CN115508489A - Preparation method of tumor cell specific modified polypeptide/protein, antibody and tumor diagnosis and treatment method - Google Patents
Preparation method of tumor cell specific modified polypeptide/protein, antibody and tumor diagnosis and treatment method Download PDFInfo
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- G—PHYSICS
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Abstract
The application discloses a preparation method of a specific modified polypeptide/protein of a tumor cell, an antibody and a tumor diagnosis and treatment method. The target protein or polypeptide which is newly modified is prepared, and the target protein or polypeptide is used for preparing the antibody for diagnosing the tumor and the antibody for treating the tumor. When the library is searched, the modified polypeptide and protein are searched according to the difference of the amino acid sites, the connected molecular sites and the modification groups of the polypeptide/protein, and the biological behavior of the tumor can be explained by utilizing the function of the modified protein. Antibodies can be prepared with the clear structures of the modified polypeptides and proteins. The antibody can be used for tumor diagnosis after being prepared, and the humanized antibody can kill tumor cells due to antigen-antibody reaction, and can also carry cytotoxic drugs to directionally kill tumors.
Description
Technical Field
The application relates to the technical field of biological genetic engineering, in particular to a preparation method of a specific modified polypeptide/protein of a tumor cell, an antibody and a tumor diagnosis and treatment method.
Background
Most tumors are still incurable at present, in that no specific antigens of the tumor are found. The currently discovered tumor markers are of various types and are partially used for clinical diagnosis. However, these antigens are present not only in tumor cells but also in normal cells, so that diagnosis is only of reference value, and chemotherapy and radiotherapy destroy normal cells and tumor cells together because the specific antigens of the tumor cells cannot be accurately positioned.
According to the characteristics of tumors, the theory of tumor pathogenesis is established through 10 years of research. The core of theory is that the onset of tumors is the development of new modifying groups by proteins. The new modified group theory can explain tumorigenesis, metabolic characteristics, metastasis, calcification, immobility, immune evasion and the like.
According to this theory, it is found that only tumor cells have this new modifier group on the infrared spectrum, and normal cells do not have this new modifier group. Further mass spectrometry proves that the modified group exists, and the modified polypeptide and protein are found, so that the tumorigenesis, the metabolic characteristics, the transfer, the tumor occurrence immune evasion and the like can be completely explained according to the functions of the protein.
By finding out the specific modification of tumor cells, the modified polypeptide and protein can be used for producing corresponding antibodies; with the monoclonal antibody, the tumor cell therapy can be directionally killed without affecting normal cells, and the tumor can be cured.
After the specific protein is modified, the abnormal spatial position causes the abnormal protein function, so that the tumor is generated and developed, such as tumor immunity evades the functional protein, the discovered signal-proliferation pathway and other functional protein groups have important values for the future medical application, and corresponding antibodies can be prepared according to the protein or the polypeptide, thereby providing a basis for realizing the behavior of controlling cytology in the future.
Therefore, it is necessary to provide a method for preparing the novel modified gene protein to obtain the modified polypeptide or protein in tumor cells.
Disclosure of Invention
The main objective of the present application is to provide a preparation method of a tumor cell specific modified polypeptide/protein, an antibody and a tumor diagnosis and treatment method, so as to solve the current problems.
In order to achieve the above object, the present application provides the following techniques:
the first aspect of the application provides a preparation method of a tumor cell specific modified polypeptide/protein, which comprises the following steps:
extracting cell membrane protein from tumor tissue;
carrying out enzymolysis on the cell membrane protein by adopting a protein enzymolysis method to obtain target polypeptide;
enriching the target polypeptide by adopting a strong anion exchange chromatography method to obtain the enriched target polypeptide;
carrying out mass spectrum analysis and identification on the enriched target polypeptide to determine whether a target modification group exists or not;
according to the modification type and the modification site, and through PD library checking, the sequence of the modification gene is determined, and the preparation method of the specific modified polypeptide/protein of the tumor cell is obtained.
