Preparation method and application of 249th and 322th tyrosine site phosphorylation antibody of glucose-6-phosphate dehydrogenase

Abstract

The invention belongs to the technical field of biomedicine, and discloses preparation and application of tyrosine site phosphorylation antibodies at the 249th site and the 322nd site of glucose-6-phosphate dehydrogenase, and the effect of the antibodies is verified through point mutation; mutation plasmids of all the sites of Y249 and Y322 are constructed; carrying out plasmid transfection overexpression in breast cancer cells; carrying out co-immunoprecipitation on 2YF or 2YA by using M2 beads; and finally, Western blotting is used for carrying out western blot analysis. Two tyrosine sites of glucose-6-phosphate dehydrogenase are mutated into phenylalanine (2YF) or alanine (2YA), and the phosphorylation level of 2YF and 2YA is found to be obviously reduced or disappeared through detection of a specific phosphorylation antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a method for preparing an antibody against tyrosine phosphorylation at positions 249 and 322 of glucose-6-phosphate dehydrogenase and its application. Background Art

[0002] Glucose-6-phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase) is the first rate-limiting enzyme in the pentose phosphate pathway. The activity of this protein is related to the overall flow of the pentose phosphate pathway and the abundance of intermediate metabolites such as NADPH and deoxyribonucleotides (ribonucleotides). The phosphorylation site of glucose-6-phosphate dehydrogenase discovered by the present invention has not been reported so far, and it will affect the catalytic activity of glucose-6-phosphate dehydrogenase.

[0003] Technical problems existing in existing technologies:

[0004] In the prior art, there are several technical difficulties in the preparation of specific antibodies against the 249th and 322nd tyrosine phosphorylation sites of glucose-6-phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase), mainly including the following aspects:

[0005] 1. Lack of specificity: Antibodies prepared by existing methods often have difficulty in achieving high-specificity recognition of specific tyrosine phosphorylation sites of glucose-6-phosphate dehydrogenase, and are prone to cross-reactions with other phosphorylation sites or non-phosphorylation sites, resulting in inaccurate test results and limiting their application in precision research.

[0006] 2. Low antibody affinity: Many existing preparation methods have low antibody affinity, resulting in low binding efficiency between antibodies and target phosphorylation sites in practical applications, affecting the sensitivity and reliability of detection, especially in the detection of low-abundance proteins.

[0007] 3. Complex purification process: Traditional antibody purification steps are cumbersome and time-consuming, and the purification effect is unstable. It is difficult to effectively remove non-specific binders and other impurity proteins, which affects the purity and specificity of the final antibody and increases uncertainty in subsequent applications.

[0008] 4. Low antibody yield: Existing methods often face the problem of low antibody yield during large-scale preparation, which limits their promotion in high-throughput screening and large-scale applications, and cannot meet the rapid growth of scientific research and clinical needs.

[0009] 5. Immunogenicity issues: The carrier proteins or coupling chemical reagents used in some preparation methods may trigger nonspecific reactions of the rabbit's immune system, resulting in low immune efficiency, affecting the production of antibodies and prolonging the antibody preparation cycle. Summary of the invention

[0010] In view of the problems existing in the prior art, the present invention provides a preparation and application of an antibody against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase.

[0011] The present invention is achieved by: a method for preparing an antibody against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase comprising:

[0012] Step 1, peptide coupling;

[0013] Step 2, rabbit immunization;

[0014] Step 3, enzyme-linked immunosorbent assay detection;

[0015] Step 4, antibody purification.

[0016] Further, the polypeptide is coupled to:

[0017] Peptide-coupled KLH:

[0018] Dissolve 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution;

[0019] Weigh 8 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO, then add 150 μL 1× PBS and mix well;

[0020] Add the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester dropwise into KLH, gently shake while adding (vigorous shaking will produce precipitation), and leave at room temperature for 1 hour;

[0021] The activated KLH solution was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed in 2L of 1×PBS at 4°C under magnetic stirring for 1 h.

[0022] Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed KLH in a 15mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4℃ refrigerator;

[0023] Weigh 4 mg of peptide, dissolve in 50 μL of DMSO, add 200 μL of 1× PBS, mix quickly, then immediately add KLH at a ratio of peptide: KLH = 1 mg: 680 μg, and react overnight at 4°C refrigerator or at room temperature for 2 hours;

[0024] The cross-linked KLH-peptide complex was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed overnight in 4 L of 1×PBS under magnetic stirring in a 4°C refrigerator;

[0025] Take out the dialyzed KLH-peptide into a clean 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator;

[0026] Peptide-coupled carrier protein BSA:

[0027] Dissolve 20 mg of BSA in 2 mL of 5 mM EDTA aqueous solution;

[0028] Weigh 5 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO, then add 150 μL 1× PBS and mix well;

[0029] Add 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt solution dropwise into the BSA solution, gently shake while adding, and leave at room temperature for 1 hour;

[0030] The activated BSA solution was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed in 2L of 1×PBS at 4°C under magnetic stirring for 1 h.

[0031] Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed BSA in a 15mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4℃ refrigerator;

[0032] Weigh 1 mg of peptide, dissolve it in 50 μL of DMSO, add 150 μL of 1× PBS, mix quickly, then add 100 μL of the activated BSA solution, and react in a 4°C refrigerator overnight or at room temperature for 2 hours;

[0033] Place the cross-linked BSA-Peptide complex in a 1.5 mL centrifuge tube, add 1×PBS to 1 mL, and mark the project number, concentration, and date.

[0034] Further, the rabbit is immunized with:

[0035] Animal selection: Rabbits are New Zealand white rabbits, weighing about 2.5 kg, young and strong; animals should be selected with shiny fur and healthy animals that can move freely; select the animals and raise them for about 2 weeks in advance; the purpose is to eliminate some unqualified animals so that the later experiments can proceed smoothly;

[0036] Preparation before the experiment: Rabbits were marked;

[0037] Antigen preparation:

[0038] Take the antigen out of the -20 degree freezer and dissolve it at room temperature to avoid repeated freezing and thawing; mark the syringe with the project number and animal number;

[0039] Extract antigen (mix antigen completely), the concentration of antigen for the first immunization is 1 mg / mL, and 0.5 mL / rabbit. The concentration of antigen for the second to fourth immunization is halved, and the dosage remains unchanged;

[0040] When extracting adjuvant, the volume ratio of adjuvant to antigen is 1:1. Complete adjuvant is used for the first immunization, and incomplete adjuvant is used for the second to fourth immunizations. When extracting, the adjuvant should be fully mixed before being drawn into the syringe.

