSPGGTTYYASWAKGRFTISKTSTTVDLKITSLTTEDTATYFCVRGWSS SSKFMLWGQGTLVTVSS (SEQ ID NO. 7).

[0047] In some preferred embodiments, the variable region includes a light chain variable region VL having an amino acid sequence as shown in SEQ ID NO. 8.

[0048] The amino acid sequence of the light chain variable region VL in the variable region is as follows:

[0049] DVVVTQTPASVSEPVGGTVTINCQASQNVYNNLAWYQQKPGQPPK LLIYDASALASGVPSRFSGSGSGTEFTLTISGVQCDDAATYYCQGGYYYY NYIGGFGGGTEVVVK (SEQ ID NO. 8).

[0050] In some preferred embodiments, the binding protein further includes a light chain constant region and a heavy chain constant region. The combination of the constant region and the variable region yields a complete antibody.

[0051] Secondly, the present invention provides the application of the above-mentioned binding protein in the preparation of m6A detection products.

[0052] The m6A binding protein provided by this invention can specifically recognize m6A and has a high affinity for m6A, thus it can be used in the preparation of m6A detection products.

[0053] In some preferred embodiments, the products include, but are not limited to, immunochromatographic test strips and ELISA kits, or other products known to those skilled in the art.

[0054] Thirdly, the present invention provides an ELISA kit for detecting m6A, wherein the kit uses the aforementioned binding protein as a capture protein.

[0055] The ELISA kit provided by this invention has high sensitivity and accurate detection.

[0056] Fourthly, the present invention provides a gene encoding the binding protein, the gene having nucleic acid sequences as shown in SEQ ID NO. 9 and SEQ ID NO. 10:

[0057] cagtcggtggaggagtccgggggtcgcctggtcacgcctgggacacccctgacactcacctgcacagcctctggattctccaccagttataccacatgagttgggtccgccaggttccagggaaggggctggaatggatcggaatcagtagtcctggtggtaccacatactatg cgagctgggcaaaaggccgattcaccatctccaaaacctcgactacggtggatctgaaaatcaccagtctgacaaccgaggacacggccacctatttctgtgtcagaggatggagtagtagtagtaaatttatgttgtggggccaaggcaccctggtcaccgtctcctcag(SEQ ID NO.9).

[0058] gatgtggtggtgacccagactccagcctccgtgtctgaacctgtggggggcacagtcaccatcaattgccaggccagtcagaacgtttacaacaatttagcctggtatcagcagaaaccagggcagcctcccaagctcctgatctatgatgcatccgctctggcatc tggggtcccatcgcggttcagcggcagtggatctgggacagaattcactctcaccatcagcggcgtgcagtgtgacgatgctgccacttactactgtcaaggcggttactattattataattatattggtggttttggcggagggaccgaggtggtggtcaaag(SEQ ID NO.10).

[0059] Fifthly, the present invention provides a recombinant plasmid comprising a vector and the gene, the sequence of which is shown in SEQ ID NO. 9 and SEQ ID NO. 10.

[0060] In a sixth aspect, the present invention provides a cell line expressing the aforementioned binding protein.

[0061] This cell line can be used for the preparation of m6A-binding proteins.

[0062] In a seventh aspect, the present invention provides a method for preparing a binding protein, comprising: culturing the cell line in a culture medium, and then isolating the binding protein.

[0063] Since the cell line can express the aforementioned binding protein, culturing the cell line can yield a fermentation broth rich in the binding protein, which can then be separated to obtain the binding protein. This preparation method is simple and convenient.

