CN118598972A - Histone H4 polypeptide and histone H4 antibody - Google Patents
Histone H4 polypeptide and histone H4 antibody Download PDFInfo
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Abstract
The invention discloses a histone H4 polypeptide and a histone H4 antibody. The anti-histone H4 antibody prepared by the histone H4 polypeptide provided by the invention can specifically identify endogenous H4 protein or immunogen H4 polypeptide protein in a tissue in immunoblotting analysis, and has extremely important significance for detecting the existence of histone H4 in blood circulation, pathogenesis and prognosis of diseases.
Description
Technical Field
The invention relates to the technical field of polypeptide and antibody preparation thereof, in particular to a histone H4 polypeptide and a histone H4 antibody.
Background
Histones (Histone) are strongly cationic proteins within the eukaryotic nucleus, including H2A, H2B, H, H4, which are highly conserved across species. In the nucleus, histones are the major protein component of chromatin and play an important role in gene regulation as bobbins for DNA winding. Histones may be released into the extracellular space in three forms: free release, as DNA-entangled nucleosomes or as part of neutrophil extracellular traps, all three forms can be detected in serum following severe cell death (such as sepsis, trauma, ischemia/reperfusion injury and autoimmune disease). Once the histone enters the extracellular space, it acts as a damage-associated molecular pattern (DAMP) protein, activating the immune system and causing further cytotoxicity. Blocking histone release, neutralizing circulating histones, or blocking histone signaling, etc. may be effective in reducing mortality in animal models of acute organ injury.
Extracellular histones have been found to be potential mediators of death in sepsis and the like, and in particular circulating histone H4 levels are closely related to sepsis, sepsis and other inflammatory conditions. Therefore, detection and recognition of histone H4 is particularly important for medical detection.
Disclosure of Invention
First, the technical problem to be solved
The invention aims to provide a histone H4 polypeptide and a histone H4 antibody.
(II) technical scheme
In order to solve the technical problems, in a first aspect, the invention provides a histone H4 polypeptide, wherein the histone H4 polypeptide has an amino acid sequence shown as SEQ ID NO. 1.
In a second aspect, the present invention provides a recombinant protein obtainable by cross-linking a histone H4 polypeptide as described above with a carrier protein.
Preferably, the recombinant protein is obtained by cross-linking the histone H4 polypeptide described above with Keyhole Limpet Hemocyanin (KLH) using 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester coupling method.
In a third aspect, the present invention provides a histone H4 antibody recognizing histone H4, which histone H4 antibody is prepared from the recombinant protein of any one of the above-described methods of immunizing animals.
In a fourth aspect, the present invention provides a method for preparing the histone H4 antibody, which comprises:
S1: crosslinking the histone H4 polypeptide and the carrier protein keyhole limpet hemocyanin by adopting a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester coupling method;
S2: dissolving the crosslinked KLH-peptide dry powder in a phosphate buffer solution to prepare mother liquor 1 with the concentration of 800 mug/mL, mother liquor 2 with the concentration of 400 mug/mL and mother liquor 3 with the concentration of 200 mug/mL respectively;
s3: adding 0.75mL of mother solution 1 into an equal volume of Freund's complete adjuvant, fully emulsifying, and subcutaneously injecting 1mL into the neck and back of a white rabbit to perform primary immunization;
s4: after 3 weeks of primary immunization, 0.75mL of mother liquor 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken to be subcutaneously injected into a white rabbit for the 2 nd immunization;
s5: after immunization for 2 weeks, mixing 0.75mL of mother liquor 2 with an equal volume of Freund's incomplete adjuvant, fully emulsifying, taking 1mL of the mixture into a white rabbit subcutaneous multipoint injection, and performing immunization for 3 times;
S6: after immunization for 2 weeks at 3 times, mixing 0.75mL of mother liquor 2 with an equal volume of Freund's incomplete adjuvant, fully emulsifying, taking 1mL of the mixture into white rabbits for subcutaneous multipoint injection, and performing immunization for 4 times;
S7: after the 4 th immunization for 2 weeks, directly injecting 1mL of mother solution 3 into the auricular vein for the 5 th immunization;
S8: on day 4 after the 5 th immunization, 10mL of blood is taken from the auricular vein, the multi-antiserum is collected for identification, the serum titer reaches 1:64000, and the whole body blood of the rabbit can be collected and obtained by adopting the carotid artery.