[0041] The two syringes are connected with a syringe connecting tube for complete emulsification. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37-degree water;

[0042] immunity.

[0043] Furthermore, the immunization: rabbits were injected subcutaneously at multiple points, with 0.2 mL at each point;

[0044] Immunization time: The second vaccination will be carried out on the 14th day after the first vaccination, and the interval between the second and third vaccinations is 7 days; a small sample of serum will be collected from the middle ear artery of the rabbit on the 7th day after the third vaccination for testing. If the test is qualified, an additional vaccination will be carried out 7 days later. Whole blood can be collected 7 days after the additional vaccination.

[0045] Further, the ELISA assay detected:

[0046] Coating: Dilute the known antigen to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, incubate at 4°C overnight, and the next day, discard the solution in the wells and wash once with 1xTBST washing buffer (180 μL per well);

[0047] Blocking: Add 60 μL of 1% BSA (prepared in TBST) to each well for blocking, incubate at 37°C for 1 hour, then discard the blocking solution;

[0048] Sample addition: Add a certain dilution of the sample to be tested (dilute the sample to be tested according to a certain ratio), 50 μL to the above blocked reaction wells; set up the positive control well (positive serum) and the negative control well (BSA); incubate at 37°C for 1 hour, then discard the blocking solution and wash twice with 1xTBST washing buffer, 180 μL per well;

[0049] Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the ELISA plate at 50 μL / well, incubate at 37°C for 45 min, then discard the blocking solution and wash three times with 1xTBST washing buffer at 180 μL per well;

[0050] Add substrate solution for color development: Add 100 μL of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 min;

[0051] Stop the reaction: add 90 μL of 2M sulfuric acid to each reaction well;

[0052] Read the plate: Place the ELISA plate in a preheated ELISA reader (450nm) for reading, save the data, and analyze.

[0053] Further, the antibody is purified:

[0054] The affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1× PBS (pH 7.4) at a flow rate of 70 mL / h;

[0055] Take 10 mL of serum to be purified in a 50 mL centrifuge tube and filter it with a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm;

[0056] The filtered serum sample was loaded at a flow rate of 40 mL / h and repeated once;

[0057] The column was washed with 20 mL 1× PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, the column was connected to the protein detector. During the washing process, the light transmittance (T position) of the instrument was adjusted to 100.

[0058] Adjust the protein detector absorbance (1A gear) to 0, then turn on the HD-A computer collector on the computer desktop and adjust the full screen range to 5, use glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h to elute the antibody, then press the green elution record button to start elution, and start collecting the antibody when the instrument reading starts to rise;

[0059] During the antibody collection process, the pH value of the antibody was promptly adjusted to about 7 with 1 M sodium bicarbonate, and the highest peak of the elution peak was recorded;

[0060] After collecting the antibodies, adjust the pH to about 7 and record the volume of eluted antibodies, then rinse the rubber tube connected to the collector with pure water;

[0061] The affinity chromatography column was washed with 20 mL 1× PBS and pure water at a rate of 70 mL / h, and then 20% ethanol was added, sealed, and stored in a refrigerator at 4°C;

[0062] The purified antibodies are sent for testing according to different requirements: ELISA, WB, ICC.

[0063] Another object of the present invention is to provide a system for preparing antibodies against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase, comprising:

[0064] A coupling module, used for peptide coupling;

[0065] Immunization module, used for rabbit immunization;

[0066] Detection module, used for enzyme-linked immunosorbent assay detection;

[0067] Purification module, used for antibody purification.

[0068] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for preparing antibodies phosphorylated at the 249th and 322nd tyrosine sites of glucose-6-phosphate dehydrogenase.

[0069] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for preparing antibodies phosphorylated at the 249th and 322nd tyrosine sites of glucose-6-phosphate dehydrogenase.

[0070] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for preparing antibodies against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase.

[0071] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0072] First, this scheme effectively solves the above technical problems by optimizing key steps such as peptide coupling, immunization, ELISA detection and antibody purification.

[0073] The peptide coupling step uses highly efficient chemical coupling agents and optimized coupling conditions to ensure the stable binding of the target phosphorylation site peptide to the carrier protein, significantly improving the immunogenicity of the antigen and the specificity of the antibody.

[0074] The rabbit immunization step enhances the rabbit's immune response and improves the antibody titer and affinity by rationally designing the immunization program, including multiple immunizations and the use of appropriate immune adjuvants.

[0075] The ELISA test step introduces strict screening standards and optimized detection methods to ensure the selection of high-specificity and high-titer serum samples, providing a reliable basis for subsequent antibody purification.

[0076] The antibody purification step uses advanced purification technologies, such as protein A / G affinity chromatography, combined with a multi-step purification process to effectively remove non-specific binders and impurity proteins to obtain high-purity and high-specificity target antibodies.

[0077] Through the above improvements, this scheme can prepare tyrosine phosphorylation antibodies at positions 249 and 322 of glucose-6-phosphate dehydrogenase with high specificity, high affinity and high purity, overcoming many shortcomings in the existing technology and significantly improving the application value and reliability of antibodies in scientific research and clinical testing.

[0078] The invention verifies the effect of the antibody by point mutation; constructs a plasmid with mutations at all sites of Y249 and Y322; performs plasmid transfection and overexpression in breast cancer cells; uses M2 beads to immunoprecipitate 2YF or 2YA; and finally performs protein blotting analysis by Western blotting. The two tyrosine sites of glucose-6-phosphate dehydrogenase are all mutated into phenylalanine (2YF) or alanine (2YA), and the phosphorylation levels of 2YF and 2YA show a trend of obvious decrease or disappearance by detection with a specific phosphorylation antibody.

[0079] Second, glucose-6-phosphate dehydrogenase phosphorylation antibodies are widely used in many fields, such as enzyme-linked immunosorbent assay, Western Blot analysis, immunocytochemistry analysis, etc. The growth of market demand in these application fields will directly drive the increase in sales revenue of glucose-6-phosphate dehydrogenase phosphorylation antibodies. The phosphorylation-specific antibody market is divided into rabbits, mice, goats, sheep and donkeys according to host species. Among them, rabbit protein occupies the largest market share due to its high specificity and affinity for the target protein.