[0064] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0065] Example 1: Preparation of Immunogen and Detection Antigen

[0066] N6-methyladenosine m6A ( Figure 1α-A is a methylation modifier of adenosine, the most common modification in eukaryotic cell mRNA, and its content or changes in mRNA are currently detected using antibodies. m6A, as a small molecule antigen, has a molecular weight of only 281.27 Daltons, and immunization alone cannot elicit a sufficient immune response. OVA (ovalbumin) is a relatively stable soluble albumin with a molecular weight of approximately 42.7 kDa, and is a commonly used carrier protein for weak antigenic compounds. This study conjugated m6A and OVA as a whole to induce an immune response in rabbits. Later, m6A was conjugated with BSA (bovine serum albumin) to verify whether the antibody specifically targets only the small molecule and not the carrier protein OVA.

[0067] Reagents: OVA, BSA, coupling reagent CDI (carbonyl diimidazole), DMSO, 1×PBS (pH 7.2-7.4).

[0068] Consumables: 15mL imported centrifuge tubes, 1.5mL EP tubes, pipette tips, dialysis bags.

[0069] Equipment: Electronic analytical balance, pipette, water bath, horizontal shaker, 4℃ refrigerator, -20℃ refrigerator, dialysis clamp.

[0070] Sample: m6A small molecule.

[0071] Antigen preparation:

[0072] Step a): Remove the small molecule m6A to be coupled, the coupling reagent CDI (carbonyl diimidazole), and the carrier protein OVA or BSA from the refrigerator and allow them to equilibrate to room temperature.

[0073] Step b): Weigh 5 mg of small molecule m6A, dissolve it in 0.2 ml of DMSO, and set aside.

[0074] Step c): Weigh 50 mg of CDI (carbonyl diimidazole), dissolve it in 500 μL of DMSO, add the dissolved m6A small molecule, mix, and place in a 37°C water bath for 2 hours.

[0075] Step d): Weigh 5 mg of OVA or BSA and dissolve them in 8 ml of 10 mM PBS (pH 7.2-7.4). Add the activated m6A small molecule dropwise to the OVA or BSA protein solution and react at room temperature on a horizontal shaker for 24 h.

[0076] Step e): After the reaction is complete, dialyze the solution three times with 3L of 1×PBS (pH 7.2-7.4).

[0077] Step f): Place the cross-linked m6A-BSA cross-linked complex and m6A-OVA cross-linked complex into a 1.5 mL EP tube, add 1×PBS to 1 mL, and label with the item number, concentration and date, ready for immunization and detection.

[0078] Antigen detection: Cross-linked m6A-BSA and m6A-OVA were detected by SDS-PAGE. Results: As shown. Figure 2 As shown, the molecular weights of m6A-BSA and m6A-OVA are slightly higher than those of uncrosslinked BSA and OVA.

[0079] Example 2: Rabbit immunization and serum collection

[0080] Reagent: adjuvant, 75% alcohol.

[0081] Consumables: Syringes.

[0082] Sample: m6A-OVA antigen.

[0083] Rabbit immunization:

[0084] Step a): Animal selection. Use New Zealand White rabbits, around 2.5 kg in young adulthood. Choose healthy animals with glossy fur and free movement. After selecting the animal, raise it for about two weeks to allow it to acclimatize.

[0085] Step b): Prepare for the experiment and mark the rabbits.

[0086] Step c): Remove the antigen from the -20°C freezer and thaw it at room temperature, avoiding repeated freeze-thaw cycles. Label the syringe with the project number and animal number.

[0087] Step d): Extract the antigen (the antigen should be completely mixed). The concentration of the antigen for the first immunization is 1 mg / ml, and for rabbits it is 0.5 ml / rabbit. The amount of antigen for the second to fourth immunizations is halved.

[0088] Step e): Draw the adjuvant, with a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. Ensure the adjuvant is thoroughly mixed before drawing it into the syringe.

[0089] Step f): Connect the two syringes with the syringe connecting tube and emulsify them completely. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37°C water.

[0090] Step g): Rabbits were given multiple subcutaneous injections, 0.2 ml at each point. A second immunization was given 14 days after the first, with a 7-day interval between the second and third immunizations. Blood samples were collected from the rabbits after six immunizations for titer testing.