In a fifth aspect, the present invention provides a method for affinity purification of a histone H4 antibody described above, the method comprising:
(1) Affinity purification of antibody: diluting antiserum with PBS buffer solution, filtering with a filter membrane, passing through Protein A affinity chromatography column, washing 5-10 times of the volume of the chromatography column with loading buffer solution, eluting antibody bound by the chromatography column with eluent, collecting effluent liquid, immediately adjusting pH value of the collected effluent liquid of each tube to 7.0-8.0 with neutralizing solution, and finally replacing the buffer solution of the recovered antibody solution with PBS;
(2) Peptide affinity purification: pouring the NHS activated agarose gel into a chromatography empty column, and discarding the protection solution; the cleaning solution is used for three times of rapid suction filtration and cleaning; adding a bare peptide sample solution to be coupled with the concentration of 0.75mg/mL into the chromatographic column of the washed filler; flushing the filler with a cleaning solution after coupling; washing the chromatographic column with PBS; adding the IgG antibody purified by Protein A into a chromatographic column, rotating uniformly to enable the sample and the filler to be fully contacted and adsorbed, and rotating at 4 ℃ for incubation overnight; washing the chromatographic column with PBS, eluting the target antibody with eluent, neutralizing with neutralizing solution, regulating pH value to 7.0-8.0, transferring the obtained purified antibody into ultrafiltration tube, centrifuging and concentrating the antibody, and replacing with PBS buffer solution; packaging in refrigerator for preservation.
In a sixth aspect, the present invention provides a method for identifying a histone H4 antibody as described above, comprising:
Preparing SDS-PAGE gel according to a standard method, adding histone H4 polypeptide with the protein concentration of 0.1mg/mL or 2 mug of kidney tissue nuclear protein sample or 2 mug of spleen tissue lysate into a gel hole, carrying out constant-pressure electrophoresis for 30 minutes at 80V, changing 120V voltage when the sample runs through the concentrated gel to form a straight line, stopping electrophoresis when the bromophenol blue indicator runs to two thirds of the separation gel, adopting a wet transfer membrane method to maintain constant current for 200mA for 35 minutes, and transferring the protein to a PVDF membrane; the antibody purified from the blood of the immunized rabbit is used as a primary antibody, the adopted concentration is 1mg/mL, and the dilution ratio is 1: incubating overnight at 1000,4 ℃; commercial HRP-conjugated goat anti-rabbit antibody is used as a secondary antibody, incubated for 1 hour at room temperature, and a chemiluminescent substrate is used for covering the surface of the film for color development, and a Tanon 5200 full-automatic chemiluminescent image analysis system is used for imaging.
(III) beneficial effects
The technical scheme of the invention has the following advantages:
The anti-histone H4 antibody prepared from the histone H4 polypeptide can specifically identify endogenous H4 protein or immunogen H4 polypeptide in tissues in immunoblotting analysis, and has extremely important significance for detecting the existence of histone H4 in blood circulation, the pathogenesis and prognosis of diseases.
