Mylk2 protein immunogen with specific histidine methylation modification, polyclonal antibody and application

Abstract

The application discloses a specific histidine methylation modified MYLK2 protein immunogen, a polyclonal antibody and application, and the specific histidine methylation modified MYLK2 protein immunogen comprises a polypeptide with an amino acid sequence as shown in SEQ ID No. 2. The polyclonal antibody specifically recognizing the specific histidine methylation modified MYLK2 protein can be obtained by immunizing animals with the specific histidine methylation modified MYLK2 protein immunogen, the change of MYLK2 specific histidine methylation in a biological sample can be rapidly and simply detected, and the development of the function of the specific histidine methylation MYLK2 protein in skeletal muscle and the dynamic change of MYLK2 specific histidine methylation in the development process of skeletal muscle related diseases is promoted.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immunogen of MYLK2 protein with specific histidine methylation modification, and also to a polyclonal antibody that can specifically recognize MYLK2 protein with specific histidine methylation modification, as well as the application of the polyclonal antibody. Background Technology

[0002] Myosin light chain kinase 2 (MYLK2) belongs to the myosin light chain kinase family, a class of enzymes involved in the phosphorylation of myosin light chains. MYLK2 is mainly distributed in skeletal muscle, where it primarily regulates muscle contraction by phosphorylating myosin light chains (MLCs). This process is a crucial biological event in muscle, and researchers are dedicated to understanding in detail how MYLK2 precisely regulates this step to ensure effective muscle contraction and relaxation.

[0003] The relationship between MYLK2 and skeletal muscle diseases, such as muscle atrophy and myopathy, has attracted widespread attention. Furthermore, the role of MYLK2 in adaptive changes in skeletal muscle is also a focus of research, including its impact on MYLK2 expression and activity during exercise, training, and rehabilitation. MYLK2 may be influenced by epigenetic regulation, including DNA methylation and histone modifications. Some of these regulatory mechanisms have been studied in skeletal muscle to gain a deeper understanding of MYLK2's role in gene expression regulation. Research on the role of MYLK2 in exercise physiology, including the regulation of MYLK2 expression by exercise and its potential role in exercise adaptation and performance, will contribute to understanding the practical applications of MYLK2 in sports and sports medicine. Therefore, in-depth research on MYLK2 may reveal potential therapeutic strategies, including the development of drugs, gene therapy, or other interventions targeting MYLK2, to treat skeletal muscle-related diseases and sports injuries.

[0004] Overall, research on skeletal muscle MYLK2 helps to reveal the complexities of muscle biology and can provide insights into understanding and treating skeletal muscle-related diseases and promoting exercise adaptation.

[0005] Methylation, as a biochemical strategy to enhance the characteristics of modified residues, is commonly used by cells for recognition and regulation. When discussing protein methylation, lysine or arginine methylation is the most well-known in the field; however, histidine in proteins can also be methylated. Currently, there are no specific functional studies on the histidine methylation modification of the MYLK2 protein, and no specific antibodies targeting its histidine methylation sites exist. Furthermore, the only current method for identifying histidine methylation on MYLK2 is mass spectrometry, but mass spectrometry requires complex platforms, which are generally unavailable in laboratories. Additionally, the entire mass spectrometry process is time-consuming, and the results are not readily apparent. Summary of the Invention

