CN117534753B - Polypeptide for inducing high-performance NF- κB polyclonal antibody and application thereof - Google Patents
Polypeptide for inducing high-performance NF- κB polyclonal antibody and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- Chemical & Material Sciences (AREA)
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- Health & Medical Sciences (AREA)
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- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention belongs to the technical field of antibody preparation, and in particular relates to an induced high-performance NF- κB polyclonal antibody polypeptide and application thereof, wherein the preparation method of the NF- κB polyclonal antibody induced polypeptide comprises the following steps: step 1, preparing experimental materials; step 2, preparing an experimental instrument; activation of Fmoc-AA; step 4, activating amino acid connection resin; step 5, removing Fmoc protecting groups of the resin connected with the amino acid; step 6, activating amino acid; step 7, activating carboxyl component coupling; step 8, preparing peptide resin; step 9, separating the polypeptide; step 10, separating and purifying the polypeptide and analyzing. The invention reduces the cost, the short peptide is composed of several amino acids, and can be directly synthesized by chemical method, if the whole or partial protein is synthesized by synthesis method, the invention has the advantages of long length, high cost when expressed by gene recombination method, accelerating the preparation speed, only needing short time for polypeptide synthesis, and improving the antibody quality due to shortening the length of amino acid chain.
Description
Technical Field
The invention belongs to the technical field of antibody preparation, and particularly relates to an induced high-performance NF- κB polyclonal antibody polypeptide and application thereof.
Background
Nuclear factor kappa B (nuclear factor kappa-B, NF-kappa B) protein is a protein expressed in cells, and is originally discovered by David Baltimore, so named because the protein family can selectively bind to B cell kappa-light chain enhancers to regulate the expression of a plurality of genes, and NF-kappa B can be discovered in almost all animal cells and is involved in the response of the cells to external stimuli, such as cytokines, radiation, heavy metals, viruses and the like, and the NF-kappa B plays a key role in the inflammatory response, the immune response and the like of the cells, and the error regulation of the NF-kappa B can cause autoimmune diseases, chronic inflammation and various cancers. NF-. Kappa.B is also associated with synaptic plasticity and memory.
At present, tumor targeted therapy aiming at NF- κB is one of hot spots for cancer therapy, and the main idea is to inhibit the activity of NF- κB; inhibiting phosphorylation of IκBα protein, avoiding degradation by ubiquitination; the NF- κB protein is the focus of research and anti-tumor medicine development, and the development of high quality anti-NF- κB antibody is the production and production requirement, so that it has great market value.
Disclosure of Invention
The invention aims to provide an induced high-performance NF- κB polyclonal antibody polypeptide and application thereof, which can reduce cost, wherein the short peptide is composed of a plurality of amino acids, and can be directly synthesized by a chemical method, and if a complete or partial protein is synthesized by a synthesis method, the whole or partial protein is expressed by a gene recombination method with high cost due to long length and high cost, thereby accelerating the preparation speed, and the polypeptide synthesis only needs a short time, and the antibody quality is improved due to the shortened length of an amino acid chain.
The technical scheme adopted by the invention is as follows:
a polypeptide for inducing high-performance NF- κb polyclonal antibody and application thereof, the amino acid sequence: cysteine-lysine-serine-isoleucine-glycine-histidine-lysine-proline-glutamic acid-proline-threonine-aspartic acid, beginning with a thiol-containing amino acid, constructed with an acidic amino acid at position 9, position 12, the amino acid sequence: aspartic acid-glutamic acid-leucine-phenylalanine-proline-leucine-isoleucine-phenylalanine-alanine-glutamic acid-proline, starting with two acidic amino acids, the penultimate position being filled with an acidic amino acid, the amino acid sequence: arginine-isoleucine-glutamic acid-threonine-asparagine-proline-phenylalanine-glutamine-valine-proline, with asparagine being used consecutively at positions 5-7.