Drawings
FIG. 1 is a immunoblotting diagram of a purified histone H4 polypeptide antibody according to example 1 of the present invention; the polypeptide of the invention is used for immunizing animals to obtain polyclonal antibodies, the polyclonal antibodies are combined with the immunogen histone H4 polypeptide, and the band in the lane is the signal of the histone H4 polypeptide;
FIG. 2 is a immunoblotting diagram of the purified histone H4 polypeptide antibody according to example 1 of the present invention; the polypeptide of the invention is used for immunizing animals to obtain polyclonal antibodies, the polyclonal antibodies are combined with endogenous histone H4 in the mouse kidney tissue or spleen tissue lysate, and the bands in lanes are signals of the endogenous histone H4; m represents Marker,1 represents kidney tissue lysate, 2 represents spleen tissue lysate;
FIG. 3 is an HPLC chart of a histone H4 polypeptide of example 1 of the present invention;
FIG. 4 is a mass spectrum of histone H4 polypeptide according to example 1 of the present invention.
Detailed Description
In order that the above objects, features and advantages of the invention will be more clearly understood, a further description of the invention will be made. It should be noted that, without conflict, the embodiments of the present invention and features in the embodiments may be combined with each other.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced otherwise than as described herein; it will be apparent that the embodiments in the specification are only some, but not all, embodiments of the invention.
In the present invention, unless otherwise specified, the materials and equipment used are commercially available or are commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Example 1
The embodiment provides a histone H4 polypeptide, wherein the histone H4 polypeptide has an amino acid sequence shown as SEQ ID NO. 1. The amino acid sequence is specifically AVTYTEHAKRKTVTAM.
The histone H4 antibody prepared by the histone H4 polypeptide can specifically identify endogenous H4 protein or pure H4 polypeptide protein in tissues in immunoblotting (Western blot) analysis.
The histone H4 antibody in this example is obtained by the following steps:
1. Analysis and design of polypeptide sequences: and screening out linear epitopes of histone B cells by utilizing ABCpred, bepiPred, IEDB, allerTOP, toxinPred websites and the like, carrying out antigen epitope analysis on the linear epitopes, mainly evaluating indexes such as antigenicity, hydrophilicity, accessibility, sensitization, toxicity, specificity and the like, and finally determining 16 amino acids at 70-85 positions of histone H4 as a synthetic polypeptide amino acid sequence, wherein the sequence is AVTYTEHAKRKTVTAM.
2. Polypeptide synthesis and crosslinking
To increase the immunogenicity of the polypeptide, the histone H4 polypeptide was cross-linked with the carrier protein Keyhole Limpet Hemocyanin (KLH) using SMCC (4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester) coupling. Wherein, the histone H4 polypeptide is synthesized by Shanghai peptide biotechnology Co., ltd, the HPLC spectrum of the histone H4 polypeptide is shown in figure 3, and the mass spectrum is shown in figure 4.
3. Polypeptide immunization and antiserum preparation: the animals are immunized by healthy New Zealand white rabbits with the age of more than 6 months and the weight of about 2.5 kg. Before immunization, rabbit ear vein blood is taken to separate serum before immunization, and the serum is used as negative control in the subsequent serum detection stage.
In the primary immunization, the crosslinked KLH-peptide dry powder is dissolved in phosphate buffer (PBS, pH 7.4) to form 800 mug/mL mother liquor concentration, 0.75mL of KLH-peptide with the concentration of 800 mug/mL is added into equal volume Freund complete adjuvant to fully emulsify (until the KLH-peptide does not diffuse in water), and the KLH-peptide is injected subcutaneously at the back of the neck of a white rabbit, wherein the injection volume is 1 mL/rabbit. 3 weeks after the initial immunization (21 days), the 2 nd immunization was performed, the KLH-peptide dose was halved (0.75 mL, 400. Mu.g/mL), the KLH-peptide solution was mixed with an equal volume of Freund's incomplete adjuvant, and after sufficient emulsification, 1mL of the solution was taken for subcutaneous multipoint injection. Followed by 4 booster immunizations every 2 weeks. At immunization 3 and 4, the KLH-peptide dose was halved (0.75 mL, 400. Mu.g/mL), the KLH-peptide solution was mixed with an equal volume of Freund's incomplete adjuvant, and after sufficient emulsification, 1mL of subcutaneous multipoint injection was taken. After 2 weeks of 4 th immunization, 1mL of KLH-peptide with the concentration of 200 mug/mL was directly injected into the ear margin vein for the last boost, 10mL of blood was taken from the ear margin vein on day 4 after immunization, and the polyclonal serum was collected. The ELISA plate is coated with uncrosslinked synthetic polypeptide, and immune serum titer is detected by indirect ELISA method. Serum titer reaches 1:64000, and the carotid artery blood is taken, and antiserum is obtained by standard method.