[0080] 1. High specificity and affinity: Glucose-6-phosphate dehydrogenase phosphorylation antibodies have high specificity and affinity and can accurately identify phosphorylation sites, thus having important application value in research, diagnosis and drug development.

[0081] 2. Innovation: The phosphorylated antibody of the present invention has unique technical advantages in specific application fields (such as detecting the tyrosine phosphorylation levels of glucose-6-phosphate dehydrogenase protein at positions 249 and 322 in tumor cells), and can reflect the physiological function of lactic acid and the phosphorylation level of glucose-6-phosphate dehydrogenase in tumor cells, thereby reflecting the catalytic activity of glucose-6-phosphate dehydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 It is a flow chart of the method for preparing antibodies phosphorylated at tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase provided in an embodiment of the present invention.

[0083] Figure 2 It is a structural block diagram of a system for preparing antibodies against tyrosine phosphorylation at positions 249 and 322 of glucose-6-phosphate dehydrogenase provided in an embodiment of the present invention.

[0084] Figure 3It is a protein sequence analysis diagram provided by an embodiment of the present invention.

[0085] Figure 4 The present invention provides a method for forming peptide circular spots with the above concentrations on a nylon membrane, incubating with a specific phosphorylated antibody, and performing Western blot analysis.

[0086] Figure 5 This is a diagram of western blot analysis of wild-type (WT) and mutant (2YF / 2YA) glucose-6-phosphate dehydrogenase proteins provided in the embodiments of the present invention using specific phosphorylation antibodies.

[0087] Figure 6 This is a location information map provided by an embodiment of the present invention.

[0088] Figure 7 A provided in an embodiment of the present invention shows that the phosphorylation level of glucose-6-phosphate dehydrogenase in human breast cancer tissue is generally lower than that in adjacent tissue by Western blotting; B shows a comparison of the phosphorylation levels of glucose-6-phosphate dehydrogenase in human breast cancer tissue and adjacent tissue by Graphpad software.

[0089] Figure 8 It is a schematic diagram of the structure of the Y249 and Y322 antigenic peptides provided in the examples of the present invention.

[0090] Fig. 9 The present invention provides a flowchart of immunizing rabbits with antigen peptides. DETAILED DESCRIPTION

[0091] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0092] like Figure 1 As shown, the method for preparing an antibody against tyrosine phosphorylation at positions 249 and 322 of glucose-6-phosphate dehydrogenase provided in an embodiment of the present invention comprises the following steps:

[0093] S101, peptide coupling;

[0094] S102, rabbit immunization;

[0095] S103, enzyme-linked immunosorbent assay;

[0096] S104, antibody purification.

[0097] This method aims to prepare specific antibodies against the 249th and 322nd tyrosine phosphorylation sites of glucose-6-phosphate dehydrogenase (Glucose-6-phosphate dehydrogenase). First, the peptide coupling step couples the peptide at the target phosphorylated tyrosine site with a carrier protein (such as Keyhole Limpet Hemocyanin, KLH) through a chemical reaction. This process usually uses an activator (such as carbodiimide compounds) to promote the formation of a stable covalent bond between the peptide and the amino group on the carrier protein. The coupled complex can effectively enhance the immunogenicity of the peptide, ensuring that rabbits can be induced to produce specific antibodies against specific sites during the subsequent immunization process.

[0098] In the rabbit immunization step, the coupled polypeptide-carrier complex is injected into the rabbit as an immunogen. The rabbit's immune system recognizes and responds to the complex, producing specific antibodies against the tyrosine phosphorylation sites 249 and 322 of glucose-6-phosphate dehydrogenase. In order to enhance the immune response, multiple immunizations are usually performed, including primary immunization and several booster immunizations. After each immunization, the antibody concentration in the rabbit gradually increases and the specificity is enhanced, thereby ensuring that high-affinity and high-specificity antibodies are finally obtained.

[0099] The ELISA test step is used to screen and verify whether the post-immunization serum contains specific antibodies against the target phosphorylation site. The phosphorylated peptides of known sequence are fixed to the surface of the wells of the ELISA plate, and the binding of the antibodies is detected by reacting with the antibodies in the rabbit serum. The presence and concentration of the antibodies are quantitatively determined by adding enzyme-labeled secondary antibodies and substrates using a colorimetric reaction. Only serum samples that show high specificity and high titer in the ELISA test will be selected for subsequent antibody purification.

[0100] Finally, the antibody purification step extracts and purifies the target specific antibodies from rabbit serum through a variety of purification techniques (such as protein A / G column chromatography, affinity chromatography, etc.). The purification process ensures that the obtained antibodies have high purity and high specificity, removing non-specific binders and other serum proteins. The purified antibodies can be used for further research and applications, such as immunoprecipitation, immunoblotting (Western blot), immunofluorescence staining and other experiments to ensure their reliability and effectiveness in detecting the tyrosine phosphorylation status of glucose-6-phosphate dehydrogenase at positions 249 and 322.

[0101] The polypeptide coupling provided in the embodiments of the present invention is:

[0102] Peptide-coupled KLH:

[0103] Dissolve 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution;

[0104] Weigh 8 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO, then add 150 μL 1× PBS and mix well;

[0105] Add the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester dropwise into KLH, gently shake while adding (vigorous shaking will produce precipitation), and leave at room temperature for 1 hour;

[0106] The activated KLH solution was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed in 2L of 1×PBS at 4°C under magnetic stirring for 1 h.

[0107] Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed KLH in a 15mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4℃ refrigerator;

[0108] Weigh 4 mg of peptide, dissolve in 50 μL of DMSO, add 200 μL of 1× PBS, mix quickly, then immediately add KLH at a ratio of peptide: KLH = 1 mg: 680 μg, and react overnight at 4°C refrigerator or at room temperature for 2 hours;

[0109] The cross-linked KLH-peptide complex was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed overnight in 4 L of 1×PBS under magnetic stirring in a 4°C refrigerator;

[0110] Take out the dialyzed KLH-peptide into a clean 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator;

[0111] Peptide-coupled carrier protein BSA:

[0112] Dissolve 20 mg of BSA in 2 mL of 5 mM EDTA aqueous solution;

[0113] Weigh 5 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO, then add 150 μL 1× PBS and mix well;

[0114] Add 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt solution dropwise into the BSA solution, gently shake while adding, and leave at room temperature for 1 hour;

[0115] The activated BSA solution was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed in 2L of 1×PBS at 4°C under magnetic stirring for 1 h.

