CN117866916A - MYLK2 protein immunogen modified by specific histidine methylation, polyclonal antibody and application - Google Patents
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Abstract
The invention discloses a specific histidine methylation modified MYLK2 protein immunogen, a polyclonal antibody and application thereof, wherein the specific histidine methylation modified MYLK2 protein immunogen comprises a polypeptide with an amino acid sequence shown as SEQ ID No. 2. Through the specific histidine methylation modified MYLK2 protein immunogen immune animal, the polyclonal antibody capable of specifically recognizing the specific histidine methylation modified MYLK2 protein can be obtained, the change of the specific histidine methylation of MYLK2 in a biological sample can be rapidly and simply detected, and the research on the function of the specific histidine methylation MYLK2 protein in skeletal muscle and the development of dynamic change of the specific histidine methylation of MYLK2 in the development process of skeletal muscle related diseases can be promoted.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a MYLK2 protein immunogen with specific histidine methylation modification, a polyclonal antibody capable of specifically recognizing the MYLK2 protein with specific histidine methylation modification and application of the polyclonal antibody.
Background
Myosin light chain kinase 2 (Myosin Light Chain Kinase, mylk 2) belongs to the myosin light chain kinase family, which is a class of enzymes involved in phosphorylation of the myosin light chain. MYLK2 is predominantly distributed in skeletal muscle, where muscle contraction is regulated primarily by phosphorylating myosin light chains (Myosin light chain, MLC). This process is an important biological event in the muscle and researchers are working to know in detail how MYLK2 regulates this step precisely to ensure efficient muscle contraction and relaxation.
The relationship between MYLK2 and skeletal muscle diseases is of great interest, such as muscle atrophy, muscle lesions, and the like. In addition, the role of MYLK2 in skeletal muscle adaptation is also of interest, such as the effect on MYLK2 expression and activity during exercise, training and rehabilitation. MYLK2 may be affected by epigenetic regulation, including DNA methylation and histone modification. Some of these regulatory mechanisms have been studied in skeletal muscle to understand more deeply the role of MYLK2 in the regulation of gene expression. Research of the role of MYLK2 in exercise physiology, including the regulation of MYLK2 expression by exercise, the potential role of MYLK in exercise adaptability and exercise performance, helps to understand the practical application of MYLK2 in sports and sports medicine. Thus, through intensive research into MYLK2, potential therapeutic strategies may be revealed, including the development of drugs, gene therapy or other intervention approaches for MYLK2 to treat skeletal muscle-related diseases and motor injuries.
In general, studies of skeletal muscle type MYLK2 help reveal the complexity of muscle biology and can provide insight in understanding and treating skeletal muscle related diseases and promoting motor adaptation.
Methylation, a biochemical strategy that increases the characteristics of modified residues, is commonly used by cells for recognition and regulation. Referring to protein methylation, well known in the art are lysine or arginine methylation; indeed, protein histidine can also be methylated. However, no specific functional study on the histidine methylation modified MYLK2 protein exists at present, and no specific antibody on the histidine methylation modified site exists. The current means for identifying histidine methylation on MYLK2 can only be through a mass spectrum mode, but a platform required by a mass spectrum technology is complex, and a general laboratory does not have the conditions; in addition, the whole flow of the mass spectrum method has long processing time on the sample, and the obtained result cannot be intuitively presented.