[0091] Step h): Collecting a small serum sample: Restrain the rabbit in a frame and gently tap its ear to dilate the central auricular artery. Disinfect the area with 75% alcohol. Hold the rabbit's ear with your left hand and insert the syringe with your right hand, about one-third of the way down the central auricular artery, parallel to the artery and pointing towards the heart. Collect 8 ml of blood at a time. After collection, apply pressure with a cotton ball to stop the bleeding.

[0092] Example 3: Rabbit serum titer detection

[0093] Rabbit serum titers against small molecules were detected using an indirect ELISA method. Rabbits with serum OD450nm values ​​exceeding 0.6 at a 1:64000 dilution were considered to have acceptable titers and could proceed to the next step.

[0094] Reagents: Goat anti-rabbit-HRP (Huaan Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Sangon Biotech, domestically produced analytical grade.

[0095] Consumables: Microplate (Hangzhou Shengyou).

[0096] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), ELISA reader (MD: Cmax plus).

[0097] Indirect ELISA procedure:

[0098] Step a): Dilute m6A-BSA to 2 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0099] Step b): Shake off the liquid in the wells, and add 1% [unspecified ingredient] at a rate of 150 μL / well to the microplate.

[0100] BSA / TBS were placed in a 37℃ electric thermostatic incubator and kept at a constant temperature for 1 hour.

[0101] Step c): Shake off the liquid in the wells and add 50 μL / well of serum at different dilution ratios to the microplate. Cover and incubate at 37°C for 30 min.

[0102] Step d): Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute goat anti-rabbit-HRP to the working concentration (1:30000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 45 min.

[0103] Step e): Develop color, terminate and read the value. Discard the liquid in the wells. Add washing buffer to the microplate at a rate of 180 μL / well and wash the microplate 3 times. Add 100 μL of freshly prepared TMB chromogenic substrate to each reaction well and incubate at 37°C for 10 min. Then add 90 μL / well of stop solution to terminate the reaction and measure the OD value at 450 nm on the microplate reader.

[0104] The results, as shown in Table 2, indicate that the valence (1:64000) of both rabbits met the requirements.

[0105] Table 2

[0106]

[0107] Example 4: Isolation of rabbit peripheral blood lymphocytes (PBMCs)

[0108] Reagents: Rabbit peripheral blood lymphocyte separation kit (Solarbio: P8760), red blood cell lysis buffer (Solarbio: R1010), PBS (BasalMedia: B310KJ-500ml).

[0109] Consumables: 50mL centrifuge tubes (BD: 352070), 10mL pipettes (Hangzhou Lanjieke: 352070).

[0110] Equipment: Biosafety cabinet (Sujing: BSC-1300IIB2), centrifuge (Thermo: THM#75004530).

[0111] Isolation of rabbit peripheral blood lymphocytes (PBMCs):

[0112] Step a): Take 20 ml of anticoagulated blood from the rabbit with the best titer after the sixth immunization, place it on ice, and dilute the whole blood with an equal volume of PBS.

[0113] Step b): Add an equal volume of rabbit peripheral lymphocyte separation solution to a centrifuge tube, and spread the diluted blood evenly on top of the separation solution.

[0114] Step c): Centrifuge at 1800 rpm on a horizontal rotor for 30 min at room temperature.

[0115] Step d): After centrifugation, distinct layers will appear: the top layer is diluted plasma, the middle layer is a clear separation medium, the white membrane layer between the plasma and the separation medium is the lymphocyte layer, and the bottom of the centrifuge tube contains red blood cells and granulocytes. Carefully aspirate the white membrane layer cells into a clean 50mL centrifuge tube, and wash the white membrane layer cells with 10mL PBS. Centrifuge at 1500rpm for 5min.

[0116] Step e): Discard the supernatant, resuspend the cells in 20 mL of PBS, centrifuge at 1500 rpm for 5 min. Repeat step e) and discard the supernatant for later use.