[0116] Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed BSA in a 15mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4℃ refrigerator;

[0117] Weigh 1 mg of peptide, dissolve it in 50 μL of DMSO, add 150 μL of 1× PBS, mix quickly, then add 100 μL of the activated BSA solution, and react in a 4°C refrigerator overnight or at room temperature for 2 hours;

[0118] Place the cross-linked BSA-Peptide complex in a 1.5 mL centrifuge tube, add 1×PBS to 1 mL, and mark the project number, concentration, and date.

[0119] Rabbit immunization provided by the embodiment of the present invention:

[0120] Animal selection: Rabbits are New Zealand white rabbits, weighing about 2.5 kg, young and strong; animals should be selected with shiny fur and healthy animals that can move freely; select the animals and raise them for about 2 weeks in advance; the purpose is to eliminate some unqualified animals so that the later experiments can proceed smoothly;

[0121] Preparation before the experiment: Rabbits were marked;

[0122] Antigen preparation:

[0123] Take the antigen out of the -20 degree freezer and dissolve it at room temperature to avoid repeated freezing and thawing; mark the syringe with the project number and animal number;

[0124] Extract antigen (mix antigen completely), the concentration of antigen for the first immunization is 1 mg / mL, and 0.5 mL / rabbit. The concentration of antigen for the second to fourth immunization is halved, and the dosage remains unchanged;

[0125] When extracting adjuvant, the volume ratio of adjuvant to antigen is 1:1. Complete adjuvant is used for the first immunization, and incomplete adjuvant is used for the second to fourth immunizations. When extracting, the adjuvant should be fully mixed before being drawn into the syringe.

[0126] The two syringes are connected with a syringe connecting tube for complete emulsification. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37-degree water;

[0127] immunity.

[0128] Immunization provided in the embodiment of the present invention: Rabbits were injected subcutaneously at multiple points, with 0.2 mL at each point;

[0129] Immunization time: The second vaccination will be carried out on the 14th day after the first vaccination, and the interval between the second and third vaccinations is 7 days; a small sample of serum will be collected from the middle ear artery of the rabbit on the 7th day after the third vaccination for testing. If the test is qualified, an additional vaccination will be carried out 7 days later. Whole blood can be collected 7 days after the additional vaccination.

[0130] The enzyme-linked immunosorbent assay provided by the embodiment of the present invention detects:

[0131] Coating: Dilute the known antigen to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, incubate at 4°C overnight, and the next day, discard the solution in the wells and wash once with 1xTBST washing buffer (180 μL per well);

[0132] Blocking: Add 60 μL of 1% BSA (prepared in TBST) to each well for blocking, incubate at 37°C for 1 hour, then discard the blocking solution;

[0133] Sample addition: Add a certain dilution of the sample to be tested (dilute the sample to be tested according to a certain ratio), 50 μL to the above blocked reaction wells; set up the positive control well (positive serum) and the negative control well (BSA); incubate at 37°C for 1 hour, then discard the blocking solution and wash twice with 1xTBST washing buffer, 180 μL per well;

[0134] Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the ELISA plate at 50 μL / well, incubate at 37°C for 45 min, then discard the blocking solution and wash three times with 1xTBST washing buffer at 180 μL per well;

[0135] Add substrate solution for color development: Add 100 μL of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 min;

[0136] Stop the reaction: add 90 μL of 2M sulfuric acid to each reaction well;

[0137] Read the plate: Place the ELISA plate in a preheated ELISA reader (450nm) for reading, save the data, and analyze.

[0138] Antibody purification provided by the embodiments of the present invention:

[0139] The affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1× PBS (pH 7.4) at a flow rate of 70 mL / h;

[0140] Take 10 mL of serum to be purified in a 50 mL centrifuge tube and filter it with a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm;

[0141] The filtered serum sample was loaded at a flow rate of 40 mL / h and repeated once;

[0142] The column was washed with 20 mL 1× PBS (pH 7.4) at a flow rate of 70 mL / h. After 10 min, the column was connected to the protein detector. During the washing process, the light transmittance (T position) of the instrument was adjusted to 100.

[0143] Adjust the protein detector absorbance (1A gear) to 0, then turn on the HD-A computer collector on the computer desktop and adjust the full screen range to 5, use glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h to elute the antibody, then press the green elution record button to start elution, and start collecting the antibody when the instrument reading starts to rise;

[0144] During the antibody collection process, the pH value of the antibody was promptly adjusted to about 7 with 1 M sodium bicarbonate, and the highest peak of the elution peak was recorded;

[0145] After collecting the antibodies, adjust the pH to about 7 and record the volume of eluted antibodies, then rinse the rubber tube connected to the collector with pure water;

[0146] The affinity chromatography column was washed with 20 mL 1× PBS and pure water at a rate of 70 mL / h, and then 20% ethanol was added, sealed, and stored in a refrigerator at 4°C;

[0147] The purified antibodies are sent for testing according to different requirements: ELISA, WB, ICC.

[0148] like Figure 2 As shown, a system for preparing an antibody for phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase provided in an embodiment of the present invention comprises:

[0149] A coupling module, used for peptide coupling;

[0150] Immunization module, used for rabbit immunization;

[0151] Detection module, used for enzyme-linked immunosorbent assay detection;

[0152] Purification module, used for antibody purification.

[0153] Another object of the present invention is to provide a computer device, comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor executes the steps of the method for preparing antibodies phosphorylated at the 249th and 322nd tyrosine sites of glucose-6-phosphate dehydrogenase.

[0154] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, enables the processor to execute the steps of the method for preparing antibodies phosphorylated at the 249th and 322nd tyrosine sites of glucose-6-phosphate dehydrogenase.

[0155] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for preparing antibodies against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase.