Disclosure of Invention
In view of the foregoing, there is a need for providing a specific histidine methylation-modified MYLK2 protein immunogen. The inventors found in the study that histidine 148 on the human MYLK2 (Swiss-Prot: Q9H1R 3) protein was identified as having methylation modifications and could be mediated by the histidine N1 methyltransferase CARNMT1, H being the site methylated by CARNMT1 due to the preference of the CARNMT1 modification substrate for a motif (C/P) X (F/Y) XH; in contrast, there is such a motif on MYLK2, and by reaction, CARNMT1 can transfer the methyl group on the methyl donor SAM to the N atom of the first position of the 148 histidine on the humanized MYLK2, thereby achieving the purpose of methylation modification of the MYLK2 protein. Based on the findings, the invention develops a specific histidine methylation modified MYLK2 protein immunogen, which can obtain corresponding polyclonal antibodies through immunizing animals, and the polyclonal antibodies can specifically identify the specific histidine methylation modified MYLK2 protein, so that the identification of the specific histidine methylation on the MYLK2 protein is realized, the whole detection process can be greatly simplified, the detection time is shortened, and the detection result is intuitively presented, thereby promoting the research on the functions of the specific histidine methylation MYLK2 protein in skeletal muscles and the development of the dynamic change of the specific histidine methylation of the MYLK2 in the skeletal muscle related disease development process.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides a specific histidine methylation modified MYLK2 protein immunogen comprising a polypeptide with an amino acid sequence shown as SEQ ID No. 2.
In a further aspect, the specific histidine methylation modified MYLK2 immunogen further comprises a carrier protein coupled to the polypeptide.
Further, the carrier protein is selected from the group consisting of hemocyanin.
In a second aspect, the invention provides a polyclonal antibody specifically recognizing a specific histidine methylation modified MYLK2 protein, obtained by immunizing an animal with a specific histidine methylation modified MYLK2 protein immunogen according to the first aspect of the invention.
Further, the animals used for immunization are rabbits.
Further, the animal used for immunization is New Zealand white rabbit.
According to a third aspect of the present invention there is provided a method of preparing a polyclonal antibody according to the second aspect of the present invention, comprising the steps of:
immunizing an animal by adopting the specific histidine methylation modified MYLK2 protein immunogen, collecting serum, and purifying to obtain the polyclonal antibody.
Further, the immunized animal is a rabbit.
Further, the immunized animal is a New Zealand white rabbit.
Further, the purification is performed by affinity chromatography.
Further, the time, dosage and Freund's adjuvant type of each immunization of the New Zealand white rabbits are specifically as follows:
。
in a fourth aspect, the invention provides the use of a polyclonal antibody according to the second aspect of the invention or a polyclonal antibody prepared by a preparation method according to the third aspect of the invention, for identifying a specific histidine methylation modified MYLK2 protein.
In a fifth aspect, the present invention provides the use of a polyclonal antibody according to the second aspect of the present invention or a polyclonal antibody prepared by a preparation method according to the third aspect of the present invention, in the preparation of a product for detecting a specific histidine methylation-modified MYLK2 protein.
Further, the product is a preparation, a chip or a kit.
In a sixth aspect, the invention provides a reagent for detecting a specific histidine methylation-modified MYLK2 protein, comprising a polyclonal antibody according to the second aspect of the invention or a polyclonal antibody prepared by the preparation method according to the third aspect of the invention.
In a seventh aspect, the invention provides a test strip for detecting a specific histidine methylation-modified MYLK2 protein, comprising a detection reagent according to the sixth aspect of the invention.
In an eighth aspect, the invention provides a product of a specific histidine methylation modified MYLK2 protein comprising a detection reagent as described in the sixth aspect of the invention or comprising a detection strip as described in the seventh aspect of the invention.
Further, the product is a chip or a kit.
The invention has the beneficial effects that:
the MYLK2 protein immunogen with specific histidine methylation modification can be used for preparing a polyclonal antibody, and the polyclonal antibody can be used for highly specifically recognizing the MYLK2 protein with specific histidine methylation modification and not recognizing the MYLK2 protein without methylation modification, so that the change of MYLK2 specific histidine methylation in a biological sample can be rapidly and simply detected, and a great pushing effect is exerted on the research of the function of the MYLK2 protein with specific histidine methylation in skeletal muscles and the dynamic change of MYLK2 specific histidine methylation in the development process of skeletal muscle related diseases.
Drawings
FIG. 1 shows the results of the detection of MYLK2 protein recognition at position 148 by purified polyclonal antibody in example 4.
Detailed Description
The following detailed description of embodiments of the invention is exemplary and is provided merely to illustrate the invention and is not to be construed as limiting the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The 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.