[0006] In view of this, the present invention needs to provide an immunogen of MYLK2 protein with specific histidine methylation modification. The inventors discovered in their research that histidine at position 148 of the human MYLK2 (Swiss-Prot: Q9H1R3) protein is identified as methylated, and this modification can be mediated by the histidine N1 methyltransferase CARNMT1. Since CARNMT1 modifies substrates with a specific motif (C / P)X(F / Y)XH, where H is the methylation site of CARNMT1, and MYLK2 possesses such a motif, through the reaction, CARNMT1 can transfer the methyl group from the methyl donor SAM to the N atom of the first position of histidine at position 148 of the human MYLK2 protein, thereby achieving the purpose of methylating the MYLK2 protein. Based on the above findings, this invention develops an immunogen for MYLK2 protein with specific histidine methylation modification. This immunogen can be used to obtain corresponding polyclonal antibodies by immunizing animals. These polyclonal antibodies can specifically recognize MYLK2 protein with specific histidine methylation modification, thereby enabling the identification of specific histidine methylation on MYLK2 protein. This greatly simplifies the entire detection process, shortens the detection time, and presents the detection results intuitively, thus promoting research on the function of MYLK2 protein with specific histidine methylation in skeletal muscle and the dynamic changes of MYLK2 specific histidine methylation during the development of skeletal muscle-related diseases.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a MYLK2 protein immunogen with specific histidine methylation modification, comprising a polypeptide with an amino acid sequence as shown in SEQ ID No. 2.

[0009] In a further embodiment, the MYLK2 immunogen with specific histidine methylation modification also includes a carrier protein, which is coupled to the polypeptide.

[0010] In a further embodiment, the carrier protein is selected from hemocyanin.

[0011] The second aspect of the present invention provides a polyclonal antibody that specifically recognizes MYLK2 protein with specific histidine methylation modification, obtained by immunizing animals with the MYLK2 protein immunogen with specific histidine methylation modification as described in the first aspect of the present invention.

[0012] In a further embodiment, the animal used for immunization is a rabbit.

[0013] In a further embodiment, the immunization is performed using New Zealand white rabbits.

[0014] A third aspect of the present invention provides a method for preparing a polyclonal antibody as described in the second aspect of the present invention, comprising the following steps:

[0015] Animals were immunized with the MYLK2 protein immunogen modified with the specific histidine methylation, and serum was collected and purified to obtain polyclonal antibodies.

[0016] In a further embodiment, the immunized animal is a rabbit.

[0017] In a further embodiment, the immunized animal is a New Zealand white rabbit.

[0018] In a further embodiment, the purification is performed using affinity chromatography.

[0019] A further detailed plan specifies the timing, dosage, and Freund's adjuvant type for each immunization of the New Zealand White rabbits as follows:

[0020] .

[0021] The fourth aspect of this invention provides the use of the polyclonal antibody as described in the second aspect of this invention or the polyclonal antibody prepared by the preparation method described in the third aspect of this invention in recognizing MYLK2 protein with specific histidine methylation modification.

[0022] The fifth aspect of this invention provides the use of the polyclonal antibody as described in the second aspect of this invention or the polyclonal antibody prepared by the preparation method described in the third aspect of this invention in the preparation of products for detecting MYLK2 protein with specific histidine methylation modification.

[0023] In a further embodiment, the product may be a formulation, a chip, or a reagent kit.

[0024] The sixth aspect of the present invention provides a detection reagent for MYLK2 protein with specific histidine methylation modification, comprising a polyclonal antibody as described in the second aspect of the present invention or a polyclonal antibody prepared by the preparation method described in the third aspect of the present invention.

[0025] A seventh aspect of the present invention provides a test strip for detecting MYLK2 protein with specific histidine methylation modification, comprising the detection reagents as described in the sixth aspect of the present invention.

[0026] The eighth aspect of the present invention provides a product of a MYLK2 protein with specific histidine methylation modification, comprising a detection reagent as described in the sixth aspect of the present invention or a test strip as described in the seventh aspect of the present invention.

[0027] In a further embodiment, the product may be a chip or a reagent kit.

[0028] The beneficial effects of this invention are:

[0029] Polyclonal antibodies can be prepared using the MYLK2 protein immunogen with specific histidine methylation modification provided in this invention. These polyclonal antibodies can highly specifically recognize MYLK2 proteins with specific histidine methylation modification and will not recognize unmethylated MYLK2 proteins. This enables rapid and simple detection of changes in specific histidine methylation of MYLK2 in biological samples, which plays a significant role in promoting research on the function of MYLK2 proteins with specific histidine methylation in skeletal muscle and the dynamic changes in specific histidine methylation of MYLK2 during the development of skeletal muscle-related diseases. Attached Figure Description