Specifically, the preparation method of the histone H4 antibody comprises the following steps:
S1: crosslinking the histone H4 polypeptide and carrier protein Keyhole Limpet Hemocyanin (KLH) by adopting a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester coupling method;
S2: dissolving the crosslinked KLH-peptide dry powder in a phosphate buffer solution to prepare mother liquor 1 with the concentration of 800 mug/mL, mother liquor 2 with the concentration of 400 mug/mL and mother liquor 3 with the concentration of 200 mug/mL respectively;
s3: adding 0.75mL of mother solution 1 into an equal volume of Freund's complete adjuvant, fully emulsifying, and subcutaneously injecting 1mL into the neck and back of a white rabbit to perform primary immunization;
s4: after 3 weeks of primary immunization, 0.75mL of mother liquor 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken to be subcutaneously injected into a white rabbit for the 2 nd immunization;
S5: after 2 weeks of immunization, 0.75mL of mother liquor 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken to be subcutaneously injected into a white rabbit for immunization for 3 rd time;
S6: after immunization for 2 weeks at the 3 rd time, 0.75mL of the mother solution 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken for subcutaneous multipoint injection of the white rabbits to carry out immunization for the 4 th time;
s7: after the 4 th immunization for 2 weeks, directly injecting 1mL of the mother solution 3 into the auricular vein for the 5 th immunization;
S8: on day 4 after the 5 th immunization, 10mL of blood is taken from the auricular vein, the multi-antiserum is collected for identification, the serum titer reaches 1:64000, and the whole body blood of the rabbit can be collected and obtained by adopting the carotid artery.
Fasted operation was performed on rabbits 24 hours prior to blood sampling. Blood was collected into a coagulation tube, allowed to stand at room temperature for one hour, then centrifuged in a centrifuge at 4℃and 3000rpm for 15min, and the supernatant serum was collected and collected into a 50mL centrifuge tube. Serum and PBS buffer solution are mixed according to the volume ratio of 1:1, and then filtering through a 0.45 μm filter membrane, and performing the next antibody purification step.
4. Antibody affinity purification
(1) Protein a method for IgG antibody purification: antisera were buffered with PBS buffer at 1:1 (filtered through a 0.45 μm filter), followed by washing the column with a loading buffer (PBS, pH 7.4) 10 times the column volume (i.e. complete elution of the hybrid Protein). The column bound antibody was then eluted with an eluent (0.1M glycine, pH 3.0), the effluent was collected, and the pH of the collected tube effluent was immediately adjusted to 7.6 with a neutralization solution (1M Tris-HCl, pH 8.5). Finally, the buffer solution of the recovered antibody solution was replaced with PBS.
(2) Peptide affinity purification: 3mL of NHS activated agarose gel was poured into the chromatography empty column and the protection solution was discarded. The wash was completed by three quick-pull washes (needle pull down, accelerated liquid flow) with 12mL of wash solution (10 mM HCl) and the effluent solution was reddened as measured by pH paper. To the washed packed chromatographic column was added 4mL (0.75 mg/mL) of bare peptide sample solution to be coupled and reacted at 28℃with shaking at room temperature for 4h. After coupling, the filler was washed with 50mL of washing solution (0.1M Tris-HCl,1M NaCl,pH7.0) and used. The column was then rinsed with 15mL PBS. IgG antibodies purified by Protein A were added to the column, spun well, allowed to contact and adsorb the sample and packing well, and incubated overnight at 4 ℃. The column was washed with 30mL of PBS, 15mL of eluent (0.1M glycine, pH 3.0) was used to elute the antibody of interest, and the solution was rapidly neutralized with a neutralization solution (1M Tris-HCl, pH 8.5) to adjust the pH to 7.4. The purified antibody obtained was transferred to a 100KD ultrafiltration tube, concentrated by centrifugation at 3000g at 4deg.C, and replaced with PBS (pH 7.4) buffer. The concentration of the antibody is detected by Nanodropone (the concentration is 5.943 mg/mL), the concentration is adjusted to be 1mg/mL, and the antibody is packaged in a refrigerator at the temperature of minus 80 ℃ for preservation.