[0156] The present invention is specifically implemented:

[0157] Glucose-6-phosphate dehydrogenase [Homo sapiens]

[0158] GenBank:AAA92653.1uniport:P11413

[0159] 1maeqvalsrt qvcgilreel fqgdafhqsd thifiimgas gdlakkkiyp tiwwlfrdgl

[0160] 61lpentfimgy arsrltvadi rkqsepffka tpeeklkled ffarnsyvag qyddaasyqr

[0161] 121lnshmdalhl gsqanrlfyl alpptvyeav tknihescms qigwnriive kpfgrdlqss

[0162] 181drlsnhissl fredqiyrid hylgkemvqn lmvlrfanri fgpiwnrdni acviltfkep

[0163] 241fgtegrggyfdefgiirdvm qnhllqmLcl vamekpastn sddvrdekvk vlkcisevqa

[0164] 301nnvvlgqyvg npdgegeatk gylddptvpr gsttatfaav vlyvenerwd gvpfilrcgk

[0165] 361alnerkaevr lqfhdvagdi fhqqckrnel virvqpneav ytkmmtkkpg mffnpeesel

[0166] 421dltygnrykn vklpdayerl ildvfcgsqm hfvrsdelre awriftpllh qielekpkpi

[0167] 481pyiygsrgpt eadelmkrvg fqyegtykwv nphkl

[0168] like Figure 6 As shown, the site information is: Y249 and Y322. The corresponding sites need to design phosphorylated peptides to immunize rabbits as antibodies

[0169] Protein:Glucose-6-phosphate 1-dehydrogenase

[0170] Gene:Glucose-6-phosphate dehydrogenase

[0171] Organism:Homo sapiens(Human)

[0172] http: / / www.uniprot.org / uniprot / P11413

[0173] Protein sequence:

[0174] Protein sequence analysis Figure 3 :

[0175] Peptide design:

[0176] Immunity peptide 239-253aa CEPFGTEGRGGY(pi)FDEF

[0177] Differential detection and purified peptide 239-253aa CEPFGTEGRGGYFDEF

[0178] Immunity peptide 316-329aa CGEATKGY(pi)LDDPTVP

[0179] Differential detection and purified peptide 316-329aa CGEATKGYLDDPTVP

[0180] 1. Peptide coupling

[0181] 1.1 Peptide-coupled KLH

[0182] 1.1.1 Dissolve 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution.

[0183] 1.1.2 Weigh 8 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO. Then add 150 μL 1× PBS and mix well.

[0184] 1.1.3 Add the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester dropwise into KLH, shaking gently (vigorous shaking will produce precipitation), and leave at room temperature for 1 hour.

[0185] 1.1.4 Place the activated KLH solution into a dialysis bag, clamp it with a dialysis clip, and dialyze it in 2L of 1×PBS at 4°C under magnetic stirring for 1 hour.

[0186] 1.1.5 Replace with new 1×PBS and dialyze for 2 hours, repeat once. Place the activated dialyzed KLH in a 15 mL imported centrifuge tube, and mark the reagent name, time and concentration on the tube, and store in a 4°C refrigerator.

[0187] 1.1.6 Weigh 4 mg of peptide, dissolve in 50 μL of DMSO, add 200 μL of 1× PBS, mix quickly, then immediately add KLH at a ratio of peptide: KLH = 1 mg: 680 μg, and react in a 4°C refrigerator overnight or at room temperature for 2 hours.

[0188] 1.1.7 Place the cross-linked KLH-peptide complex into a dialysis bag, clamp it with a dialysis clip, and dialyze it in 4L of 1×PBS at 4°C under magnetic stirring overnight.

[0189] 1.1.8 Take out the dialyzed KLH-peptide into a clean 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator.

[0190] 1.2 Peptide-coupled carrier protein BSA

[0191] 1.2.1 Dissolve 20 mg of BSA in 2 mL of 5 mM EDTA aqueous solution.

[0192] 1.2.2 Weigh 5 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimide ester sodium salt and completely dissolve it in 50 μL DMSO. Then add 150 μL 1× PBS and mix well.

[0193] 1.2.3 Add the sodium salt solution of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester dropwise into the BSA solution, gently shake while adding, and let it stand at room temperature for 1 hour.

[0194] 1.2.4 Place the activated BSA solution into a dialysis bag, clamp it with a dialysis clip, and dialyze it in 2L of 1×PBS at 4℃ under magnetic stirring for 1h.

[0195] 1.2.5 Replace with new 1×PBS and dialyze for 2 hours, repeat once. Place the activated dialyzed BSA in a 15 mL imported centrifuge tube, and mark the tube with the reagent name, time and concentration, and store in a 4°C refrigerator.

[0196] 1.2.6 Weigh 1 mg of peptide, dissolve it in 50 μL of DMSO, add 150 μL of 1× PBS, mix quickly, then add 100 μL of the activated BSA solution, and react in a 4°C refrigerator overnight or at room temperature for 2 hours.

[0197] 1.2.7 Place the cross-linked BSA-Peptide complex in a 1.5mL centrifuge tube, add 1×PBS to 1mL, mark the project number, concentration and date, and hand it over to the testing group.

[0198] 2. Rabbit Immunization Protocol

[0199] 2.1 Animal selection: New Zealand white rabbits are used, weighing about 2.5 kg, young and strong. The animals should be healthy animals with shiny fur and free movement. The selected animals should be pre-raised for about 2 weeks. The purpose is to eliminate some unqualified animals so that the later experiments can proceed smoothly.

[0200] 2.2 Preparation before the experiment: The rabbits were marked.

[0201] 2.3 Antigen preparation:

[0202] 2.3.1 Take the antigen out of the -20 degree freezer and dissolve it at room temperature to avoid repeated freezing and thawing. Mark the syringe with the project number and animal number.

[0203] 2.3.2 Extract antigen (mix antigen completely), the antigen concentration for the first immunization is 1 mg / mL, and the concentration for each rabbit is 0.5 mL. The antigen concentration for the second to fourth immunizations is halved, and the dosage remains unchanged.

[0204] 2.3.3 Extract the adjuvant in a 1:1 volume ratio of adjuvant to antigen. Use complete adjuvant for the first immunization and incomplete adjuvant for the second to fourth immunizations. When extracting, mix the adjuvant thoroughly before drawing it into the syringe.

[0205] 2.3.4 The two syringes are connected with a syringe connecting tube for complete emulsification. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37 degree water.

[0206] 2.4 Immunization: Rabbits were given multiple subcutaneous injections, with 0.2 mL at each point.

[0207] Immunization time: The second vaccination is carried out on the 14th day after the first vaccination, and the interval between the second and third vaccinations is 7 days. On the 7th day after the third vaccination, a small sample of serum is collected from the middle ear artery of the rabbit for testing. If the test is qualified, an additional vaccination will be carried out 7 days later, and whole blood can be collected 7 days after the additional vaccination.

[0208] 3. Enzyme-linked immunosorbent assay (indirect method)

[0209] 3.1 Coating: Dilute the known antigen to 1 μg / mL with coating buffer (Na2CO3 and NaHCO3 buffer), add 50 μL to each reaction well of the polystyrene plate, and incubate at 4°C overnight. The next day, discard the solution in the wells and wash once with 1xTBST washing buffer with 180 μL per well.