As an alternative embodiment of the present application, optionally, the extracting cell membrane protein from tumor tissue comprises:
preparing tumor tissue, placing 400-500mg of tumor tissue in a 5ml micro centrifuge tube, adding 4ml of cell cleaning solution into the tissue, whirling briefly and removing the cleaning solution;
transferring to a 2ml tissue mill and cutting the tissue into small pieces with scissors, adding 1ml of permeabilization buffer to the tissue and milling to form a uniform suspension;
adding 1ml of permeabilization buffer solution, transferring the homogenate to a new reaction tube, and incubating for 10 minutes at 4 ℃ under the condition of continuous uniform mixing;
centrifugation at 16000 × g,4 degrees for 15 minutes sedimented permeabilized cells, the supernatant containing the cytoplasmic protein was carefully removed and transferred to a new reaction tube;
resuspending the pellet in 1ml of solubilization buffer, pipetting up and down to obtain a homogeneous resuspension, and incubating at 4 ℃ for 30min with continuous mixing;
centrifugation was carried out at 16000 × g-4 deg.C for 15 minutes, transferring the supernatant containing the soluble membrane protein and the membrane-associated protein to a new protein solution sample tube, thereby obtaining the cell membrane protein.
As an alternative embodiment of the present application, optionally, the extracting cell membrane protein from tumor tissue further comprises:
and (3) measuring the protein content of the cell membrane protein in the supernatant by using a NanoDrop One A280 ultraviolet light.
As an alternative embodiment of the present application, optionally, the protein enzymatic method employs an ultrafiltration-assisted sample preparation method, FASP enzymatic method.
As an alternative embodiment of the present application, optionally, the performing enzymolysis on the cell membrane protein by using a protein enzymolysis method to obtain a target polypeptide includes:
adding dithiothreitol DTT solution into the protein solution sample test tube to obtain a solution with the final concentration of DTT being 100mM, placing the test tube in a boiling water bath for reaction for 5min, and then placing the test tube in a test tube rack to be gradually cooled to room temperature;
adding 200 mu L of UA buffer solution into the upper solution, mixing uniformly, transferring into a 10kD ultrafiltration centrifugal tube, centrifuging at high speed (14000g, 15min), discarding the filtrate, repeating the steps, and taking the supernatant;
adding 100 μ L50 mmol/L IAA buffer, oscillating at 600rpm for 1min, reacting at room temperature in dark place for 30min, centrifuging at high speed (14000g 15min), adding 100 μ L UA buffer again, centrifuging (14000g 15min), and repeating the steps twice;
add 100. Mu.L of 25mM NH4HCO3 solution, centrifuge (14000g 15min), repeat this step twice;
adding 40 mu L of Trypsin buffer, oscillating at 600rpm for 1min, and standing at 37 ℃ for 16-18h. (1;
replacing a new collecting pipe, and centrifuging (14000g 15min); adding 40 mu L of 25mM NH4HCO3, centrifuging 14000g for 15min, and collecting filtrate; wherein the filtrate contains the target protein or polypeptide.
As an alternative embodiment of the present application, optionally, the enriching the target protein or polypeptide by using a strong anion exchange chromatography method to obtain an enriched target protein or polypeptide, includes:
elution buffer a (0.05M Tris-HCl, pH = 8.5), elution buffer B (0.5m nacl,0.05m Tris-HCl, pH = 8.5), 20% ethanol, and 1M NaOH were prepared. The elution buffer, 20% ethanol, 1M NaOH and deionized water were separately filtered through a 0.45 μ M filter to remove a small amount of impurities. Placing the solution at 4 ℃ for later use;
cleaning an AKTA protein rapid purification system, connecting an AKTA, capto HiRes Q5/50 prepacked column, an ultraviolet detector and a collection device, and checking the air tightness;
adjusting the temperature of the refrigerator to 4 ℃, sequentially adding deionized water and an elution buffer solution, respectively balancing until the detector curve does not change, and adjusting the flow rate to 0.2mL/min;
fully dissolving the filtrate collected after enzymolysis by using an elution buffer solution, filtering by using a disposable injection filter of 0.45 mu m to remove insoluble substances, and accessing an AKTA protein rapid purification system for sampling;
after the sample loading is finished, accessing an elution buffer solution A, washing out unadsorbed protein, after the flow-through peak is completely washed out, performing gradient elution by using the elution buffer solution A and an elution buffer solution B, and respectively collecting each elution peak;
polypeptide peaks were collected starting at 3mAU and stored frozen at-80 ℃.