[0117] Step f): Add 5 ml of red blood cell lysis buffer and gently vortex or invert to mix. Incubate at 37°C for 5 minutes. After red blood cell lysis, the solution should be clear and transparent.

[0118] Step g): Centrifuge at 1500 rpm for 5 minutes to precipitate white blood cells, and carefully aspirate the supernatant.

[0119] Step h): Resuspend the cells in 20 mL of PBS and centrifuge at 1500 rpm for 5 min.

[0120] Step i): Repeat step h) and discard the supernatant. The peripheral blood lymphocytes obtained are then kept for later use.

[0121] Example 5 Antigen Fluorescent Labeling

[0122] The experimental protocol involved conjugating biotin to a small-molecule conjugate carrier protein (m6A-BSA), such as... Figure 3 As shown, the m6A-BSA molecule is coated with a fluorescent dye through the binding of streptavidin and biotin, and then used for cell sorting experiments.

[0123] Reagents: small molecules, BSA, DMSO, CDI, 1×PBS (pH=7.4), biotin EZ-Link Sulfo-NHS-LC-LC-Biotin.

[0124] Consumables: EP tubes, pipette tips, 30KD ultrafiltration tubes.

[0125] Equipment: Electronic analytical balance, pipette, 4℃ freezer, -20℃ freezer, sample mixer.

[0126] m6A-OVA conjugated with biotin:

[0127] Step a): Dissolve a certain amount of small molecules, such as (1 mg), in a certain amount of DMSO, such as (50 μL).

[0128] Step b): Weigh a certain amount of BSA (small molecule: BSA = 1 mg: 1 mg) and dissolve it in PBS to 2 mg / ml.

[0129] Step c): Weigh a certain amount of CDI (small molecule: BSA: CDI = 1 mg: 1 mg: 10 mg) and completely dissolve it in DMSO to a concentration of 50 mg / ml. Then add it to the small molecule solution and react for 2 hours.

[0130] Step d): Transfer the reaction solution to the BSA component dissolved in PBS and react for 24 hours. Replace the reaction solution with 1×PBS using a 30KD ultrafiltration tube for later use.

[0131] Step e): Weigh 20 times the molar amount of BSA of EZ-Link Sulfo-NHS-LC-LC-Biotin and dissolve it in ultrapure water to a concentration of 10 mg / ml. Add the solution to the mixture and react for 1 hour. Then add the reaction solution to a 30 KD ultrafiltration tube for ultrafiltration and replacement.

[0132] Preparation of fluorescently labeled m6A-BSA

[0133] Reagents: Biotinylated m6A-BSA, Streptavidin - Brilliant Violet 421 TM Streptomycin - Alexa 647, BSA-Biotin.

[0134] Consumables: EP tubing, pipette tips.

[0135] Equipment: pipette.

[0136] Step a): Take 0.8 μL of biotinylated m6A-BSA and react it with 1 μL of streptavidin-Brilliant Violet421. TM or streptavidin - Alexa Mix 647 in an EP tube, gently pipette to mix, and incubate at room temperature for 20 minutes.

[0137] Step b): Add 0.5 μL of BSA-Biotin to the mixed system to block excess streptavidin-Brilliant Violet 421. TM or streptavidin - Alexa 647. Gently mix by pipetting and incubate at room temperature for 20 minutes.

[0138] Step c): The biotinylated m6A-BSA and the streptavidin conjugate labeled with fluorescent dye in the EP tube are left to stand for subsequent experiments.

[0139] Example 6: Sorting of B cells with specific binding to m6A

[0140] Antigen-positive B cells are selected negatively by T cell surface markers, and B cell surface immunoglobulin-positive cells are selected positively. Then, specific B cells are sorted from peripheral blood mononuclear cells by sorting antigens labeled with fluorescent dyes.