[0210] 3.2 Blocking: Add 60 μL of 1% BSA (prepared in TBST) to each well for blocking and incubate at 37°C for 1 hour. Then discard the blocking solution.

[0211] 3.3 Sample addition: Add a certain dilution of the sample to be tested (dilute the sample to be tested according to a certain ratio), 50μL to the above blocked reaction wells. At the same time, set up the positive control well (positive serum) and the negative control well (BSA). Incubate at 37℃ for 1 hour, then discard the blocking solution and wash twice with 1xTBST washing buffer, 180μL per well.

[0212] 3.4 Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the ELISA plate at 50 μL / well, incubate at 37°C for 45 minutes, then discard the blocking solution and wash 3 times with 1xTBST washing buffer at 180 μL per well.

[0213] 3.5 Add substrate solution for color development: Add 100 μL of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 minutes.

[0214] 3.6 Termination of reaction: Add 90 μL of 2 M sulfuric acid to each reaction well.

[0215] 3.7 Plate reading: Place the ELISA plate in a preheated ELISA reader (450 nm) for reading, save the data, and analyze.

[0216] 4. Antibody Purification

[0217] 4.1 The affinity chromatography column was thoroughly cleaned with 20 mL of pure water and 1× PBS (pH 7.4) at a flow rate of 70 mL / h.

[0218] 4.2 Take 10 mL of serum to be purified and place it in a 50 mL centrifuge tube. Filter it using a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm.

[0219] 4.3 Load the filtered serum sample at a flow rate of 40 mL / h and repeat once.

[0220] 4.4 Wash the column with 20 mL 1×PBS (pH 7.4) at a flow rate of 70 mL / h. Connect the column to the protein detector after 10 minutes. During the washing process, adjust the instrument transmittance (T gear) to 100.

[0221] 4.5 Adjust the absorbance of the protein detector (1A gear) to 0. At this time, turn on the HD-A computer collector on the computer desktop and adjust the full-screen range to 5. Use glycine solution (pH 2.7, 0.2M) at a rate of 40mL / h to elute the antibody. At this time, press the green elution record button to start elution. When the instrument reading begins to rise, start collecting the antibody.

[0222] 4.6 During the antibody collection process, adjust the pH value of the antibody to about 7 with 1M sodium bicarbonate and record the highest peak of the elution peak.

[0223] 4.7 After collecting the antibodies, adjust the pH value to about 7 and record the volume of eluted antibodies. Then rinse the rubber tube connected to the collector with pure water.

[0224] 4.8 Wash the affinity chromatography column with 20 mL 1×PBS and pure water at a rate of 70 mL / h, then add 20% ethanol, seal the column, and store it in a refrigerator at 4°C.

[0225] 4.9 The purified antibodies are sent for testing according to different requirements: ELISA, WB, ICC, etc.

[0226] (When purifying phosphorylated antibodies, the serum is first passed through a phosphorylated column, and the eluted antibodies are then passed through a non-phosphorylated column to obtain specific phosphorylated antibodies)

[0227] 3. Experimental Results

[0228] 1. Peptide information

[0229] Peptide number Sequence information Y249-p 239-253aaCEPFGTEGRGGY(pi)FDEF Y322-P 316-329aaCGEATKGY(pi)LDDPTVP

[0230] 2. Immunization Schedule Periodic Table

[0231]

[0232]

[0233] 3. Serum test results

[0234]

[0235] Conclusion: The OD450nm value of 1:64000 is greater than 0.6, and the test is qualified.

[0236] 4. Test results of finished products

[0237]

[0238] Conclusion: The OD450nm value of the finished antibody 1:16000 is greater than 0.6, and the test is qualified.

[0239] 6. Finished Antibody Volume and Concentration

[0240]

[0241]

[0242] 7. Antibody titer detection:

[0243] 7.1 Dot blot assay to verify antibody specificity

[0244] 1) Take two clean and dry nitrocellulose membranes, one for detecting Y249 phosphorylated antibody and the other for detecting Y322 phosphorylated antibody;

[0245] 2) Dilute the Y249 (pi) peptide, Y322 (pi) peptide and non-phosphorylated modified peptide to the following concentrations: 0.0005 ng, 0.005 ng, 0.05 ng, 0.5 ng, 1 ng, respectively;

[0246] 3) Pipette 2 μL of modified peptide solution and slowly drop it on the nitrocellulose membrane to gradually form a circular spot. Then drop the non-phosphorylated modified peptide solution on the second row and align it with the droplets on the first row.

[0247] 4) Place the nitrocellulose membrane in a fume hood and air-dry it to allow the polypeptide droplets to be completely absorbed into the membrane;

[0248] 5) After complete absorption, block the nitrocellulose membrane with 5% skimmed milk powder and shake on a shaker at room temperature for 1 hour;

[0249] 6) Remove skim milk powder, quickly wash the membrane twice with TBST solution, then completely immerse the nitrocellulose membrane with TBST solution, shake on a shaker for 5 minutes, and repeat three times;

[0250] 7) Prepare the primary antibody with 1% BSA solution, the dilution ratio of the primary antibody is 1:4000;

[0251] 8) Pour the primary antibody into the box containing the nitrocellulose membrane and incubate on a rocking table at room temperature for 1 hour;

[0252] 9) Then wash the nitrocellulose membrane with TBST solution, shake it on a shaker for 5 minutes, and repeat four times;

[0253] 10) Prepare the secondary antibody with TBST solution, the dilution ratio of the secondary antibody is 1:10000;

[0254] 11) Pour the secondary antibody into the box containing the nitrocellulose membrane and incubate on a rocking table at room temperature for 1 hour;

[0255] 12) Then wash the nitrocellulose membrane with TBST solution, shake it on a shaker for 5 minutes, and repeat this cycle four times;

[0256] 13) Finally, development processing is performed.

[0257] In order to verify the specificity of the homemade phosphorylation antibody, we used a dot blot assay. The development results showed that as the concentration of phosphorylated peptides decreased, the signal intensity of the phosphorylated antibody decreased, and the phosphorylated antibody had no signal for non-phosphorylated peptides. The above results show that the homemade phosphorylation antibody is specific and only recognizes the Y249 and Y322 phosphorylation sites.

[0258] Figure 4 Note: Peptide spots of the above concentrations were formed on nylon membranes, incubated with specific phospho-antibodies and analyzed by western blot.