As an alternative embodiment of the present application, optionally, mass spectrometric analysis and identification of the enriched target polypeptide are performed to determine whether the target modification group is present or not, including:
collecting the enriched target polypeptide as an analysis sample;
performing chromatographic separation on each sample by adopting an HPLC liquid phase system Easy nLC with a flow rate of nanoliters;
and (3) carrying out mass spectrometry on the sample by using a Q-active mass spectrometer after chromatographic separation, and realizing the identification of the post-translational modification type and the determination of the modification site.
As an optional embodiment of the present application, optionally, the preparation method for obtaining the tumor cell specific modified polypeptide/protein by determining the sequence of the modified gene according to the modification type and modification site and by PD library survey comprises:
preparing protein identification software: mascot and Proteome discover;
and performing library-searching identification and quantitative analysis on the modification type and the modification site by using protein identification software, determining the type and the modification site of the modification group, and determining the modification molecular weight and the structure of the modification group to obtain the preparation method of the specific modified polypeptide/protein of the tumor cell.
In a second aspect, the present application provides an antibody, wherein the antibody is prepared according to the method for preparing the tumor cell specific modified polypeptide/protein, which is prepared by the method for preparing the tumor cell specific modified polypeptide/protein.
The third aspect of the application provides a tumor diagnosis and treatment method, which utilizes the antibody to be applied to the preparation of an in vitro diagnosis kit; or the antibody is used for killing tumor cells through the antigen-antibody cytotoxicity.
Compared with the prior art, this application can bring following technological effect:
based on the scheme of the embodiment of the application, cell membrane protein is prepared from tumor tissue cells, after proteolysis, target polypeptide is enriched by strong anion exchange chromatography, and the polypeptide is subjected to mass spectrum to determine a modification site, modify a molecular structure and identify the correctness of the modified polypeptide/protein. In the library search, the amino acid sites, the connected molecular sites and the modification groups of the polypeptides/proteins are different, so that the searched modified polypeptides and proteins are not existed before. The modified protein or polypeptide and gene are identified by mass spectrum, the target protein or polypeptide which is newly modified and the gene thereof are subjected to antibody preparation, and the target protein or polypeptide is utilized to prepare an antibody for diagnosing tumor and an antibody for treating tumor. And the modified protein function can be used for explaining the biological behavior of the tumor, preparing a corresponding antibody and controlling the cell biological behavior by using the antibody. The prepared antibody can be used for diagnosis, and the humanized antibody can carry cytotoxic drugs to kill tumor directionally, and can also kill tumor cells due to antigen-antibody reaction. Therefore, the method can be used for discovering and accurately extracting modified proteins by using modified polypeptides or proteins in tumor cells.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, serve to provide a further understanding of the application and to enable other features, objects, and advantages of the application to be more apparent. The drawings and their description illustrate the embodiments of the invention and do not limit it. In the drawings:
FIG. 1 is a schematic diagram of the process for preparing the tumor cell-specific modified polypeptide/protein of the present invention.
Detailed Description
In order to make the technical solutions better understood by those skilled in the art, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only partial embodiments of the present application, but not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort shall fall within the protection scope of the present application.
It should be noted that the terms "first," "second," and the like in the description and claims of this application and in the drawings described above are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order. It should be understood that the data so used may be interchanged under appropriate circumstances in order to facilitate the description of the embodiments of the application herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present application will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
Example 1
In a first aspect, the present application provides a method for preparing a tumor cell specific modified polypeptide/protein, comprising the following steps:
1. extracting cell membrane protein from tumor tissue;
2. carrying out enzymolysis on the cell membrane protein by adopting a protein enzymolysis method to obtain target protein or polypeptide;
the membrane protein is preferably enzymatically hydrolyzed into polypeptide by FASP enzymolysis; the target polypeptide needs to be determined according to a target modification group deduced theoretically;
3. carrying out enrichment treatment on the target polypeptide by using strong anion exchange chromatography to obtain the enriched target polypeptide; enrichment of the polypeptide of interest will allow the modification to be found in the mass spectrum.