In a first aspect, the present invention provides a specific histidine methylation modified MYLK2 protein immunogen useful for preparing polyclonal antibodies specifically recognizing specific histidine methylation modified MYLK2 proteins. Specifically, the specific histidine methylation modified MYLK2 protein immunogen comprises a polypeptide with an amino acid sequence shown as SEQ ID No. 2.
The MYLK2 protein as described herein refers to a human MYLK2 protein (Swiss-Prot: Q9H1R 3), and the specific histidine refers to histidine 148 in the human MYLK2 protein. The unmethylated peptide fragment of the humanized MYLK2 protein comprising histidine 148 is herein designated as polypeptide 1, which has the amino acid sequence: RRGSPAFLHSPSCPAII (SEQ ID No. 1), wherein the underline indicates that the 148 th histidine in the human MYLK2 protein, C is used for coupling carrier protein; methylation modification on N of specific histidine pi position in polypeptide 1 to obtain methylation modified peptide RRGSPAFLH(methyl)SPSCPAII, named polypeptide 2, has the amino acid sequence: RRGSPAFLXSPSCPAII (SEQ ID No. 2), wherein X in the sequence represents an N-methylation modification of N1-histidine methylation, i.e.histidine pi.
In this example, the 148 th histidine methylation modification of MYLK2 protein is mediated by histidine N1 methyltransferase CARNMT1, and specific methods are described in the applicant's research paper METTL 9-mediated N1-histidine methylation of zinc transporters is required for tumor growth.M Lv et al protein & cell 2021.
The polyclonal antibody obtained after immunization of animals by using the specific histidine methylation modified MYLK2 protein immunogen comprising the polypeptide 2 with the amino acid sequence shown in SEQ ID No.2 can specifically identify the specific histidine methylation modified MYLK2 protein and not identify the unmethylated modified MYLK2 protein, and can realize rapid identification of the specific histidine methylation modified MYLK2 protein.
Further, specific histidine methylation-modified MYLK2 proteins herein also include carrier proteins coupled to polypeptide 2, thereby facilitating stimulation of helper T cells, further inducing B cell immune responses. In particular, the carrier protein may be selected from carrier proteins conventionally employed in the art, preferably from hemocyanin (KLH) or Bovine Serum Albumin (BSA); more preferably, the carrier protein is selected from the group consisting of hemocyanin.
In a second aspect the invention provides a polyclonal antibody specifically recognizing a specific histidine methylation modified MYLK2 protein obtained by immunizing an animal with a specific histidine methylation modified MYLK2 immunogen as described above, wherein the animal used for immunization may be a rabbit or a mouse, preferably the animal is a rabbit, and in one or more embodiments of the invention the immunized animal used is a new zealand white rabbit.
In a third aspect, the present invention provides a method for preparing a polyclonal antibody specifically recognizing a specific histidine methylation-modified MYLK2 protein, the preparation method comprising steps S101 to S105.
Step S101, providing MYLK2 protein specific histidine methylation modified polypeptide.
Specifically, polypeptide 2 is artificially synthesized to obtain specific histidine methylation modified polypeptide, and the amino acid sequence of the polypeptide is RRGSPAFLXSPSCPAII(SEQ ID No.2)。
Step S102, coupling the polypeptide with specific histidine methylation modification with carrier protein to obtain the immunogen.
Specifically, the polypeptide 2 in the step S101 is coupled with carrier protein through a protein coupling technology, more specifically, the carrier protein is activated firstly, then the activated carrier protein and the polypeptide 2 are mixed and reacted, and the mixture is dialyzed overnight to obtain immunogen; it will be appreciated that the choice of carrier protein is similar to that described in the first aspect of the invention and will not be specifically described herein.
Step S103, immunizing animals by using the immunogen.