The preparation method of the NF- κB polyclonal antibody polypeptide comprises the following steps:
step 1, preparing experimental materials;
Step 2, preparing an experimental instrument;
activation of Fmoc-AA;
Step 4, activating amino acid connection resin;
Step 5, removing Fmoc protecting groups of the resin connected with the amino acid;
Step 6, activating amino acid;
step 7, activating carboxyl component coupling;
Step 8, preparing peptide resin;
Step 9, separating the polypeptide;
step 10, separating and purifying the polypeptide and analyzing.
In a preferred embodiment, the assay material preparation comprises preparing P-hydroxymethylphenoxymethyl polyethylene resin, wang resin, fmoc-AA, NMP azamethylpyrrolidone, DCM dichloromethane, meoH methanol, PIPERIDINE piperidine, DMAP dimethylaminopyridine, HOBT hydroxybenzotriazole, DCCN, N' -dicyclohexylcarbodiimide, TFA trifluoroacetic acid, EDT1, 2-ethanedithiol, thioanisole, crystalline phenol, acetonitrile, and the assay device preparation comprises a polypeptide autosynthesizer, rotary evaporator, high performance liquid chromatograph, freeze dryer, vacuum circulating water pump, centrifuge.
In a preferred embodiment, the Fmoc-AA is activated by reaction with DCC and HOBT.
In a preferred embodiment, the activated amino acid-linked resin is obtained by reacting the activated amino acid with a P-hydroxymethylphenoxymethyl polyethylene resin under DMAP conditions.
In a preferred embodiment, the Fmoc protecting group of the amino acid-linked resin is removed by the action of PIPERIDINE piperidine.
In a preferred embodiment, the amino acid is activated by reacting the amino acid with DCC and HOBT.
In a preferred embodiment, the activated carboxylic component is coupled by coupling the resin from step 4 with an excess of activated carboxylic component and removing the amino protecting group of the activated amino component after coupling.
In a preferred embodiment, the peptide resin is prepared by repeating steps 3 to 7 above, 21 times, and each coupling of the activated carboxylic components from left to right in sequence: IEQPKQRGMRFR to obtain peptide resin, similar to the preparation method of NF-KB polypeptide fragment, the polypeptide fragment of comparative example repeats the above steps 15 times, and the coupling activated carboxyl component is as follows in sequence from left to right: cysteine-lysine-serine-isoleucine-glycine-histidine-lysine-proline-glutamic acid-proline-threonine-aspartic acid.
In a preferred embodiment, the separation of the polypeptide is by reacting trifluoroacetic acid in combination with a scavenger mixed with 1, 2-ethanedithiol, thioanisole, and water with the peptide resin to separate the NF-kb or comparative polypeptide fragment from the peptide resin; filtering out resin after reaction, and removing the scavenger by reduced pressure distillation; dissolving the NF-KB or the comparative example polypeptide fragment with water, and extracting with diethyl ether to obtain a crude NF-KB or comparative example polypeptide compound.
In a preferred embodiment, the separation and purification and analysis of the polypeptide are performed by using high performance liquid chromatography to separate and purify the NF-kb or comparative polypeptide compound crude product, specifically, the operation conditions are as follows: chromatographic column: c1825×250mm, chromatograph: waters, mobile phase: a-0.1% TFAH2O, B-0.1% TFA in 60% acetonitrile, detection wavelength: 214nm, flow rate: 10ml/min, eluting gradient from 20-60% B to obtain polypeptide with purity >90%, and meeting immune requirement; further, the separated and purified NF-KB polypeptide is subjected to high performance liquid chromatography under the following analysis conditions: chromatographic column: c184.6x150 mm, mobile phase: a-0.1% TFA, H2O, B-0.1% TFA, acetonitrile, detection wavelength: 214nm, flow rate: elution gradient from 0-60% B was 1 ml/min.