After the membrane protein is subjected to enzymolysis to form polypeptide, the identification of post-translational modification types and the determination of modification sites can be realized through enrichment, reversed phase chromatography and mass spectrum. However, the posttranslational modified proteins have low abundance and low stoichiometric number, are easily covered by high-abundance proteins without enrichment, and cannot obtain experimental results. Therefore, the target polypeptide must be enriched to find the modification in the mass spectrum.
In this example, the amino group of the strong anion exchange column was adsorbed to the carboxyl group of the polypeptide by strong anion exchange chromatography, and the polypeptide was eluted from the column with high sodium after washing with a buffer solution to obtain a carboxyl-rich polypeptide.
4. Carrying out mass spectrum analysis and identification on the enriched target protein or polypeptide to clearly determine whether a target modification group exists;
the polypeptide can be further purified by liquid chromatography, separated, and then introduced into a mass spectrometer to determine whether the immobilized molecular weight is absent and whether the target modifying group is present. For proteins, mass spectrometry can determine not only the molecular weight of polypeptides and proteins, but also the amino acid sequence and post-translational modifications of polypeptides. Modifying the protein after translation, namely firstly carrying out enzyme digestion on the protein to obtain a peptide fragment, and then carrying out mass spectrum analysis; and obtaining the relative molecular mass information of a series of peptide fragments through mass spectrometry.
5. According to the modification type and modification site, and through PD library search, the sequence of the modification group is determined, and the preparation method and gene of the specific modified polypeptide/protein of the tumor cell are obtained
The RAW data of mass spectrometry are RAW files, and library checking identification and quantitative analysis are carried out by using protein identification software Mascot2.2 and protein discovery 1.4.
The relevant parameters and specifications are as follows, trypsin digestion, maximum allowed maximum number of missed cleavage sites 2, immobilization of modified Carbammidomethyl (C);
(ii) a variable modification of Oxidation (M), acetyl (Protein N-term); primary ion mass capacity): ± 20ppm, secondary ion mass tolerance: 0.1Da.
Database used for database search: self-selection is carried out;
screening criteria for authentic proteins: high. The database structure is as follows:
for example, performing a library search, the final polypeptide, and matching the corresponding Protein in the library and the corresponding GO analysis in the Protein pathway to obtain the variable modification, oxidation (M), acetyl (Protein N-term): modifying the molecular weight of 72, wherein the amino acid is serine and the hydroxyl of the side chain of threonine is connected with C 3 H 5 O 2 。
As an alternative embodiment of the present application, optionally, the extracting of the cell membrane protein from the tumor tissue comprises:
preparing tumor tissue, placing 400-500mg of tumor tissue in a 5ml micro centrifuge tube, adding 4ml of cell cleaning solution into the tissue, whirling briefly and removing the cleaning solution;
transferring to a 2ml tissue mill and cutting the tissue into small pieces with scissors, adding 1ml of permeabilization buffer to the tissue and milling to form a uniform suspension;
adding 1ml of permeabilization buffer solution, transferring the homogenate to a new reaction tube, and incubating for 10 minutes at 4 ℃ under the condition of continuous uniform mixing;
the permeabilized cells were pelleted by centrifugation at 16000 × g,4 ℃ for 15 minutes, the supernatant containing the cytoplasmic protein was carefully removed and transferred to a new reaction tube;
resuspending the pellet in 1ml of solubilization buffer, pipetting up and down to obtain a homogeneous resuspension, and incubating at 4 ℃ for 30min with continuous mixing;
the cell membrane protein was obtained by centrifugation at 16000 × g-4 deg.C for 15 minutes and transferring the supernatant containing the soluble membrane protein and membrane-associated protein to a new protein solution sample tube.
As an alternative embodiment of the present application, optionally, the extracting cell membrane protein from tumor tissue further comprises:
and (3) measuring the protein content of the cell membrane protein in the supernatant by using a NanoDrop One A280 ultraviolet light.
As an alternative embodiment of the present application, optionally, the protein enzymatic method employs an ultrafiltration-assisted sample preparation method, FASP enzymatic method.