The immunized animal described herein may be a rabbit or a mouse, preferably, the immunized animal is a rabbit; in one embodiment, the animals are new zealand white rabbits, the new zealand white rabbits are immunized by the immunogen obtained in the step S102 for a plurality of times, the new zealand white rabbits are selected to require healthy animals which have a weight of about 2.5kg and are bright in hair and free to move, and the selected animals are kept for about 2 weeks, so that unqualified animals are eliminated, and the smooth performance of subsequent experiments is ensured; in one embodiment, the immunogen is injected into an animal by mixing the immunogen with an adjuvant to enhance the immune response of the body to the antigen or change the type of immune response, and the adjuvant is of a variety, and in one embodiment of the invention, freund's complete adjuvant and Freund's incomplete adjuvant which are most commonly used in animal tests at present are adopted, and preferably, in one embodiment, the volume ratio of the adjuvant to the immunogen is 1:1, the time, dosage and Freund's adjuvant species of each immunization are specifically:
in this embodiment, the immunization is performed by multipoint subcutaneous immunization, and it is understood that the immunized site may be subcutaneous in the back, subcutaneous in the abdomen, subcutaneous in the armpit, or subcutaneous in the four limbs, but is not limited thereto.
Step S104, serum is collected from the immunized animal to determine the antibody titer.
Specifically, after the immunization is finished, blood is firstly collected for carrying out Elisa detection to determine whether the serum contains corresponding antibodies and whether the titers of the antibodies reach the standard, wherein the serum is collected by adopting a conventional method in the field.
And step S105, after the detection titer reaches the standard, taking blood for antibody purification.
Specifically, in one embodiment, the antibody purification in the present invention is preferably affinity chromatography purification, wherein the antibody containing the peptide fragment corresponding to the specific histidine methylation modification (polypeptide 2) is first subjected to primary purification, and then the antibody containing the peptide fragment corresponding to the same peptide fragment containing the unmethylation modification (polypeptide 1) is subjected to secondary purification to remove the identified antibody; the purified antibodies were identified by Elisa assay.
In some embodiments, the purification specific steps are: pretreating the affinity chromatographic column, adding serum containing polyclonal antibody into the pretreated affinity chromatographic column for loading, and eluting with eluent.
In some embodiments, the Elisa assay specifically comprises the steps of:
(1) coating the enzyme-linked plate with specific histidine methylation modified polypeptide, and washing the plate;
(2) closing the coated enzyme-linked plate, incubating, and discarding the sealing liquid;
(3) adding the polyclonal antibody obtained by purification, incubating, discarding the sealing solution, and washing the plate;
(4) adding enzyme-labeled secondary antibodies, incubating, discarding sealing liquid, and washing the plate;
(5) adding substrate liquid for color development, and reading the plate after stopping the reaction.
The polyclonal antibody sample to be tested is diluted in a ratio, and in one or more embodiments of the invention, diluted in a ratio of 1:250 to 1:1024000.
Experimental results show that the polyclonal antibody prepared by the preparation method disclosed by the invention has the following structure that: the antibody titer was above 1 at 250 dilutions, and the Elisa assay was above the positive standard of 1.
The fourth aspect of the present invention provides an application of the polyclonal antibody according to the second aspect of the present invention or the polyclonal antibody prepared by the preparation method according to the third aspect of the present invention in identifying a specific histidine methylation-modified MYLK2 protein, and in particular, the polyclonal antibody obtained in the present invention may be applied to Westernblot, or immunofluorescence, immunohistochemistry, flow detection, ELISA kit, etc. for detecting a specific histidine methylation-modified MYLK2 protein, thereby realizing rapid and simple detection of a change in MYLK2 specific histidine methylation in a biological sample, and playing a great role in researching a function of a specific histidine methylation MYLK2 protein in skeletal muscle and a dynamic change in MYLK2 specific histidine methylation in a skeletal muscle related disease development process.
In a fifth aspect, the present invention provides the use of a polyclonal antibody according to the second aspect of the present invention or a polyclonal antibody prepared by a preparation method according to the third aspect of the present invention, for the preparation of a product recognizing a specific histidine methylation-modified MYLK2 protein. The product can be a preparation, a chip or a kit.