[0141] Reagents: PBS (Basal Media: B310KJ-500ml), fetal bovine serum (Sijiqing: 11011-8611), mouse anti-rabbit CD4 antibody-FITC, mouse anti-rabbit IgG-PE, mouse anti-rabbit IgM-Biotin, streptavidin-PE / Cy7, Brilliant Violet 421 TM -Streptavidin, Alexa 647-Streptavidin, biotin-labeled m6A-BSA.

[0142] Consumables: 15mL centrifuge tubes (BD: 352097), EP tubes (AXYGEN: MCT-150-C).

[0143] Equipment: Biosafety cabinet (Sujing: BSC-1300IIB2), flow cytometer (Sony: MA900), centrifuge (Thermo: THM#75004530).

[0144] m6A specifically binds to B cell sorting

[0145] Step a): Set the flow cytometry sorting indicators (IgG, IgM, m6A-BSA) and experimental groups (blank cell control group, compensated single positive tube control group, reduced fluorescence control group, and experimental group).

[0146] Step b): For IgM and antigen (m6A-BSA) positive indicators, IgM-Biotin and antigen (m6A-BSA)-Biotin must first be reacted with streptavidin-PE / Cy7 and Brilliant Violet 421, respectively. TM / Alexa After pre-incubation with 647-streptavidin for 20 min, followed by blocking with excess BSA-Biotin for 20 min, the sample was used for sorting and detection and added to the corresponding experimental group in step e.

[0147] Step c): Take peripheral blood lymphocytes, resuspend them in 10 mL PBS, count the cells, aliquot the cells into 1.5 mL EP tubes and centrifuge at 1500 rpm for 5 min.

[0148] Step d): Carefully discard the supernatant and resuspend in 100 μL PBS.

[0149] Step e): Add the corresponding antibody to each experimental group set in step a, and gently vortex to mix.

[0150] Step f): After incubating at room temperature for 20 min, resuspend in 800 uL PBS, centrifuge at 1500 rpm for 5 min.

[0151] Step g): Carefully discard the supernatant, resuspend the cells in 1 mL of PBS, and centrifuge at 1500 rpm for 5 min.

[0152] Step h): After repeating step g, carefully discard the supernatant. Add 1 mL of PBS containing 2% FBS to the experimental group for resuspension, and add 200 μL of PBS containing 2% FBS to the other groups for resuspension.

[0153] Step i): Place on ice and load onto the machine.

[0154] Step j): Adjust fluorescence compensation on the flow cytometer using a blank cell control group and a compensated single-positive control group, and then gate the flow cytometer by subtracting one fluorescence control group. Finally, load the experimental group samples and sort out the antigen-double-positive cell population, such as... Figure 4 As shown in Figure A: Selected rabbit PBMC lymphocyte populations were subjected to FSC-W and FSC-A to remove adhesion cells; Figure B: FITC and PE-Cy7 double-negative cells were obtained; Figure D: PE positive cells were selected; Figure E: Antigen positive cells were selected for single-cell isolation and collection.

[0155] Example 7: Lysis of a single B cell

[0156] Reagents: DTT, Digitonin, Rnase Inhibitor, Oligo(dT)20VN, Random hexamers, DEPC water.

[0157] Consumables: PCR plates, pipette tips.

[0158] Equipment: PCR instrument.

[0159] Step a): Prepare single-cell lysis buffer according to the table and dispense it into the wells of the PCR plate.

[0160] Table 3 Single-cell lysate

[0161] Components Final concentration DTT 5mM Digitonin 0.10％ Rnase Inhibitor 5U Oligo(dT)20VN 5μM Random hexamers 5μM DEPC H2O Make up to 10μL

[0162] Step b): Sort single cells and inject them into the lysis buffer of the PCR plate wells, then keep on ice for later use.

[0163] Step c): Quick-freeze the 96-well PCR plate at -80°C until all samples are frozen, then place it in a 37°C metal bath until thawed. Repeat this freeze-thaw cycle three times, then place it on ice.