As an alternative embodiment of the present application, optionally, the performing enzymolysis on the cell membrane protein by using a protein enzymolysis method to obtain a target polypeptide includes:
adding dithiothreitol DTT solution into the protein solution sample test tube to obtain a solution with the final concentration of DTT being 100mM, placing the test tube in a boiling water bath for reaction for 5min, and then placing the test tube in a test tube rack to be gradually cooled to room temperature;
adding 200 mu L of UA buffer solution into the upper solution, mixing uniformly, transferring into a 10kD ultrafiltration centrifugal tube, performing high-speed centrifugation (14000g, 15min), discarding the filtrate, repeating the steps, and taking the supernatant;
adding 100 mu L50 mmol/L IAA buffer, oscillating at 600rpm for 1min, reacting in dark at room temperature for 30min, centrifuging at high speed (14000g 15min), adding 100 mu L UA buffer again, centrifuging (14000g 15min), and repeating the steps twice;
add 100. Mu.L of 25mM NH4HCO3 solution, centrifuge (14000g 15min), repeat this step twice;
adding 40 mu L of Trypsin buffer, oscillating at 600rpm for 1min, and standing at 37 ℃ for 16-18h. (1;
replacing a new collecting pipe, and centrifuging (14000g 15min); adding 40 mu L of 25mM NH4HCO3, centrifuging for 14000g for 15min, and collecting filtrate; wherein the filtrate contains the target protein or polypeptide.
As an alternative embodiment of the present application, optionally, the enriching the target protein or polypeptide to obtain an enriched target polypeptide includes:
elution buffer a (0.05M Tris-HCl, pH = 8.5), elution buffer B (0.5m nacl,0.05m Tris-HCl, pH = 8.5), 20% ethanol, and 1M NaOH were prepared. The elution buffer, 20% ethanol, 1M NaOH and deionized water were separately filtered through a 0.45 μm filter to remove a small amount of impurities. Placing the solution at 4 ℃ for later use;
cleaning an AKTA protein rapid purification system, connecting the AKTA, a Capto HiRes Q5/50 prepacked column, an ultraviolet detector and a collection device, and checking the air tightness;
adjusting the temperature of a refrigerator to 4 ℃, sequentially adding deionized water and an elution buffer solution, respectively balancing until the curve of a detector does not change, and adjusting the flow rate to be 0.2mL/min;
fully dissolving the filtrate collected after enzymolysis by using an elution buffer solution, filtering by using a 0.45-micrometer disposable filter for injection to remove insoluble substances, and accessing an AKTA protein rapid purification system for sampling;
after the sample loading is finished, accessing an elution buffer solution A, washing out unadsorbed protein, after a flow-through peak is completely washed out, performing gradient elution by using the elution buffer solution A and an elution buffer solution B, and respectively collecting each elution peak, wherein the elution peak is 3mAU initial collection; and stored frozen at-80 ℃. After the peptide fragment is freeze-dried, 40 mu L of 0.1% formic acid solution is added for redissolving, and the peptide fragment is quantified by adopting an OD280 protein determination method.
Enrichment:
the protein is a 2-type protein, which has different charges at different pH values, and is known to be weakly acidic, and the isoelectric point of the protein is between 3 and 6. The weak acidic protein is obtained by simply purifying the protein, but the basic protein cannot be completely determined to have no amino acid modified, so that the modified polypeptide is lost when the protein is purified and then subjected to enzymolysis analysis. The polypeptide is produced in a solution with the pH of about 8.5 after enzymolysis, the N-terminal amino group on the polypeptide and any histidine in the peptide are not protonated and exist in a neutral form, and Asp, glu and tyrosine residues in the peptide are all anions, lysine is partially protonated, arginine is completely protonated, and the polypeptide rich in Asp, glu and tyrosine residues is easy to retain through ion exchange at the pH. Therefore, strong anion exchange chromatography is carried out after selective membrane proteolysis, and the polypeptide containing-COOH can be enriched. The target polypeptide has modified amino acid side chain, contains-COOH at the terminal, and is anionic in a solution with pH of 8.5. Therefore, strong anion exchange chromatography was chosen.