The product contains the polyclonal antibody of the second aspect of the invention or the polyclonal antibody prepared by the preparation method of the third aspect of the invention. To enable detection of specific histidine methylation-modified MYLK2 proteins.
In a sixth aspect, the present invention provides a reagent for detecting a specific histidine methylation-modified MYLK2 protein, comprising a polyclonal antibody according to the second aspect of the present invention or a polyclonal antibody prepared by the preparation method according to the third aspect of the present invention.
In a seventh aspect, the invention provides a test strip for detecting a specific histidine methylation-modified MYLK2 protein, comprising a detection reagent according to the sixth aspect of the invention.
In an eighth aspect, the invention provides a product of a specific histidine methylation modified MYLK2 protein comprising a detection reagent as described in the sixth aspect of the invention or comprising a detection strip as described in the seventh aspect of the invention.
Further, the product is a chip or a kit.
It will be appreciated that reagents such as diluents, converting solutions, buffers, washing solutions, eluents and purifying solutions, which are conventional in the art, may also be included in the specific products, and specific reagent types will be different depending on the detection technique and will not be specifically described herein.
The present invention will be illustrated by the following examples, which are given for illustrative purposes only and are not intended to limit the scope of the present invention in any way, and unless otherwise specified, the conditions or procedures not specifically described are conventional and the reagents and materials employed are commercially available.
Example 1 preparation of Polypeptides
The following polypeptides were synthesized by the assigned Hangzhou Hua An Biotechnology Co., ltd:
a polypeptide with an amino acid sequence shown as SEQ ID No.1, which is named as polypeptide 1;
the polypeptide with the amino acid sequence shown as SEQ ID No.2 is named as polypeptide 2.
EXAMPLE 2 preparation of histidine methylation-modified human MYLK2 protein immunogen at position 148
Coupling the polypeptide 2 of example 1 with hemocyanin (KLH) by protein coupling technology to obtain the 148 th histidine methylation modified humanized MYLK2 immunogen, which comprises the following specific steps:
1. polypeptide 2 conjugated KLH
(1) 20mg of KLH was dissolved in 2mL of 5mM aqueous EDTA;
(2) 8mg of Sulfo-SMCC is weighed and completely dissolved in 50 mu L of DMSO, then 150 mu L of 1XPBS is added and uniformly mixed;
(3) Dropwise adding the Sulfo-SMCC solution into the KLH solution, gently shaking while adding, and standing at room temperature for 1h;
(4) Placing the activated KLH solution into a dialysis bag, clamping the dialysis bag, and dialyzing the dialysis bag for 1h in 2L of 1XPBS under magnetic stirring of a refrigerator at 4 ℃;
(5) The new 1XPBS was exchanged for dialysis for 2h and repeated once; placing activated and dialyzed KLH into a 15mL inlet centrifuge tube, marking the names, time and concentration of the reagents on the tube, and preserving the tube in a refrigerator at 4 ℃;
(6) 4mg of polypeptide 2 was weighed, dissolved in 50. Mu.L of LDMSO, and 200. Mu.L of 1XPBS was added to the solution, followed by rapid mixing according to the polypeptide: klh=1 mg: immediately adding KLH in 680 mug proportion, and reacting for 2 hours at 4 ℃ overnight or at room temperature;
(7) Placing the crosslinked KLH-peptide crosslinked compound into a dialysis bag, clamping the crosslinked KLH-peptide crosslinked compound by a dialysis clamp, and dialyzing the crosslinked KLH-peptide crosslinked compound in 4L of 1XPBS under magnetic stirring at a temperature of 4 ℃ for overnight;
(8) And taking out the dialyzed KLH-peptide into a clean 1.5mL centrifuge tube, subpackaging according to the immune dose, and preserving in a refrigerator at-20 ℃.