5. The anti-histone H4 antibody is identified by the following steps:
(1) Immunoblotting analysis:
SDS-PAGE gel was prepared according to standard methods, 5. Mu.L of histone H4 polypeptide (2. Mu.g of kidney tissue nucleoprotein sample or 2. Mu.g of spleen tissue lysate) with protein concentration of 0.1mg/mL was added to the gel well, electrophoresis was carried out at constant pressure of 80V for about 30 minutes, when the sample ran through the gel concentrate in a substantially straight line, the 120V voltage was changed, electrophoresis was stopped until the bromophenol blue indicator was run to two thirds of the separation gel (about 50 minutes), and the protein was transferred to PVDF membrane by wet transfer method with constant flow of 200mA for 35 minutes. Antisera (polyclonal antibody against histone H4) purified from the blood of immunized rabbits were used as primary antibody at a concentration of 1mg/mL, with a dilution ratio of 1: incubated overnight at 1000,4 ℃. Commercial HRP conjugated goat anti-rabbit antibody (1:30000 dilution) was used as secondary antibody and incubated for 1 hour at room temperature. The surface of the cover film was developed using a chemiluminescent substrate, tanon 5200,5200 full-automatic chemiluminescent image analysis system was used for imaging.
FIG. 1 is a WB pattern of the purified histone H4 polypeptide antibody according to example 1 of the present invention. The target band of the immunoblot is histone H4 polypeptide immunogen.
FIG. 2 is a WB pattern of the purified histone H4 polypeptide antibody according to example 1 of the present invention. The detection sample is mouse kidney tissue or spleen tissue lysate, and the band in the lane is the signal of endogenous histone H4. M represents Marker,1 represents kidney tissue lysate, 2 represents spleen tissue lysate.
The anti-histone H4 antibody prepared from the histone H4 polypeptide can specifically identify endogenous H4 protein or pure H4 polypeptide protein in tissues in immunoblotting analysis, and has extremely important significance for detecting the existence of histone H4 in blood circulation, the pathogenesis and prognosis of diseases.
It is to be understood that this invention is not limited to the particular steps and structures described above. Also, a detailed description of known method techniques is omitted here for the sake of brevity.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A histone H4 polypeptide, characterized by: the histone H4 polypeptide has an amino acid sequence shown as SEQ ID NO. 1.
2. A recombinant protein, characterized in that: the recombinant protein is obtained by crosslinking the histone H4 polypeptide according to claim 1 with a carrier protein.
3. The recombinant protein according to claim 1, wherein: the recombinant protein is obtained by crosslinking the histone H4 polypeptide and keyhole limpet hemocyanin according to claim 1 by adopting a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester coupling method.
4. A histone H4 antibody recognizing histone H4, wherein the histone H4 antibody is prepared from the recombinant protein according to any one of claims 2 to 3 by a method of immunizing an animal.