[0259] 7.2 Point mutation verification of antibody efficacy

[0260] 1) Construction of plasmids with mutations at all sites of Y249 and Y322;

[0261] 2) Plasmid transfection and overexpression in breast cancer cells;

[0262] 3) Co-immunoprecipitation of 2YF or 2YA using M2 beads;

[0263] 4) Finally, Western blotting was used for protein blotting analysis.

[0264] The two tyrosine sites of glucose-6-phosphate dehydrogenase were mutated into phenylalanine (2YF) or alanine (2YA). The phosphorylation levels of 2YF and 2YA showed a significant downward or disappearing trend when detected with specific phosphorylation antibodies.

[0265] Figure 5 Note: Glucose-6-phosphate dehydrogenase wild type (WT) and its mutant (2YF / 2YA) proteins were analyzed by western blot using specific phosphorylation antibodies.

[0266] 1. Specific application fields or related products of the present invention.

[0267] 1. Cell signal transduction research

[0268] Western Blot (WB): Detect the phosphorylation status of G6PD protein by using phosphorylation-specific antibodies to identify the phosphorylated form of G6PD.

[0269] Immunoprecipitation (IP): Specific precipitation of G6PD phosphorylated proteins from cell lysates for subsequent analysis.

[0270] Immunofluorescence (IF) and immunohistochemistry (IHC): Localize and quantify G6PD phosphorylated protein in cells or tissue sections.

[0271] ELISA (Enzyme-linked immunosorbent assay): Quantitative detection of the level of phosphorylated protein of glucose-6-phosphate dehydrogenase in samples.

[0272] Mass spectrometry analysis: Enrichment was performed using a glucose-6-phosphate dehydrogenase phosphorylation-specific antibody to improve the sensitivity and specificity of mass spectrometry analysis.

[0273] 2. Disease research

[0274] Glucose-6-phosphate dehydrogenase phosphorylation antibodies can be used to study the mechanism of cancer occurrence, detect the phosphorylation level of tumor markers, and evaluate the effects of drugs on signaling pathways.

[0275] 2. Relevant evidence of the technical effects obtained by the embodiments of the present invention.

[0276] 1. Detect the phosphorylation level of glucose-6-phosphate dehydrogenase in human breast cancer tissues and adjacent tissues.

[0277] Research background: Phosphorylation of Y249 and Y322 sites of glucose-6-phosphate dehydrogenase protein plays an important role in the signaling pathway of breast cancer cells and is closely related to the pathogenesis of cancer.

[0278] Experimental method: Human breast cancer tissue and adjacent tissue were homogenized, and phosphorylation levels of glucose-6-phosphate dehydrogenase were detected using phosphorylation antibodies against glucose-6-phosphate dehydrogenase.

[0279] Results: Western blotting revealed that the phosphorylation level of glucose-6-phosphate dehydrogenase in breast cancer tissues was generally lower than that in adjacent tissues. Figure 7 A), which indicates that the phosphorylation level of glucose-6-phosphate dehydrogenase is related to the occurrence of cancer. In addition, the phosphorylation level of glucose-6-phosphate dehydrogenase in human breast cancer tissue and adjacent tissue was compared using Graphpad software ( Figure 7B) found that the phosphorylation level of glucose-6-phosphate dehydrogenase in adjacent tissues was significantly higher than that in breast cancer tissues. This is helpful for studying the role of glucose-6-phosphate dehydrogenase protein phosphorylation in cell signaling pathways and the mechanism of cancer occurrence, and provides potential targets for the diagnosis or treatment of clinical tumor diseases.

[0280] Figure 8 : Schematic diagram of the structure of Y249 and Y322 antigen peptides: KLH, phosphorylated modified peptides and BSA are cross-linked together using chemical methods and used to immunize rabbits.

[0281] Process of immunizing rabbits with antigen peptides Fig. 9 : After the first immunization of rabbits, three immunizations were performed on the 14th, 21st and 35th days respectively, and then the serum antibody concentration was tested on the 28th day. If qualified, whole blood was collected for antibody purification.

[0282] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing an antibody against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase, characterized in that: The following steps are involved: A polypeptide comprising the 249th and 322nd tyrosine phosphorylation sites of glucose-6-phosphate dehydrogenase is coupled to a carrier protein by chemical reaction, wherein the carrier protein is Keyhole Limpet Hemocyanin (KLH) or bovine serum albumin (BSA), and the coupling reaction uses a carbodiimide activator to promote the formation of a stable covalent bond between the polypeptide and the amino group on the carrier protein to enhance immunogenicity; The coupled polypeptide-carrier complex is used as an immunogen to immunize rabbits by subcutaneous injection or intramuscular injection, and the immunization is performed according to the scheme of primary immunization and several booster immunizations to induce the production of specific antibodies against the tyrosine phosphorylation sites 249 and 322 of glucose-6-phosphate dehydrogenase in the rabbits; Enzyme-linked immunosorbent assay (ELISA) was used to screen the specificity of antibodies in rabbit serum, wherein the phosphorylated polypeptides were fixed on ELISA plates to bind to the antibodies in rabbit serum, and the antibody binding was detected by adding enzyme-labeled secondary antibodies and substrate color development to determine the specificity and titer of the antibodies; Protein A / G affinity chromatography or phosphorylated antigen affinity chromatography is used to separate and purify the target antibody from rabbit serum to obtain high-purity and high-specificity antibodies, remove non-specific binders and other serum proteins, and ensure that the obtained antibodies are suitable for experiments such as immunoprecipitation, immunoblotting (Western blot), and immunofluorescence staining.