The invention has the technical effects that:
The cost is reduced, the short peptide is composed of a plurality of amino acids, and can be directly synthesized by a chemical method, if the whole or partial protein is synthesized by a synthesis method, the preparation speed is increased by using a gene recombination method due to the fact that the length is long, the cost is high, the preparation speed is increased, the polypeptide synthesis only needs a short time, and the antibody quality is improved due to the fact that the length of an amino acid chain is shortened;
through the accurate operation of a plurality of steps and the strict preparation of experimental materials, the reliability and the accuracy of experiments are ensured, and the synthesis and preparation methods of the polypeptides are optimized, so that the required polypeptides can be efficiently obtained;
Through the steps of high performance liquid chromatography separation and purification, polypeptide with purity higher than 90% can be obtained, the requirements of immune experiments are met, and the sensitivity and the specificity of the antibody are proved to be higher through detection methods such as ELISA, western blot and the like;
The method can be used for preparing antibodies with various peptide fragments, has wider application fields, and ensures the reliability and accuracy of experimental results through the detection and verification of various experimental methods.
Drawings
FIG. 1 is a schematic illustration of a preparation process for inducing high-performance NF- κB polyclonal antibody polypeptide and application thereof according to the present invention;
FIG. 2 is a schematic representation of activation of Fmoc-AA by reaction with DCC and HOBT for inducing high performance NF- κB polyclonal antibody polypeptide and its use according to the present invention;
FIG. 3 is a schematic representation of an activated amino acid ligation resin for inducing high performance NF- κB polyclonal antibody polypeptides and uses thereof according to the present invention;
FIG. 4 is a schematic representation of the reaction scheme for inducing high performance NF- κB polyclonal antibody polypeptide and Fmoc protecting groups of amino acid-linked resin used in the same;
FIG. 5 is a schematic representation of a reaction for inducing coupling of activated carboxyl components of a high performance NF- κB polyclonal antibody polypeptide and its use in accordance with the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
Example 1
Referring to FIGS. 1-5, the invention provides a polypeptide for inducing high-performance NF- κB polyclonal antibody and application thereof, and the amino acid sequence: cysteine-lysine-serine-isoleucine-glycine-histidine-lysine-proline-glutamic acid-proline-threonine-aspartic acid, beginning with a sulfhydryl-containing amino acid, constructed with an acidic amino acid at position 9, position 12, amino acid sequence: aspartic acid-glutamic acid-leucine-phenylalanine-proline-leucine-isoleucine-phenylalanine-alanine-glutamic acid-proline, beginning with two acidic amino acids, filling the penultimate position with an acidic amino acid, amino acid sequence: arginine-isoleucine-glutamic acid-threonine-asparagine-proline-phenylalanine-glutamine-valine-proline, asparagine being used consecutively at positions 5-7.
The preparation method of the NF- κB polyclonal antibody polypeptide comprises the following steps:
step 1, preparing experimental materials;
Step 2, preparing an experimental instrument;
activation of Fmoc-AA;
Step 4, activating amino acid connection resin;
Step 5, removing Fmoc protecting groups of the resin connected with the amino acid;
Step 6, activating amino acid;
step 7, activating carboxyl component coupling;
Step 8, preparing peptide resin;
Step 9, separating the polypeptide;
step 10, separating and purifying the polypeptide and analyzing;
the preparation of experimental materials comprises the preparation of P-methylol phenoxymethyl polyethylene resin, wang resin, fmoc-AA, NMP nitrogen methyl pyrrolidone, DCM dichloromethane, meoH methanol, PIPERIDINE piperidine, DMAP dimethyl amino pyridine, HOBT hydroxybenzotriazole, DCCN, N' -dicyclohexylcarbodiimide, TFA trifluoroacetic acid, EDT1, 2-ethanedithiol, thioanisole, crystalline phenol and acetonitrile, and the preparation of experimental instruments comprises a polypeptide automatic synthesizer, a rotary evaporator, a high performance liquid chromatograph, a freeze dryer, a vacuum circulating water pump and a centrifuge;