[0030] Figure 1 This is the detection result of the purified polyclonal antibody used in Example 4 to recognize the MYLK2 protein modified by histidine methylation at position 148. Detailed Implementation

[0031] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0032] Unless otherwise defined, 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 pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0033] The first aspect of this invention provides an immunogen of MYLK2 protein with specific histidine methylation modification, which can be used to prepare polyclonal antibodies that can specifically recognize MYLK2 protein with specific histidine methylation modification. Specifically, the immunogen of MYLK2 protein with specific histidine methylation modification comprises a polypeptide with the amino acid sequence shown in SEQ ID No. 2.

[0034] The MYLK2 protein mentioned in this article refers to the human MYLK2 protein (Swiss-Prot: Q9H1R3), and the specific histidine mentioned refers to histidine 148 in the human MYLK2 protein. The unmethylated peptide of the human MYLK2 protein containing this histidine 148 is named peptide 1, and its amino acid sequence is: RRGSPAFL H SPSCPAII (SEQ ID No. 1), where the underlined part represents histidine 148 in the human MYLK2 protein, and C is used for coupling the carrier protein; methylation modification at the N of a specific histidine π position in peptide 1 yields the methylated peptide RRGSPAFL. H(methyl) SPSCPAII, named polypeptide 2, has the following amino acid sequence: RRGSPAFL X SPSCPAII (SEQ ID No. 2), where X in the sequence represents N1-histidine methylation, i.e., N-methylation modification at the π position of histidine.

[0035] In this embodiment, the methylation modification of histidine at position 148 of the MYLK2 protein is mediated by the histidine N1 methyltransferase CARNMT1. For details, please refer to the applicant's research paper METTL9 mediated N1-histidine methylation of zinc transporters is required for tumor growth. M Lvet. Protein & Cell. 2021.

[0036] In this paper, a polyclonal antibody was obtained by immunizing animals with a MYLK2 protein immunogen containing a specific histidine methylation modification of polypeptide 2 as shown in SEQ ID No. 2. This antibody can specifically recognize MYLK2 proteins with specific histidine methylation modification but not unmethylated MYLK2 proteins, thus enabling rapid identification of MYLK2 proteins with specific histidine methylation modification.

[0037] Furthermore, the MYLK2 protein with specific histidine methylation modification described herein also includes a carrier protein, which is coupled to peptide 2, thereby facilitating the stimulation of helper T cells and further inducing a B cell immune response. Specifically, the carrier protein can be selected from carrier proteins conventionally used in the art; preferably, the carrier protein is selected from hemocyanin (KLH) or bovine serum albumin (BSA); more preferably, the carrier protein is selected from hemocyanin.

[0038] A second aspect of the present invention provides a polyclonal antibody that specifically recognizes a MYLK2 protein with a specific histidine methylation modification, obtained by immunizing an animal with a MYLK2 immunogen with a specific histidine methylation modification as described above. The animal used for immunization can be a rabbit or a mouse, preferably a rabbit. In one or more embodiments of the present invention, the immunized animal is a New Zealand white rabbit.

[0039] The third aspect of the present invention provides a method for preparing a polyclonal antibody that specifically recognizes a MYLK2 protein modified with a specific histidine methylation, the method comprising steps S101-S105.

[0040] Step S101: Provide a polypeptide with specific histidine methylation modification of the MYLK2 protein.

[0041] Specifically, polypeptide 2 is artificially synthesized to obtain a polypeptide with specific histidine methylation modification, the amino acid sequence of which is RRGSPAFL. X SPSCPAII (SEQ ID No. 2).

[0042] Step S102: Couple the peptide modified with specific histidine methylation to the carrier protein to obtain the immunogen.

[0043] Specifically, the polypeptide 2 in step S101 is coupled to the carrier protein using protein conjugation technology. More specifically, the carrier protein is first activated, and then the activated carrier protein is mixed with polypeptide 2 and reacted, followed by overnight dialysis to obtain the immunogen. It is understood that the selection of the carrier protein is similar to that described in the first aspect of this invention, and will not be elaborated here.