2. Polypeptide 2 coupled to carrier protein BSA (polypeptide 2 coupled to BSA for use in post purification antibodies and antibody detection)
(1) 20mg of BSA was dissolved in 2mL of 5mM EDTA aqueous solution;
(2) Weighing 5mg of Sulfo-SMCC, completely dissolving in 50 mu L of DMSO, adding 150 mu L of 1XPBS, and uniformly mixing;
(3) Dropwise adding the Sulfo-SMCC solution into the BSA solution, gently shaking while adding, and standing at room temperature for 1h;
(4) Placing the activated BSA solution into a dialysis bag, clamping the BSA solution by a dialysis clamp, and dialyzing the BSA solution for 1h in 2L of 1XPBS under magnetic stirring of a refrigerator at 4 ℃;
(5) The dialysis was repeated once by replacing fresh 1XPBS for 2 h. Placing activated and dialyzed BSA into a 15mL inlet centrifuge tube, marking the names, time and concentration of the reagents on the tube, and preserving the BSA in a refrigerator at 4 ℃;
(6) 1mg of polypeptide 2 is weighed, dissolved in 50 mu L of DMSO, added with 150 mu L of 1XPBS, quickly mixed, then added with 100 mu L of the activated BSA solution, and reacted for 2 hours at 4 ℃ in a refrigerator overnight or at room temperature;
(7) The crosslinked BSA-Peptide complex is added into a 1.5mL centrifuge tube, 1XPBS is added to 1mL, the project number, the concentration and the date are marked, and the BSA-Peptide complex is crossed with a detection group.
EXAMPLE 3 preparation of polyclonal antibody specifically recognizing the 148 th histidine methylation-modified human MYLK2 protein
Animal immunization:
taking the 148 th histidine methylation modified humanized MYLK2 immunogen in the example 2 out of a refrigerator at the temperature of minus 20 ℃, dissolving at normal temperature, avoiding repeated freeze thawing, and marking a syringe; according to the configuration in table 1, the volume ratio of adjuvant to the 148 th histidine methylation modified human MYLK2 immunogen is 1:1 extracting an adjuvant, fully emulsifying, wherein the emulsification standard is as follows: the emulsified immunogen is dripped into 37 ℃ water and is not dispersed as being qualified.
Before primary immunization, numbering all New Zealand white rabbits, wherein the concentration of primary antigen is 1mg/mL, the concentration of rabbits is 0.5 mL/rabbit, the amount of secondary-quaternary antigen is halved, immunization is carried out according to specific time and dosage shown in table 1, and multipoint subcutaneous injection is adopted during immunization, wherein each point is 0.2mL; and 3, detecting small sample serum of middle ear artery at 7 days after three-immunity of the rabbit, wherein the detection is qualified, and taking whole blood after 7 days after the three-immunity of the rabbit and the three-immunity of the rabbit.
TABLE 1 immunization times, times and doses
Antibody titer detection:
after three immunizations, the Elisa test is carried out on whether the serum contains the corresponding antibody and whether the titer of the antibody reaches the standard, and the specific steps are as follows:
(1) And (3) wrapping the plate: with coating buffer (coating buffer: na 2 CO 3 And NaHCO 3 Buffer) the known antigen polypeptides (polypeptide 1 and polypeptide 2, respectively) were diluted to 1. Mu.g/mL, 50. Mu.l were added to the reaction wells of each polystyrene plate, overnight at 4 ℃, the next day the in-well solution was discarded, and 1XPBST wash buffer was used to wash 1 time at 180. Mu.l per well;
(2) Closing: blocking with 150 μl of 1% BSA (prepared by PBST) per well, incubating at 37deg.C for 1 hr, and discarding blocking solution;
(3) Sample adding: diluting the sample to be detected according to a certain proportion (the dilution proportion is shown in Table 2), taking 50 mu L of diluted antibody into the closed reaction hole, setting a negative control hole (PBS), incubating for 1 hour at 37 ℃, discarding the sealing liquid, and washing with 150 mu L of 1xPBST washing buffer solution for 3 times;
(4) Adding enzyme-labeled antibody: fresh diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) was added to the wells of the enzyme-labeled plate at 50. Mu.L/well, incubated at 37℃for 45min, after which the blocking solution was discarded and washed 3 times with 150. Mu.L of 1xPBST buffer per well;
(5) Adding substrate liquid for color development, and reading a plate: 50 μl of TMB substrate solution prepared temporarily was added to each reaction well, the reaction was stopped by adding 50 μl of 1M sulfuric acid to each reaction well, and the ELISA plate was read in a preheated ELISA (450 nm), the results are shown in Table 2.