As an alternative embodiment of the present application, optionally, mass spectrometric analysis and identification of the enriched target polypeptide are performed to determine whether the target modification group is present or not, including:
collecting the enriched target polypeptide as an analysis sample;
performing chromatographic separation on each sample by adopting an HPLC liquid phase system Easy nLC with a flow rate of nanoliters;
and (3) carrying out mass spectrometry on the sample by using a Q-active mass spectrometer after chromatographic separation, and realizing the identification of the post-translational modification type and the determination of the modification site.
For proteins, mass spectrometry can determine not only the molecular weight of polypeptides and proteins, but also polypeptide peptide amino acid sequences and post-translational modifications. Modifying the protein after translation, namely firstly carrying out enzyme digestion on the protein to obtain a peptide fragment, and then carrying out mass spectrum analysis; and obtaining the relative molecular mass information of a series of peptide fragments through mass spectrometry. For a specific peptide fragment, the sequence information and the molecular weight are determined under the condition that no post-translational modification is generated, when the certain post-translational modification is generated, the molecular weight of part of the peptide fragment is found to be just increased or reduced by the molecular weight of a modification group in the mass spectrum detection process, the peptide fragment can be assumed to be newly modified, and the post-translational modification type identification, modification site analysis and the like can be realized by secondary confirmation through a spectrogram of a secondary or multistage mass spectrum.
As an optional embodiment of the present application, optionally, the preparation method for obtaining the tumor cell specific modified polypeptide/protein by determining the sequence of the modification group according to the modification type and modification site and by PD library survey comprises:
preparing protein identification software: mascot and Proteome discover;
and performing library-searching identification and quantitative analysis on the modification type and the modification site by using protein identification software, determining the type and the modification site of the modification group, and determining the modification molecular weight and the structure of the modification group to obtain the preparation method of the specific modified polypeptide/protein of the tumor cell.
Example 2
In a second aspect, the present application provides an antibody, wherein the antibody is prepared according to the method for preparing the tumor cell specific modified polypeptide/protein, which is prepared by the method for preparing the tumor cell specific modified polypeptide/protein.
With the modified polypeptide, the antibody can be prepared by searching the library to obtain the modified protein. The prepared antibody can be used for preparing an in vitro diagnostic kit, and the humanized antibody can carry cytotoxic drugs to kill tumors directionally and possibly kill tumor cells due to antigen-antibody reaction. The manner of antibody production is not limited. For example, a humanized antibody or the like can be obtained by inoculating a preparation method (antigen) of a tumor cell-specific modified polypeptide/protein for preparing a tumor cell.
Because the modified polypeptide/protein is not existed in normal cells, but existed in tumor cells, just like penicillin treatment bacteria, only bacteria have cell walls, and human bodies do not have penicillin only aims at the bacteria, and the influence on the human bodies is very little.
Therefore, the antibody prepared by the new modification of the amino acid is brand new, and the tumor-specific antibody has great possibility of curing the tumor. The modified protein groups are respectively used for preparing antibodies, so that the cell behavior can be controlled, and the possibility is provided for the regeneration of tissues and organs of bionics.
Example 3
In a third aspect, the present application provides a method for diagnosing and treating tumors, wherein the antibody prepared by the method is applied to an in vitro diagnostic kit: comprises preparing reagents by an enzyme-linked immunosorbent assay, a chemiluminescence assay, a colloidal gold assay and the like; or the antibody is used for killing tumor cells through the antigen-antibody cytotoxicity.
Example 4
In addition to the separation and extraction of the modified protein in the cell membrane, the new modification of the protein/polypeptide should be theoretically at the Golgi internal O-junction, but the lactic acid product should not enter the Golgi, so that the modified protein/polypeptide should be present in the plasma of the endoplasmic reticulum, but in a small amount. Antibodies can also be made to these modified polypeptides or proteins.
Thus, this example also provides an isolation method for extracting modified proteins in the cytoplasm.