[0044] Step S103: Immunize the animals with the immunogen.

[0045] The immunized animals described herein can be rabbits or mice, preferably rabbits. In one embodiment, New Zealand white rabbits are used as the immunized animals. The New Zealand white rabbits are immunized multiple times with the immunogen obtained in step S102. The New Zealand white rabbits are selected from young, healthy animals weighing around 2.5 kg, with glossy fur and good mobility. Selected animals are pre-fed for about two weeks to eliminate unqualified animals and ensure the smooth progress of subsequent experiments. In one embodiment, the immunogen is injected into the animal after being mixed with an adjuvant to enhance the body's immune response to the antigen or change the type of immune response. There are many types of adjuvants. In one embodiment of the present invention, Freund's complete adjuvant and Freund's incomplete adjuvant, which are currently the most commonly used in animal experiments, are used as preferred. In one embodiment, the volume ratio of adjuvant to immunogen is 1:1. The specific time, dosage, and type of Freund's adjuvant for each immunization are as follows:

[0046]

[0047] In this embodiment, immunization is performed using a multi-site subcutaneous immunization method. It can be understood that the immunization site can be subcutaneous on the back, abdomen, armpit, or limbs, but is not limited to these.

[0048] Step S104: Collect serum from immunized animals to determine antibody titer.

[0049] Specifically, after immunization, blood is first collected for ELISA testing to determine whether the serum contains the corresponding antibodies and whether the antibody titer meets the standard. The serum collection can be carried out using conventional methods in this field.

[0050] Step S105: After the titer meets the standard, blood is collected for antibody purification.

[0051] Specifically, in one embodiment, the antibody purification in this invention is preferably affinity chromatography purification. First, a peptide containing a specific histidine methylation modification (peptide 2) is used for preliminary purification. Then, the same peptide containing the same unmethylated modification (peptide 1) is used for secondary purification to remove the antibody that recognizes the peptide containing the unmethylated modification. The purified antibody is then identified by ELISA detection.

[0052] In some specific embodiments, the purification steps are as follows: pretreating the affinity chromatography column, then adding serum containing polyclonal antibodies into the pretreated affinity chromatography column for loading, and eluting with elution buffer.

[0053] In some specific implementations, ELISA detection includes the following steps:

[0054] ① Coat an enzyme-linked plate with a polypeptide modified by specific histidine methylation, and wash the plate;

[0055] ② Block the coated ELISA plate, incubate, and discard the blocking solution;

[0056] ③ Add the purified polyclonal antibody, incubate, discard the blocking solution, and wash the plate;

[0057] ④ Add enzyme-labeled secondary antibody, incubate, discard blocking solution, and wash plate;

[0058] ⑤ Add substrate solution to develop color, stop the reaction, and then read the plate.

[0059] The polyclonal antibody sample to be tested is diluted at a certain ratio. In one or more embodiments of the present invention, the dilution ratio is from 1:250 to 1:1024000.

[0060] Experimental results show that the polyclonal antibody prepared by the method described in this invention has an antibody titer of over 1 when diluted at a ratio of 1:250, and the ELISA detection value is higher than the positive standard of 1.

[0061] The fourth aspect of this invention provides the application of the polyclonal antibody described in the second aspect of this invention or the polyclonal antibody prepared by the preparation method described in the third aspect of this invention in recognizing MYLK2 protein with specific histidine methylation modification. Specifically, the polyclonal antibody obtained in this invention can be applied to Western blot, or immunofluorescence, immunohistochemistry, flow cytometry, ELISA kits, etc., to detect MYLK2 protein with specific histidine methylation modification, thereby achieving rapid and simple detection of changes in specific histidine methylation of MYLK2 in biological samples. This plays a significant role in promoting the study of the function of MYLK2 protein with specific histidine methylation in skeletal muscle and the dynamic changes in specific histidine methylation of MYLK2 during the development of skeletal muscle-related diseases.