[0164] Example 8: Acquisition of cDNA from a single B cell and preparation of TAP fragment for antibody expression

[0165] Reagents: PrimeScript TMⅡ1st Strand cDNASynthesis Kit (TAKARA: 6210A), RNaseinhibitor, dNTP, H2O.

[0166] Consumables: PCR plates, pipette tips.

[0167] Equipment: PCR instrument.

[0168] Acquisition of cDNA from a single B cell:

[0169] Step a): Place the sample from the previous step in the PCR instrument and incubate at 65°C for 5 minutes. Immediately after incubation, place it on ice for at least 2 minutes.

[0170] Step b): Prepare the reaction system according to the table below, cDNA synthesis system

[0171] Table 4 cDNA Synthesis System

[0172] Components volume Single-cell lysate 10μl 5xbuffer (Takara) 4μl PrimeScript II RTase(200U / μL) 1μl RNase inhibitor (40 U / μL) 1μl dNTP (Final conc 0.5mM) 1μl H2O 3μl

[0173] Step c): Place the sample in a PCR instrument, incubate at 42°C for 1 hour, and inactivate at 80°C for 5 seconds. After the incubation, store in a -20°C freezer for a short period; for long-term storage, store in a -80°C freezer.

[0174] Reagents: Taq DNA Polymerase (TAKARA: R001A); 2× Phanta Flash Master Mix (Dyeplus / minus) (Novazia: P520 / P510); 6× Loading Buffer (TAKARA: 9156); DL2000 DNA Marker (TAKARA: 3427A); TAE buffer, agarose.

[0175] Consumables: PCR plates, pipette tips.

[0176] Equipment: PCR instrument, horizontal electrophoresis apparatus, gel imaging system.

[0177] Preparation of TAP fragments for antibody expression:

[0178] The components required for antibody expression (such as promoter and terminator sequences) are integrated and amplified with the antibody expression sequence by PCR. The PCR product containing the splicing fragment (TAP fragment) can be directly used for transfection in the suspension 293F system.

[0179] Example 9: TAP fragment cell transfection and ELISA assay

[0180] Reagent: Expi293 TM Expression system kit (Thermo, A14635).

[0181] Consumables: 96-well deep well plate, pipette tips.

[0182] Equipment: constant temperature shaker, electric pipette.

[0183] TAP fragment cell transfection:

[0184] Step a): Incubate the Expi293F medium in a 37°C water bath beforehand. Observe the cell state and growth under a microscope. Determine the passage ratio for the cell expansion experiment based on the cell growth volume and subsequent transfection requirements. Generally, the transfection density is controlled at 2x10⁻⁶ cells / day the day before transfection. 6 Approximately 100 live cells / mL. Remove the cell slurry from the CO2 cell shaker, count the cells, and calculate the required number of cells to transfer.

[0185] Select the optimal cell culture flask size based on the amount of cells transferred. Generally, the culture volume should not exceed 1 / 3 of the flask volume and should not be less than 1 / 5 of the flask volume. Add preheated Expi 293 medium according to the cell density to achieve a final cell density of 2 x 10⁻⁶ cells / mL. 6 Approximately 100 live cells / mL, for use in the transfection procedure the following day.