the Fmoc-AA is activated by reacting with DCC and HOBT, the activated amino acid connecting resin is obtained by reacting activated amino acid with P-hydroxymethyl phenoxymethyl polyethylene resin under DMAP condition, the Fmoc protecting group of the resin connected with amino acid is removed by removing Fmoc protecting group of the resin connected with amino acid under PIPERIDINE piperidine effect, the amino acid is activated by reacting with DCC and HOBT, the activated carboxyl component is coupled to couple the resin obtained in step 4 with excessive activated carboxyl component, and the amino protecting group of the activated amino component after decoupling is removed, the peptide resin is prepared by repeating the steps 3 to 7 for 21 times, and the activated carboxyl component after each coupling is sequentially from left to right: IEQPKQRGMRFR to obtain peptide resin, similar to the preparation method of NF-KB polypeptide fragment, the polypeptide fragment of comparative example repeats the above steps 15 times, and the coupling activated carboxyl component is as follows in sequence from left to right: cysteine-lysine-serine-isoleucine-glycine-histidine-lysine-proline-glutamic acid-proline-threonine-aspartic acid, the separation of the polypeptide by reacting trifluoroacetic acid in combination with a scavenger mixed with 1, 2-ethanedithiol, thioanisole, and water with the peptide resin to separate NF- ≡b or the comparative polypeptide fragment from the peptide resin; filtering out resin after reaction, and distilling out scavenger by decompression; dissolving NF-KB or comparative example polypeptide fragments with water, and extracting with diethyl ether to obtain crude NF-KB or comparative example polypeptide compounds;
The separation and purification of the polypeptide and the analysis are to adopt high performance liquid chromatography to separate and purify crude NF-KB or comparative example polypeptide compounds, and the operation conditions are as follows: chromatographic column: c1825×250mm, chromatograph: waters, mobile phase: a-0.1% TFAH2O, B-0.1% TFA in 60% acetonitrile, detection wavelength: 214nm, flow rate: 10ml/min, eluting gradient from 20-60% B to obtain polypeptide with purity >90%, and meeting immune requirement; further, the separated and purified NF-KB polypeptide is subjected to high performance liquid chromatography under the following analysis conditions: chromatographic column: c184.6x150 mm, mobile phase: a-0.1% TFA, H2O, B-0.1% TFA, acetonitrile, detection wavelength: 214nm, flow rate: elution gradient from 0-60% B was 1 ml/min.
Example two
Experimental materials
NF-KB polypeptides and comparative polypeptides prepared according to example 1;
new Zealand white rabbits were used for the experiment.
Experimental procedure
Because polypeptides are not fully immunogenic, coupling carrier proteins is required to prepare a complete immunogen. Thus, the NF-KB polypeptide is first coupled to the carrier protein KLH (hemocyanin or keyhole limpet hemocyanin, keyhole limpet hemocyanin) with the coupling agent being 3-maleimidobenzoic acid succinimidyl ester (MBS, CAS: 58626-38-3).
After basic immunization (complete adjuvant+polypeptide antigen is fully emulsified), the experimental New Zealand white rabbits are subjected to multiple boosting (incomplete adjuvant+polypeptide antigen is fully emulsified) on the back and foot pads for multiple times in weeks 3, 5 and 7 respectively. The 7 th day after immunization, the ear margin vein was bled, serum was isolated, and the antibody titer of NF-KB was identified by ELISA. When the concentration of NF-KB antibody in serum reaches the peak, the white rabbit with the best detection data is taken, the carotid artery is taken for blood collection, and the serum is separated. The anti-NF-KB rabbit polyclonal antibody is obtained through Protein A/G affinity purification.
Similar to NF-KB polypeptide coupling and immunization methods, anti-NF-KB rabbit polyclonal antibodies were prepared from the comparative polypeptides.
The sensitivity, i.e. the titer, of the antibodies was detected by ELISA methods.
The specificity of the antibodies was detected by Western blot.
The expression site of this antibody in the cells was detected by immunohistochemistry.
The recognition specificity of the antibodies to NF-KB positive and negative expressing living cells is detected by a flow cytometry method respectively.
3. Experimental results
Sensitivity of detection of antibodies by ELISA method: the detection result shows that the antibody titer of NF- κB reaches 1:128000, and the sensitivity of the antibody is very high. And comparing the anti-NF- κB rabbit polyclonal antibody obtained after the polypeptide fragment immunization, wherein the titer is lower than 1:16000, and no qualified antibody is obtained.