[0062] The fifth aspect of this invention provides the use of the polyclonal antibody described in the second aspect of this invention or the polyclonal antibody prepared by the method described in the third aspect of this invention in the preparation of products that recognize MYLK2 proteins with specific histidine methylation modifications. The products may be formulations, chips, or kits.

[0063] The product contains the polyclonal antibody described in the second aspect of this invention or the polyclonal antibody prepared by the method described in the third aspect of this invention, to achieve the detection of MYLK2 protein with specific histidine methylation modification.

[0064] The sixth aspect of the present invention provides a detection reagent for MYLK2 protein with specific histidine methylation modification, comprising a polyclonal antibody as described in the second aspect of the present invention or a polyclonal antibody prepared by the preparation method described in the third aspect of the present invention.

[0065] The seventh aspect of the present invention provides a test strip for detecting MYLK2 protein with specific histidine methylation modification, comprising the detection reagents as described in the sixth aspect of the present invention.

[0066] The eighth aspect of the present invention provides a product of a MYLK2 protein with specific histidine methylation modification, comprising a detection reagent as described in the sixth aspect of the present invention or a test strip as described in the seventh aspect of the present invention.

[0067] In a further embodiment, the product may be a chip or a reagent kit.

[0068] It is understood that specific products may also include reagents commonly used in the field, such as diluents, conversion solutions, buffers, washing solutions, elution solutions, and purification solutions. The specific types of reagents vary depending on the detection technology, and will not be elaborated here.

[0069] The present invention will be described below through specific embodiments. It should be noted that the specific embodiments below are for illustrative purposes only and do not limit the scope of the present invention in any way. In addition, unless otherwise specified, methods that do not specifically describe conditions or steps are conventional methods, and the reagents and materials used can be obtained commercially.

[0070] Example 1: Preparation of polypeptides

[0071] The following peptides were synthesized by Hangzhou Huaan Biotechnology Co., Ltd.:

[0072] The polypeptide with the amino acid sequence shown in SEQ ID No. 1 is named polypeptide 1;

[0073] The polypeptide with the amino acid sequence shown in SEQ ID No. 2 is named polypeptide 2.

[0074] Example 2: Preparation of human MYLK2 protein immunogen modified with histidine methylation at position 148

[0075] Using protein conjugation technology, peptide 2 from Example 1 was conjugated with hemocyanin (KLH) to obtain a human MYLK2 immunogen modified with histidine methylation at position 148. The specific steps are as follows:

[0076] 1. Peptide 2 conjugated with KLH

[0077] (1) Dissolve 20 mg KLH in 2 mL of 5 mM EDTA aqueous solution;

[0078] (2) Weigh 8 mg of Sulfo-SMCC and dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well;

[0079] (3) Add the Sulfo-SMCC solution dropwise to the KLH solution while gently shaking, and let it stand at room temperature for 1 hour;

[0080] (4) Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clamp, and dialyze it in 2L of 1×PBS at 4°C with magnetic stirring for 1h.

[0081] (5) Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed KLH into a 15mL imported centrifuge tube, and label the tube with the reagent name, time and concentration, and store in a refrigerator at 4℃;

[0082] (6) Weigh 4 mg of peptide 2, dissolve it in 50 μL LDMSO, add 200 μL of 1×PBS, mix quickly, and then immediately add KLH according to the ratio of peptide:KLH = 1 mg: 680 μg. Incubate overnight at 4°C or react at room temperature for 2 h.

[0083] (7) Place the cross-linked KLH-peptide complex into a dialysis bag, clamp it with a dialysis clamp, and dialyze overnight in 4L of 1×PBS at 4°C with magnetic stirring.

[0084] (8) Take the dialyzed KLH-peptide into a clean 1.5mL centrifuge tube, dispense it according to the immunization dose, and store it in a -20℃ refrigerator.

[0085] 2. Peptide 2 conjugated with carrier protein BSA (peptide 2 is conjugated with BSA for subsequent antibody purification and detection).