[0186] Step b): On the day of transfection, preheat the Expi 293 medium to room temperature in the dark. Determine the amount of cells to be transfected and prepare the corresponding number of 96-well plates. Determine the cell density using cell counting techniques, and dilute the cells with the preheated medium to a final cell density of 3 x 10⁻⁶ cells / well. 6For live cells / mL, aliquot 1ml / well into deep-well plates using a pipette and carefully place them on a cell culture shaker: 37℃, 8% CO2, 900rpm, 80% humidity. Take the appropriate number of 96-well cell culture plates and aliquot 1ug DNA + 60ul Opti-MEM (cold reagent) into each plate, mixing gently with a pipette (recommended antibody ratio HC:LC = 1:2). Separately, take the optimal volume of centrifuge tube and dilute sufficient transfection reagent to 3.2ul Expifectamine 293 + 60ul Opti-MEM (cold reagent) per ml of cells. Gently invert to mix and incubate at room temperature for 5 minutes. After 5 minutes, transfer the diluted transfection reagent to a disposable reservoir and pipette into the corresponding wells of the 96-well cell culture plates, incubating for 10-20 minutes. Using a pipette, add the settled ExpiFectamine 293 / DNA complex to the cell suspension and carefully place the mixture on a shaker: incubate at 37°C, 8% CO2, 900 rpm, and 80% humidity. 18-22 hours post-transfection, add 6 μL of Enhancer 1 and 60 μL of Enhancer 2 to 1 ml of the solution. Harvest the supernatant after 4-7 days for analysis.

[0187] Step c): The specificity of the supernatant for recognizing the small molecule m6A was determined by indirect ELISA and competitive ELISA. The results are shown in Table 5. Figure 5 As shown. Indirect ELISA and competitive ELISA results show that these clones are specific for recognizing m6A small molecules, while other structural analogs (such as...) Figure 6 (As shown) It was almost unrecognizable, especially 1G1, which had the highest OD450 value. Finally, 1G1 was cloned for antibody heavy and light chain variable region sequencing, plasmid construction, and antibody production.

[0188] Table 5

[0189]

[0190] Example 10: Sequencing of the variable region of the heavy and light chains

[0191] Reagents: TAE buffer, agarose, nucleic acid dye.

[0192] Consumables: pipette tips.

[0193] Equipment: Nucleic acid electrophoresis instrument, nucleic acid imaging instrument.

[0194] Sequencing of heavy and light chain variable regions:

[0195] The antibody nucleic acid sequence was extracted and sent to a gene sequencing company to obtain the variable region nucleic acid and protein sequences of the 1G1 heavy and light chains.

[0196] The amino acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.7, the amino acid sequence of the light chain variable region VL is shown in SEQ ID NO.8, the nucleic acid sequence of the heavy chain variable region VH is shown in SEQ ID NO.9, and the nucleic acid sequence of the light chain variable region VL is shown in SEQ ID NO.10.

[0197] Example 11 Construction and Production of Cloning 1G1 Expression Plasmid

[0198] Based on the sequencing results of the variable regions of the heavy and light chains, the samples were sent to a gene synthesis company to construct them into an expression vector containing a signal peptide and a constant region. The synthesized plasmids were then mass-produced using the transfection method described in Example 9, with the amount of DNA added adjusted according to the production volume. The supernatant was harvested and purified after 4-7 days of culture.

[0199] Example 12 1G1 Antibody Specificity Detection

[0200] Competitive ELISA assays using m6A small molecules and other structural analogs confirmed that the 1G1 antibody specifically binds to m6A small molecules, while exhibiting almost no cross-binding to other structural analogs.

[0201] Reagents: Goat anti-rabbit-HRP (Huaan Biotechnology: HA1001), TMB substrate (Sigma: T2885), Tris (Shanghai Sangon Biotech: A501492), glycine (Shanghai Sangon Biotech: GB0235), BSA (Shanghai Sangon Biotech: A500023-0100), Tween-20 (Shanghai Sangon Biotech: A600560), NaHCO3 (Shanghai Sangon Biotech: A610482-0500); Na2CO3, Na2HPO4·12H2O, NaH2PO4·2H2O, citric acid, glycerol, DMSO, and concentrated sulfuric acid were purchased from Hangzhou Shuangmu Chemical; hydrogen peroxide and EDTA were purchased from Shanghai Sangon Biotech, domestically produced analytical grade.

[0202] Consumables: Microplate (Hangzhou Shengyou).

[0203] Equipment: Electric thermostatic incubator (Shanghai Senxin: DRP-9162), enzyme-linked immunosorbent assay (ELISA) instrument (MD: Cmax plus).