Preparing tumor tissue, placing 400-500mg of tumor tissue in a 5ml micro centrifuge tube, adding 4ml of cell cleaning solution into the tissue, whirling briefly and removing the cleaning solution;
transferring to a 2ml tissue mill and cutting the tissue into small pieces with scissors, adding 1ml of permeabilization buffer to the tissue and milling to form a uniform suspension;
adding 1ml of permeabilization buffer solution, transferring the homogenate to a new reaction tube, and incubating for 10 minutes at 4 ℃ under the condition of continuous uniform mixing;
the permeabilized cells were pelleted by centrifugation at 16000 × g,4 degrees for 15 minutes and the supernatant containing the cytoplasmic protein was transferred to a new reaction tube. And (4) measuring the protein content by using NanoDrop One A280 ultraviolet light.
Carrying out enzymolysis on the cell cytoplasmic protein by adopting a protein enzymolysis method to obtain target polypeptide;
enriching the target polypeptide by adopting a strong anion exchange chromatography method to obtain the enriched target polypeptide;
carrying out mass spectrum analysis and identification on the enriched target polypeptide to clearly determine whether a target modification group exists;
and determining the sequence of the modified gene according to the modification type and the modification site through PD library searching to obtain the preparation method of the specific modified polypeptide/protein of the tumor cell.
The method does not need to:
resuspend the pellet in 1ml of solubilization buffer, aspirate up and down with a pipettor to obtain a homogenous resuspension, incubate for 30min at 4 ℃ under continuous homogenization;
the cell membrane protein was obtained by centrifugation at 16000 × g-4 deg.C for 15 minutes and transferring the supernatant containing the soluble membrane protein and membrane-associated protein to a new protein solution sample tube.
Comparative examples can be carried out in combination with the procedure of example 1.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims (10)
1. A preparation method of the tumor cell specific modified polypeptide/protein is characterized by comprising the following steps:
extracting cell membrane protein from tumor tissue;
carrying out enzymolysis on the cell membrane protein by adopting a protein enzymolysis method to obtain target polypeptide;
enriching the target polypeptide by adopting a strong anion exchange chromatography method to obtain the enriched target polypeptide;
carrying out mass spectrum analysis and identification on the enriched target polypeptide to clearly determine whether a target modification group exists;
according to the modification type and the modification site, and through PD library checking, the sequence of the modification gene is determined, and the preparation method of the specific modified polypeptide/protein of the tumor cell is obtained.
2. The method for preparing the tumor cell specific modified polypeptide/protein according to claim 1, wherein the extracting of the cell membrane protein from the tumor tissue comprises:
preparing tumor tissue, placing 400-500mg of tumor tissue in a 5ml micro centrifuge tube, adding 4ml of cell cleaning solution into the tissue, whirling briefly and removing the cleaning solution;
transferring to a 2ml tissue mill and cutting the tissue into small pieces with scissors, adding 1ml of permeabilization buffer to the tissue and milling to form a uniform suspension;
adding 1ml of permeabilization buffer solution, transferring the homogenate to a new reaction tube, and incubating for 10 minutes at 4 ℃ under the condition of continuous uniform mixing;
centrifugation at 16000 × g,4 degrees for 15 minutes sedimented permeabilized cells, the supernatant containing the cytoplasmic protein was carefully removed and transferred to a new reaction tube;
resuspending the pellet in 1ml of solubilization buffer, pipetting up and down to obtain a homogeneous resuspension, and incubating at 4 ℃ for 30min with continuous mixing;
the cell membrane protein was obtained by centrifugation at 16000 × g-4 deg.C for 15 minutes and transferring the supernatant containing the soluble membrane protein and membrane-associated protein to a new protein solution sample tube.
3. The method for preparing a tumor cell-specific modified polypeptide/protein according to claim 2, wherein the extracting of the cell membrane protein from the tumor tissue further comprises:
and (3) measuring the supernatant by using a NanoDrop OneA280 ultraviolet light, and determining the protein content of the cell membrane protein in the supernatant.
4. The method for preparing a tumor cell-specific modified polypeptide/protein according to claim 1, wherein the protein enzymolysis method is a FASP enzymolysis method, which is an ultrafiltration-assisted sample preparation method.