[0086] (1) Dissolve 20 mg BSA in 2 mL of 5 mM EDTA aqueous solution;

[0087] (2) Weigh 5 mg of Sulfo-SMCC and dissolve it completely in 50 μL of DMSO, then add 150 μL of 1×PBS and mix well;

[0088] (3) Add the Sulfo-SMCC solution dropwise to the BSA solution while gently shaking, and let it stand at room temperature for 1 hour;

[0089] (4) Place the activated BSA solution into a dialysis bag, clamp it with a dialysis clamp, and dialyze it in 2L of 1×PBS at 4°C with magnetic stirring for 1h.

[0090] (5) Replace with fresh 1×PBS and dialyze for 2 hours, repeating once. Place the activated dialyzed BSA into a 15mL imported centrifuge tube, label the tube with the reagent name, time, and concentration, and store at 4℃.

[0091] (6) Weigh 1 mg of polypeptide 2, dissolve it in 50 μL of DMSO, add 150 μL of 1×PBS, mix quickly, then add 100 μL of the above activated BSA solution, and react overnight at 4°C or at room temperature for 2 h.

[0092] (7) Place the cross-linked BSA-Peptide complex into a 1.5 mL centrifuge tube, add 1×PBS to 1 mL, label the item number, concentration and date, and hand it over to the detection group.

[0093] Example 3: Preparation of a polyclonal antibody that specifically recognizes human MYLK2 protein modified by histidine methylation at position 148.

[0094] Animal immunization:

[0095] Take the human MYLK2 immunogen modified with histidine methylation at position 148 from the -20°C freezer in Example 2, thaw it at room temperature, avoid repeated freeze-thaw cycles, and label the syringe. According to the configuration in Table 1, extract the adjuvant at a volume ratio of 1:1 between the adjuvant and the human MYLK2 immunogen modified with histidine methylation at position 148, and emulsify it thoroughly. The emulsification standard is that the emulsified immunogen does not disperse when dropped into 37°C water, which is considered qualified.

[0096] Before the initial immunization, all New Zealand white rabbits were numbered. The initial antigen concentration was 1 mg / mL, with each rabbit receiving 0.5 mL. For the second to fourth immunizations, the antigen concentration was halved. Immunization was performed according to the specific time and dosage described in Table 1. Multiple subcutaneous injections were used for immunization, with 0.2 mL injected at each point. On the 7th day after the third immunization, a small serum sample was collected from the middle ear artery for testing. If the test was satisfactory, a booster immunization was performed 7 days later. Whole blood could be collected 7 days after the booster immunization.

[0097] Table 1. Number, timing, and dosage of immunizations

[0098]

[0099] Antibody titer testing:

[0100] After three immunizations, an ELISA test is performed to determine whether the serum contains the corresponding antibodies and whether the antibody titer meets the target. The specific steps are as follows:

[0101] (1) Plate coating: Dilute the known antigen peptides (peptide 1 and peptide 2, respectively) to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μl to each reaction well of the polystyrene plate, incubate overnight at 4°C, and the next day, discard the solution in the well and wash once with 1xPBST washing buffer at 180 μl per well.

[0102] (2) Blocking: Add 150 μL of 1% BSA (prepared with PBST) to each well for blocking, incubate at 37°C for 1 hour, and then discard the blocking solution;

[0103] (3) Sample addition: Dilute the sample to be tested according to a certain ratio (see Table 2 for dilution ratio), take 50 μL of the diluted antibody into the above-mentioned blocked reaction wells, and set up the negative control wells (PBS) at the same time. Incubate at 37°C for 1 hour, then discard the blocking solution and wash 3 times with 1xPBST washing buffer at 150 μL per well.

[0104] (4) Add enzyme-labeled antibody: Add 50 μL of freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the enzyme-labeled plate and incubate at 37°C for 45 min. Then discard the blocking solution and wash 3 times with 150 μL of 1xPBST buffer per well.