5. The method of producing the histone H4 antibody according to claim 4, which comprises:
S1: crosslinking the histone H4 polypeptide and the carrier protein keyhole limpet hemocyanin by adopting a 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester coupling method;
S2: dissolving the crosslinked KLH-peptide dry powder in a phosphate buffer solution to prepare mother liquor 1 with the concentration of 800 mug/mL, mother liquor 2 with the concentration of 400 mug/mL and mother liquor 3 with the concentration of 200 mug/mL respectively;
S3: adding 0.75mL of the mother liquor 1 into an equal volume of Freund's complete adjuvant, fully emulsifying, and subcutaneously injecting 1mL into the neck and back of a white rabbit to perform primary immunization;
S4: after 3 weeks of primary immunization, 0.75mL of the mother liquor 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken to be subcutaneously injected into a white rabbit for the 2 nd immunization;
S5: after 2 weeks of immunization, 0.75mL of the mother liquor 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken to be subcutaneously injected into a white rabbit for immunization for 3 rd time;
S6: after immunization for 2 weeks at the 3 rd time, 0.75mL of the mother solution 2 is mixed with an equal volume of Freund's incomplete adjuvant, and after full emulsification, 1mL of the mixture is taken for subcutaneous multipoint injection of the white rabbits to carry out immunization for the 4 th time;
s7: after the 4 th immunization for 2 weeks, directly injecting 1mL of the mother solution 3 into the auricular vein for the 5 th immunization;
S8: on day 4 after the 5 th immunization, 10mL of blood is taken from the auricular vein, the multi-antiserum is collected for identification, the serum titer reaches 1:64000, and the whole body blood of the rabbit can be collected and obtained by adopting the carotid artery.
6. The method of affinity purification of the histone H4 antibody of claim 4, comprising:
(1) Affinity purification of antibody: diluting antiserum with PBS buffer solution, filtering with a filter membrane, passing through Protein A affinity chromatography column, washing 5-10 times of the volume of the chromatography column with loading buffer solution, eluting antibody bound by the chromatography column with eluent, collecting effluent liquid, immediately adjusting pH value of the collected effluent liquid of each tube to 7.0-8.0 with neutralizing solution, and finally replacing the buffer solution of the recovered antibody solution with PBS;
(2) Peptide affinity purification: pouring the NHS activated agarose gel into a chromatography empty column, and discarding the protection solution; the cleaning solution is used for three times of rapid suction filtration and cleaning; adding a bare peptide sample solution to be coupled with the concentration of 0.75mg/mL into the chromatographic column of the washed filler; flushing the filler with a cleaning solution after coupling; washing the chromatographic column with PBS; adding the IgG antibody purified by Protein A into a chromatographic column, rotating uniformly to enable the sample and the filler to be fully contacted and adsorbed, and rotating at 4 ℃ for incubation overnight; washing the chromatographic column with PBS, eluting the target antibody with eluent, neutralizing with neutralizing solution, regulating pH value to 7.0-8.0, transferring the obtained purified antibody into ultrafiltration tube, centrifuging and concentrating the antibody, and replacing with PBS buffer solution; packaging in refrigerator for preservation.
7. A method of identifying a histone H4 antibody according to claim 4, comprising:
Preparing SDS-PAGE gel according to a standard method, adding histone H4 polypeptide with the protein concentration of 0.1mg/mL or 2 mug of kidney tissue nuclear protein sample or 2 mug of spleen tissue lysate into a gel hole, carrying out constant-pressure electrophoresis for 30 minutes at 80V, changing 120V voltage when the sample runs through the concentrated gel to form a straight line, stopping electrophoresis when the bromophenol blue indicator runs to two thirds of the separation gel, adopting a wet transfer membrane method to maintain constant current for 200mA for 35 minutes, and transferring the protein to a PVDF membrane; the antibody purified from the blood of the immunized rabbit is used as a primary antibody, the adopted concentration is 1mg/mL, and the dilution ratio is 1: incubating overnight at 1000,4 ℃; commercial HRP-conjugated goat anti-rabbit antibody is used as a secondary antibody, incubated for 1 hour at room temperature, and a chemiluminescent substrate is used for covering the surface of the film for color development, and a Tanon 5200 full-automatic chemiluminescent image analysis system is used for imaging.
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