5. The method for preparing the tumor cell-specific modified polypeptide/protein according to claim 2, wherein the step of performing enzymolysis on the cell membrane protein by using a protein enzymolysis method to obtain the target protein or polypeptide comprises:
adding dithiothreitol DTT solution into the protein solution sample test tube to obtain a solution with the final concentration of DTT being 100mM, placing the test tube in a boiling water bath for reaction for 5min, and then placing the test tube in a test tube rack to be gradually cooled to room temperature;
adding 200 mu LUAbuffer solution into the upper solution, mixing uniformly, transferring into a 10kD ultrafiltration centrifugal tube, centrifuging at high speed (14000g, 15min), removing the filtrate, repeating the steps, and taking the supernatant;
adding 100 μ L50 mmol/L IAAbuffer, oscillating at 600rpm for 1min, reacting at room temperature in dark place for 30min, centrifuging at high speed (14000g 15min), adding 100 μ L UA buffer again for centrifugation (14000g 15min), and repeating the steps twice;
add 100. Mu.L of 25mM NH4HCO3 solution, centrifuge (14000g 15min), repeat this step twice;
adding 40 mu of LTrypsinbuffer, shaking at 600rpm for 1min, and standing at 37 ℃ for 16-18h. (1;
replacing a new collecting pipe, and centrifuging (14000g 15min); adding 40 mu L of 25mM NH4HCO3, centrifuging 14000g for 15min, and collecting filtrate; wherein the filtrate contains the polypeptide of interest.
6. The method for preparing the tumor cell-specific modified polypeptide/protein according to claim 5, wherein the enriching of the target protein or polypeptide by strong anion exchange chromatography to obtain the enriched target protein/polypeptide comprises:
elution buffer a (0.05M Tris-HCl, pH = 8.5), elution buffer B (0.5m nacl,0.05m Tris-HCl, pH = 8.5), 20% ethanol and 1M NaOH were prepared, and the elution buffer, 20% ethanol, 1M NaOH and deionized water were suction filtered with a 0.45 μ M filter to remove a small amount of impurities, respectively. Placing the solution at 4 ℃ for later use;
cleaning an AKTA protein rapid purification system, connecting the AKTA, a Capto HiRes Q5/50 prepacked column, an ultraviolet detector and a collection device, and checking the air tightness;
adjusting the temperature of the refrigerator to 4 ℃, sequentially adding deionized water and an elution buffer solution, respectively balancing until the detector curve does not change, and adjusting the flow rate to 0.2mL/min;
fully dissolving the filtrate collected after enzymolysis by using an elution buffer solution, filtering by using a 0.45-micrometer disposable filter for injection to remove insoluble substances, and accessing an AKTA protein rapid purification system for sampling;
after the sample loading is finished, accessing an elution buffer solution A, washing out unadsorbed protein, after the flow-through peak is completely washed out, performing gradient elution by using the elution buffer solution A and an elution buffer solution B, and respectively collecting each elution peak; polypeptide peaks were collected starting from 3mAU and stored frozen at-80 ℃.
7. The method for preparing the tumor cell-specific modified polypeptide/protein according to claim 1, wherein mass spectrometry and identification of the enriched target polypeptide are performed to determine whether the target modified group exists, and the method comprises the following steps:
collecting the enriched target polypeptide as an analysis sample;
carrying out chromatographic separation on each sample by adopting an HPLC liquid phase system EasynLC with a flow rate of nanoliters;
and (3) carrying out mass spectrometry on the sample by using a Q-active mass spectrometer after chromatographic separation, and realizing the identification of the post-translational modification type and the determination of the modification site.
8. The method according to claim 1, wherein the method for preparing the tumor cell-specific modified polypeptide/protein comprises the following steps:
preparing protein identification software: mascot and Proteome discover;
and performing library search identification and quantitative analysis on the modification type and the modification site by using protein identification software, determining the type and the modification site of the modification group, determining the modification molecular weight and the structure of the modification group, and obtaining the preparation method of the specific modified polypeptide/protein of the tumor cell.
9. An antibody produced by the method for producing a tumor cell-specific modified polypeptide/protein according to any one of claims 1 to 8.
10. The tumor diagnosis and treatment method is characterized in that the antibody is applied to the preparation of an in vitro diagnosis kit; or the antibody is used for killing tumor cells through the antigen-antibody cytotoxicity.
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