[0105] (5) Add substrate solution for color development and plate reading: Add 50 μl of the temporarily prepared TMB substrate solution to each reaction well, incubate at 37℃ for 5 min, add 50 μL of 1M sulfuric acid to each reaction well to terminate the reaction, place the microplate in a preheated microplate reader (450 nm) for reading, and the results are shown in Table 2.

[0106] Table 2. Results of serum antibody titer detection

[0107]

[0108] Note: In Table 2, 1# and 2# are parallel tests of rabbits numbered RB5234, and 3# and 4# are parallel tests of rabbits numbered RB5235.

[0109] Antibody purification:

[0110] Once the antibody titer meets the standard, immunization is performed 7 days later. Seven days after immunization, whole blood can be collected for antibody purification. The specific steps are as follows:

[0111] (1) The affinity chromatography column was thoroughly washed with 20 mL of pure water and 1×PBS (pH 7.4) at a flow rate of 70 mL / h.

[0112] (2) Take 10 mL of the serum to be purified into a 50 mL centrifuge tube and filter it using a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0113] (3) Load the filtered serum sample at a flow rate of 40 mL / h and repeat once;

[0114] (4) Wash the column with 20 mL of 1×PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, connect the protein analyzer. During the washing process, adjust the instrument transmittance (T setting) reading to 100.

[0115] (5) Adjust the absorbance of the protein detector (1A range) to 0. At this time, turn on the HD-A computer acquisition device on the computer desktop and adjust the full screen range to 5. Use glycine solution (pH 2.7, 0.2M) to elute the antibody at a rate of 40mL / h. At this time, press the green elution record button to start elution. When the instrument reading starts to rise, start collecting the antibody.

[0116] (6) During antibody collection, the pH value of the antibody was adjusted to about 7 with 1M sodium bicarbonate in a timely manner, and the highest peak value of the elution peak was recorded.

[0117] (7) After the antibody collection is complete, adjust the pH value to about 7 and record the volume of eluted antibody. Then rinse the rubber tube connected to the collector with pure water.

[0118] (8) Wash the affinity chromatography column with 20 mL of 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store it at 4°C.

[0119] (9) The purified antibodies were sent for testing according to different requirements. When purifying methylated antibodies, the serum was first passed through a methylation column, and the eluted antibodies were then passed through a non-methylation column to obtain specific methylated antibodies. The purification of other modified antibodies was similar, and ELISA detection was performed. The results are shown in Table 3.

[0120] Table 3 Results of purified antibody titer assay

[0121]

[0122] Note: In Table 3, 1# and 2# are parallel tests of rabbits numbered RB5234, and 3# and 4# are parallel tests of rabbits numbered RB5235.

[0123] Finished product antibody concentration testing:

[0124] (1) After the amount of purified antibody is sufficient for delivery and the titer of the semi-finished product is qualified, mix all the antibodies and concentrate them using an ultrafiltration concentrator to achieve a certain concentration and volume.

[0125] (2) Place the concentrated antibody in 1L 0.01M PBS (pH7.4) and dialyze at room temperature. Change the medium every 3 hours for a total of 3 times (for overnight dialyze, place in a refrigerator at 2-8℃).

[0126] (4) Take the dialyzed antibody into a clean centrifuge tube, filter the antibody with a 0.22μm disposable low adsorption filter in a clean bench, take a small sample for testing, and take another 5μl to test the concentration.

[0127] (5) Concentration was detected using the Protein A280 on the denovix DS-11 micro-volume spectrophotometer.

[0128] After testing, the antibody concentrations of 0.7 mL (RB5234) and 1.2 mL (RB5235) were found to be 0.52 mg / mL (RB5234) and 0.96 mg / mL (RB5235), respectively.

[0129] Example 4: Identification of human MYLK2 protein modified with histidine methylation at position 148

[0130] Unmethylated Flag-MYLK2 fusion protein was obtained using human Flag-MYLK2 protein expressed in 293T cells;

[0131] Using the histidine N1 methyltransferase CARNMT1, a Flag-MYLK2 fusion protein with histidine methylation at position 148, namely the protein Flag-MYLK2 H148me with H148 methylation, was co-expressed in 293T cells.