[0204] Indirect ELISA procedure:

[0205] The binding specificity of the 1G1 antibody to the m6A small molecule was confirmed using a competitive ELISA method.

[0206] Indirect competition ELISA operation:

[0207] Step a): Coating: Dilute m6A-BSA to 2 μg / mL with coating buffer, add 50 μL / well to the microplate, cover and coat overnight at 4°C.

[0208] Step b): Blocking: Shake off the liquid in the wells, add 1% BSA / TBS to the microplate at a rate of 150 μL / well, and place it in a 37°C thermostatic incubator for 1 hour for blocking.

[0209] Step c): Sample loading: Shake off the liquid in the well, serially dilute the anti-m6A monoclonal antibody with PBS, and then mix it with an equal volume of serially diluted competitive agent (m6A small molecule and other structural analog molecules are diluted with PBS, PBS is used as the control group), and let it stand at room temperature for 10 min; take 100 μL of the mixture and add it to the microplate, and react at 37℃ for 45 min.

[0210] Step d): Add secondary antibody: Shake off the primary antibody mixture, add washing buffer (1×TBST) to the microplate at a rate of 180 μL / well, and wash the microplate twice. Dilute goat anti-rabbit-HRP to the working concentration (1:30000) with 1% BSA, add 50 μL / well to the microplate, cap it, and incubate at 37°C for 45 min.

[0211] Step e): Color development, termination and reading: Discard the liquid in the wells, add washing buffer to the microplate at a rate of 180 μL / well, and wash the microplate 3 times; add 100 μL of freshly prepared TMB chromogenic substrate to each reaction well, and incubate at 37°C for 10 min; then add 90 μL / well of stop solution to terminate the reaction, and measure the OD value at 450 nm on the microplate reader.

[0212] The results showed that, Figure 7 As shown, only the m6A small molecule competitively binds to the 1G1 antibody, and the OD value is significantly reduced, indicating that the 1G1 antibody specifically recognizes the m6A small molecule.

[0213] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A binding protein of m6A, characterized in that, The variable region of the binding protein comprises: a complementarity determining region CDR1-VH of an amino acid sequence as shown in SEQ ID NO. 1, a complementarity determining region CDR2-VH of an amino acid sequence as shown in SEQ ID NO. 2, a complementarity determining region CDR3-VH of an amino acid sequence as shown in SEQ ID NO. 3, a complementarity determining region CDR1-VL of an amino acid sequence as shown in SEQ ID NO. 4, a complementarity determining region CDR2-VL of an amino acid sequence as shown in SEQ ID NO. 5, and a complementarity determining region CDR3-VL of an amino acid sequence as shown in SEQ ID NO.

6.

2. The binding protein of claim 1, characterized in that, The variable region comprises a heavy chain variable region VH of an amino acid sequence as shown in SEQ ID NO.

7.

3. The binding protein of claim 1, wherein The variable region comprises a light chain variable region VL of an amino acid sequence as shown in SEQ ID NO.

8.

4. Use of the binding protein according to any one of claims 1-3 in the preparation of an m6A detection product.

5. Use according to claim 4, characterized in that, The product comprises an immunochromatography test paper or an ELISA kit.

6. An ELISA kit for detecting m6A, characterized in that, The kit uses the binding protein according to any one of claims 1-3 as a capture protein.

7. A gene encoding the binding protein of any one of claims 1-3. The nucleic acid sequence of the gene is shown in SEQ ID NO. 9 and SEQ ID NO.

10.

8. A recombinant plasmid, characterized in that, The recombinant plasmid comprises a vector and the gene according to claim 7.

9. A cell line, characterized in that, The cell line expresses the binding protein according to any one of claims 1-3.

10. A method of producing the binding protein according to any one of claims 1 to 3, characterized in that, Comprise: The cell line according to claim 9 is cultured in a culture medium, and then the binding protein is isolated.

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