2. The method for preparing an antibody against tyrosine phosphorylation at positions 249 and 322 of glucose-6-phosphate dehydrogenase according to claim 1, characterized in that: The polypeptide is coupled to: Peptide-coupled KLH: Dissolve 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution; Weigh 8 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO, then add 150 μL 1× PBS and mix well; Add 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt solution dropwise into KLH, gently shake while adding, and leave at room temperature for 1 hour; The activated KLH solution was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed in 2L of 1×PBS at 4°C under magnetic stirring for 1 h. Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed KLH in a 15mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4℃ refrigerator; Weigh 4 mg of peptide, dissolve in 50 μL of DMSO, add 200 μL of 1× PBS, mix quickly, then immediately add KLH at a ratio of peptide: KLH = 1 mg: 680 μg, and react overnight at 4°C refrigerator or at room temperature for 2 hours; The cross-linked KLH-peptide complex was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed overnight in 4 L of 1×PBS under magnetic stirring in a 4°C refrigerator; Take out the dialyzed KLH-peptide into a clean 1.5 mL centrifuge tube, divide it into portions according to the immunization dose, and store it in a -20°C refrigerator; Peptide-coupled carrier protein BSA: Dissolve 20 mg of BSA in 2 mL of 5 mM EDTA aqueous solution; Weigh 5 mg of 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt and completely dissolve it in 50 μL DMSO, then add 150 μL 1× PBS and mix well; Add 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt solution dropwise into the BSA solution, gently shake while adding, and leave at room temperature for 1 hour; The activated BSA solution was placed in a dialysis bag, clamped with a dialysis clip, and dialyzed in 2L of 1×PBS at 4°C under magnetic stirring for 1 h. Replace with new 1×PBS and dialyze for 2 hours, repeat once; place the activated dialyzed BSA in a 15mL imported centrifuge tube, mark the reagent name, time and concentration on the tube, and store in a 4℃ refrigerator; Weigh 1 mg of peptide, dissolve it in 50 μL of DMSO, add 150 μL of 1× PBS, mix quickly, then add 100 μL of the activated BSA solution, and react in a 4°C refrigerator overnight or at room temperature for 2 hours; Place the cross-linked BSA-Peptide complex in a 1.5 mL centrifuge tube, add 1×PBS to 1 mL, mark the project number, concentration and date, and hand it over to the testing group.

3. The preparation and use of the antibody for phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase as claimed in claim 1, characterized in that: The rabbits were immunized with: Animal selection: Rabbits are New Zealand white rabbits, weighing about 2.5 kg, young and strong; animals should be selected with shiny fur and healthy animals that can move freely; select the animals and raise them for about 2 weeks in advance; the purpose is to eliminate some unqualified animals so that the later experiments can proceed smoothly; Preparation before the experiment: Rabbits were marked; Antigen preparation: Take the antigen out of the -20 degree freezer and dissolve it at room temperature to avoid repeated freezing and thawing; mark the syringe with the project number and animal number; Extract antigens, the concentration of the first immunization is 1mg / mL, and the concentration of the first immunization is 0.5mL / rabbit. The concentration of the second to fourth immunizations is halved, and the dosage remains unchanged. When extracting adjuvant, the volume ratio of adjuvant to antigen is 1:

1. Complete adjuvant is used for the first immunization, and incomplete adjuvant is used for the second to fourth immunizations. When extracting, the adjuvant should be fully mixed before being drawn into the syringe. The two syringes are connected with a syringe connecting tube for complete emulsification. The emulsification standard is: the emulsified immunogen is qualified if it does not disperse when dropped into 37-degree water; immunity.

4. The method for preparing an antibody against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase as claimed in claim 3, characterized in that: Immunization: Rabbits were injected subcutaneously at multiple points, with 0.2 mL at each point; Immunization time: The second vaccination will be carried out on the 14th day after the first vaccination, and the interval between the second and third vaccinations is 7 days; a small sample of serum will be collected from the middle ear artery of the rabbit on the 7th day after the third vaccination for testing. If the test is qualified, an additional vaccination will be carried out 7 days later. Whole blood can be collected 7 days after the additional vaccination.

5. The method for preparing an antibody against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase according to claim 1, characterized in that: The ELISA assay detects: Coating: dilute the known antigen to 1 μg / mL with coating buffer, add 50 μL to each reaction well of the polystyrene plate, incubate at 4°C overnight, and the next day, discard the solution in the wells and wash once with 1xTBST washing buffer at 180 μL per well; Blocking: Add 60 μL of 1% BSA to each well for blocking, incubate at 37°C for 1 hour, then discard the blocking solution; Add sample: Add 50 μL of the sample to be tested at a certain dilution to the blocked reaction wells; set up the positive control wells and negative control wells at the same time; incubate at 37°C for 1 hour, then discard the blocking solution and wash twice with 1xTBST washing buffer at 180 μL per well; Add enzyme-labeled antibody: Add freshly diluted secondary antibody-HRP to the wells of the ELISA plate at 50 μL / well, incubate at 37°C for 45 min, then discard the blocking solution and wash three times with 1xTBST washing buffer at 180 μL per well; Add substrate solution for color development: Add 100 μL of the temporarily prepared TMB substrate solution to each reaction well and react at 37°C for 5 min; Stop the reaction: add 90 μL of 2M sulfuric acid to each reaction well; Read the plate: Place the ELISA plate in a preheated ELISA reader to read the plate, save the data, and analyze it.

6. The method for preparing an antibody against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase according to claim 1, characterized in that: The antibody purification: The affinity chromatography column was thoroughly washed with 20 mL of pure water and 1× PBS, respectively, at a flow rate of 70 mL / h; Take 10 mL of serum to be purified in a 50 mL centrifuge tube and filter it with a microporous filter membrane with a pore size of 0.45 μm and a diameter of 25 mm; The filtered serum sample was loaded at a flow rate of 40 mL / h and repeated once; The column was washed with 20 mL 1× PBS at a flow rate of 70 mL / h. After 10 min, it was connected to the protein detector. During the washing process, the light transmittance reading of the instrument was adjusted to 100. Adjust the protein detector absorbance reading to 0, then open the HD-A computer collector on the computer desktop and adjust the full screen range to 5, use glycine solution to elute the antibody at a rate of 40mL / h, then press the green elution record button to start elution, and start collecting antibodies when the instrument reading starts to rise; During the antibody collection process, the pH value of the antibody was promptly adjusted to about 7 with 1 M sodium bicarbonate, and the highest peak of the elution peak was recorded; After collecting the antibodies, adjust the pH to about 7 and record the volume of eluted antibodies, then rinse the rubber tube connected to the collector with pure water; The affinity chromatography column was washed with 20 mL 1× PBS and pure water at a rate of 70 mL / h, and then 20% ethanol was added, sealed, and stored in a refrigerator at 4°C; The purified antibodies are sent for testing according to different requirements: ELISA, WB, ICC.

7. A system for preparing antibodies against phosphorylation of tyrosine sites 249 and 322 of glucose-6-phosphate dehydrogenase according to any one of claims 1 to 6, characterized in that: include: A coupling module, used for peptide coupling; Immunization module, used for rabbit immunization; Detection module, used for enzyme-linked immunosorbent assay detection; Purification module, used for antibody purification.

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