The specificity of the NF- κB immune antibody is detected by a Western blot method: as reported in the literature, the NF-KB protein has a molecular weight of about 65kDa. The WB results of the antibody multi-sample (14 samples are respectively from different species of human, rat and mouse) prepared by the invention show that the detection band is positioned at 65kDa, which is completely the same as the reported actual molecular weight, and the impurity band is less, thus proving that the anti-NF- κB rabbit polyclonal antibody prepared by the embodiment of the invention can identify human, rat and mouse, NF- κB and has higher specificity.
And detecting whether the target tissue identification positioning of the anti-NF- κB rabbit polyclonal antibody is accurate or not by an IHC method. The operation flow is as follows: dewaxing paraffin sections of different tissues to water, thermally repairing by 0.01M citric acid repair liquid (pH 6.0) and blocking endogenous peroxide by 3% hydrogen peroxide, blocking by normal goat serum, incubating by diluted primary antibody (Anti-NF-K B), overnight at 4 ℃, sequentially adding biotin-labeled goat Anti-rabbit IgG and HRP-labeled streptavidin, DAB color development, and sealing by neutral resin.
Expression position: NF- κB has low tissue specificity and is mainly localized to the nuclear region of secretory nerve cells, oligodendrocytes, basophils, some cancer cells, etc. The detection result shows that the expression and the positioning are accurate in liver tissues of rats and mice. The antibody according to the embodiment of the invention has accurate expression and positioning in liver tissue cells of rats and mice and higher specificity.
The specificity of the antibodies was detected by flow cytometry through NF- κb expressing positive and negative cell lines. The main operation flow is as follows: culturing an NF-kb expression positive cell line: k562, siha, a431, NF-kb expressing negative cell line: hl-60, molt-4, thp-1. Cells were fixed with 4% PFA and 90% ice methanol permeabilized for 20 minutes. Blocking with 5% BSA was performed for 30 min. The anti-NF-KB rabbit polyclonal antibody is incubated for 30 minutes at room temperature, and the AF 488-labeled goat anti-rabbit secondary antibody is incubated for 40 minutes at room temperature. The flow cytometer collected 20000 event detections.
The results show that the anti-NF- κB rabbit polyclonal antibody prepared by the method can be detected in NF- κB expression positive cells, but not in NF-KB expression negative cells (HL-60 cells), and the anti-NF- κB rabbit polyclonal antibody prepared by the method has higher streaming application specificity and can be used for identifying and screening NF- κB expression positive cells. Lays a foundation for further researching the functions of NF- κB expression positive cells.
Compared with other immune stimulation means, the invention can effectively stimulate the generation of NF-kappa B polyclonal antibody, reduce the cost, the short peptide is composed of a plurality of amino acids, can be directly synthesized by a chemical method, and can meet the requirements of immune experiments if a complete or partial protein is synthesized by a synthesis method, the method is also characterized by time and labor consuming, complex approach and high cost due to long length and high cost, the gene recombination method is used for expressing the polypeptide, the preparation speed is accelerated, the polypeptide synthesis is only required for a short time, compared with other methods, the method is obviously faster, the method is prepared by a plurality of steps of accurate operations and strict experimental materials, the reliability and the accuracy of experiments are ensured, the polypeptide synthesis and the preparation method are optimized, the polypeptide with the purity higher than 90% can be obtained by the separation and purification steps of high performance liquid chromatography, the requirements of immune experiments are met, the sensitivity and the specificity of the antibody are proved to be higher by ELISA and Western blot and other detection methods, the method can be used for preparing antibodies with different peptide segments, and the application fields are wider, and the reliability and the accuracy of the results are ensured by the detection and verification of a plurality of methods.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention. Structures, devices and methods of operation not specifically described and illustrated herein, unless otherwise indicated and limited, are implemented according to conventional means in the art.
Claims (1)
1. A polypeptide for inducing high performance NF- κb polyclonal antibody, characterized in that: the amino acid sequence: cysteine-lysine-serine-isoleucine-glycine-histidine-lysine-proline-glutamic acid-proline-threonine-aspartic acid.
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