TABLE 2 serum antibody titre detection results
Note that: in Table 2, 1# and 2# are rabbit parallel test number RB5234, and 3# and 4# are rabbit parallel test number RB 5235.
Antibody purification:
after the titer of the antibody meets the standard, the antibody is added for 7 days, and whole blood can be collected after the addition of the antibody for 7 days, and the antibody is purified, specifically comprising the following steps:
(1) The affinity column was washed thoroughly with 20mL of pure water and 1XPBS (pH 7.4) in this order, at a flow rate of 70 mL/h;
(2) Taking 10mL of serum to be purified in a 50mL centrifuge tube, and carrying out suction filtration by using a microporous filter membrane with the aperture of 0.45 mu m and the diameter of 25 mm;
(3) Loading the filtered serum sample at a flow rate of 40mL/h, and repeating the steps once;
(4) Washing the column with 20mL 1XPBS (pH 7.4) at a flow rate of 70mL/h, connecting the protein detector after 10min, and adjusting the light transmittance (T grade) of the instrument to be 100 in the washing process;
(5) Adjusting the absorbance (1A grade) indication of the protein detector to 0, opening an HD-A computer collector on a computer desktop, adjusting the full screen range to 5, eluting the antibody at a speed of 40mL/h by using glycine solution (pH 2.7,0.2M), pressing a green elution record button to start eluting, and collecting the antibody when the indication of the instrument starts to rise;
(6) In the process of collecting the antibody, adjusting the pH value of the antibody to about 7 in time by using 1M sodium bicarbonate, and recording the highest peak value of the elution peak;
(7) After the antibody is collected, the pH value is regulated to about 7, the volume of the eluted antibody is recorded, and then the rubber tube connected with the collector is washed by purified water;
(8) Sequentially cleaning the affinity chromatography column with 20mL of 1XPBS and pure water at a speed of 70mL/h, adding 20% ethanol, sealing, and storing in a refrigerator at 4 ℃;
(9) The purified antibodies were submitted to different tests according to different requirements, when the methylated antibodies were purified, serum was passed through a methylation column, the eluted antibodies were passed through a non-methylation column to obtain specific methylated antibodies, other modified types of antibodies were purified similarly, and Elisa was performed, with the results shown in Table 3.
TABLE 3 purification of antibody titre detection results
Note that: in Table 3, 1# and 2# are rabbit parallel test number RB5234, and 3# and 4# are rabbit parallel test number RB 5235.
And (3) detecting the concentration of the finished antibody:
(1) After the amount of the antibody which can be delivered is purified and the detection titer of the semi-finished product is qualified, mixing all the antibodies, concentrating by using an ultrafiltration concentration tube to reach a certain concentration and volume;
(2) Placing the concentrated antibody in 1L 0.01M PBS (pH 7.4), dialyzing at room temperature, changing liquid once every 3h, and changing liquid for 3 times (overnight dialyzing in refrigerator at 2-8deg.C);
(4) Taking out the dialyzed antibody to a clean centrifuge tube, filtering the antibody in an ultra-clean workbench by using a disposable low-adsorption filter head with the thickness of 0.22 mu m, taking a small sample for inspection, and taking 5 mu l of detection concentration;
(5) The concentration was measured on an ultra-micro spectrophotometer (densovix DS-11) Protein A280 application.
After detection, the volumes of the antibodies were 0.7mL (RB 5234) and 1.2mL (RB 5235), respectively, and the antibody concentrations were 0.52mg/mL (RB 5234) and 0.96mg/mL (RB 5235), respectively.