[0132] The purified polyclonal antibody prepared in Example 3 was used for Western blotting detection, and the results are as follows: Figure 1 As shown, MYLK2 represents the level of Flag-MYLK2, and CARNMT1-mut-HA and CARNMT1-WT-HA represent the inactive and enzymatically active proteins of the histidine N1 methyltransferase CARNMT1, respectively. H148me represents the methylation level of histidine at position 148 of MYLK2 (detected using antibody purified from rabbit RB5235). Figure 1 The detection results show that the polyclonal antibody prepared in Example 3 can recognize MYLK2 with methylation at histidine position 148 but cannot recognize MYLK2 without methylation.

[0133] Based on the above results, it can be seen that the immunogen provided in this invention can be used to prepare a highly specific polyclonal antibody that recognizes human MYLK2 protein with histidine methylation at position 148. Moreover, this polyclonal antibody does not recognize unmethylated MYLK2 protein. It can be widely used in immunohistochemistry, Western blot, immunofluorescence, flow cytometry, in vitro immunoassay, etc., to achieve rapid and simple detection of changes in specific histidine methylation of MYLK2 in biological samples, and to promote the study of the function of MYLK2 protein with specific histidine methylation in skeletal muscle and the dynamic changes of specific histidine methylation of MYLK2 in the development of skeletal muscle-related diseases.

[0134] Furthermore, the polyclonal antibody preparation method in this invention is simple to operate and can be used for the large-scale preparation of antibodies that specifically recognize the human MYLK2 protein modified by histidine methylation at position 148.

[0135] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0136] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An immunogen of MYLK2 protein with specific histidine methylation modification, characterized in that, The MYLK2 protein immunogen is a polypeptide with the amino acid sequence shown in SEQ ID No.

2.

2. The MYLK2 protein immunogen with specific histidine methylation modification as described in claim 1, characterized in that, The MYLK2 immunogen with specific histidine methylation modification also includes a carrier protein, which is coupled to the polypeptide.

3. The MYLK2 protein immunogen with specific histidine methylation modification as described in claim 2, characterized in that, The carrier protein is selected from hemocyanin.

4. A method for preparing a polyclonal antibody that specifically recognizes MYLK2 protein with specific histidine methylation modification, characterized in that, Includes the following steps: Animals were immunized with the MYLK2 protein immunogen according to any one of claims 1-3, serum was collected, and polyclonal antibodies were obtained by purification. The immunized animals are New Zealand white rabbits, and the specific timing, dosage, and Freund's adjuvant type for each immunization of the New Zealand white rabbits are as follows: 。 5. The method as described in claim 4, characterized in that, The purification was performed using affinity chromatography.

6. A polyclonal antibody that specifically recognizes MYLK2 protein with specific histidine methylation modifications, characterized in that, It is prepared by the preparation method described in claim 4 or 5.

7. The use of the polyclonal antibody prepared by the method according to any one of claims 4-5 or the polyclonal antibody according to claim 6 in recognizing a specific histidine methylation modified MYLK2 protein, wherein the recognition is for in vitro non-disease diagnostic or therapeutic purposes.

8. The use of the polyclonal antibody prepared by the method according to any one of claims 4-5 or the polyclonal antibody according to claim 6 in the preparation of a product for detecting MYLK2 protein with specific histidine methylation modification.

9. The application as described in claim 8, characterized in that, The product is a formulation, chip, or reagent kit.

10. A reagent for detecting MYLK2 protein with specific histidine methylation modification, characterized in that, This includes polyclonal antibodies prepared by the method described in any one of claims 4-5 or polyclonal antibodies as described in claim 6.

11. A test strip for detecting MYLK2 protein with specific histidine methylation modification, characterized in that, Includes the detection reagent as described in claim 10.

12. A product containing a MYLK2 protein with specific histidine methylation modification, characterized in that, Includes the test reagent as described in claim 10 or the test strip as described in claim 11.

13. The product as described in claim 12, characterized in that, The product is a chip or a reagent kit.

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