EXAMPLE 4 identification of histidine-methylation-modified human MYLK2 protein at position 148
Adopting human Flag-MYLK2 protein expressed in 293T cells to obtain unmethylated Flag-MYLK2 fusion protein;
the 148 th histidine methylation modified Flag-MYLK2 fusion protein is obtained by coexpression in 293T cells by utilizing histidine N1 methylation transferase CARNMT1, namely the H148 th methylation modified protein Flag-MYLK 2H 148me;
western blotting was performed using the purified polyclonal antibody prepared in example 3, and the results are shown in FIG. 1, wherein MYLK2 represents the level of Flag-MYLK2, and CARNMT1-mut-HA and CARNMT1-WT-HA represent proteins with histidine N1 methyltransferase CARNMT1 having no enzymatic activity and enzymatic activity, respectively. H148me represents the level of histidine methylation at position 148 of MYLK2 (detected using antibodies purified from rabbit No. RB 5235). From the results of the test in FIG. 1, it can be seen that the polyclonal antibody prepared in example 3 can recognize MYLK2 with methylation modification of histidine 148 but cannot recognize MYLK2 with non-methylation modification.
According to the results, the immunogen provided by the invention can be used for preparing the polyclonal antibody capable of highly specifically recognizing the 148 th histidine methylation modified human MYLK2 protein, and the polyclonal antibody can not recognize the unmethylated MYLK2 protein, and can be widely used for immunohistochemistry, westernblot, immunosilver light, flow detection, in-vitro immunoassay and the like, so that the change of MYLK2 specific histidine methylation in a biological sample can be rapidly and simply detected, and the function of the specific histidine methylation MYLK2 protein in skeletal muscles and the development of the MYLK2 specific histidine methylation dynamic change in the skeletal muscle related disease development process can be promoted and studied.
The preparation method of the polyclonal antibody is simple to operate, and can be used for preparing a large amount of antibodies for specifically recognizing 148 th histidine methylation modified humanized MYLK2 protein.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. A specific histidine methylation modified MYLK2 protein immunogen is characterized by comprising a polypeptide with an amino acid sequence shown as SEQ ID No. 2.
2. The specific histidine-methylation-modified MYLK2 protein immunogen of claim 1, further comprising a carrier protein coupled to said polypeptide;
preferably, the carrier protein is selected from hemocyanin.
3. A polyclonal antibody specifically recognizing a specific histidine-methylation-modified MYLK2 protein, characterized in that it is obtained by immunizing an animal with the specific histidine-methylation-modified MYLK2 protein immunogen of claim 1 or 2;
preferably, the animal used for immunization is a rabbit;
preferably, the animal used for immunization is New Zealand white rabbit.
4. A method of preparing a polyclonal antibody according to claim 3, comprising the steps of:
immunizing an animal by adopting the specific histidine methylation modified MYLK2 protein immunogen, collecting serum, and purifying to obtain a polyclonal antibody;
preferably, the immunized animal is a rabbit;
preferably, the immunized animal is a New Zealand white rabbit;
preferably, the purification is performed by affinity chromatography.
5. The preparation method of claim 4, wherein the time, the dose and the Freund's adjuvant type of each immunization of the New Zealand white rabbits are as follows:
6. use of the polyclonal antibody of claim 3 or the polyclonal antibody produced by the production method of any one of claims 4-5 for the recognition of specific histidine methylation-modified MYLK2 proteins.
7. Use of the polyclonal antibody of claim 3 or the polyclonal antibody prepared by the preparation method of any one of claims 4-5 for the preparation of a product for detecting a specific histidine methylation-modified MYLK2 protein;
preferably, the product is a formulation, chip or kit.
8. A reagent for detecting a specific histidine methylation-modified MYLK2 protein, comprising the polyclonal antibody according to claim 3 or the polyclonal antibody produced by the production method according to any one of claims 4 to 5.
9. A test strip for detecting a specific histidine methylation modified MYLK2 protein comprising the detection reagent of claim 8.
10. A product of a specific histidine methylation modified MYLK2 protein comprising the detection reagent of claim 8 or comprising the detection strip of claim 9;
preferably, the product is a chip or a kit.
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