Rabbit polyclonal antibody targeting tyrosine phosphorylation at position 962 of human pi3k beta and preparation method and application thereof

Abstract

The present application relates to a rabbit polyclonal antibody targeting human PI3K beta tyrosine phosphorylation at position 962, and a preparation method and application thereof. The present application first identifies that PI3K beta protein Y962 site is phosphorylated and modified and the phosphorylation modification of the site significantly affects the function of PI3K beta protein in PTEN knockout cells through mass spectrometry combined with a series of molecular biology experiments, so an antigen peptide containing Y962 site phosphorylation is synthesized, an immunogen is obtained by coupling a carrier protein hole hemocyanin, a Japanese white rabbit is immunized six times combined with an adjuvant, and a PI3K beta Y962 site specific phosphorylation antibody is obtained after purification and identification. The present application can specifically detect the PI3K beta Y962 site phosphorylation level in cells and tissue samples, and provides an important tool for studying the role and mechanism of PI3K beta phosphorylation in tumors.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta and a preparation method and application thereof. BACKGROUND

[0002] The type I PI3K signaling pathway is one of the most important cell signal transduction pathways, PI3K phosphorylates PIP2 to PIP3, activates the phosphorylation of downstream AKT, and regulates a series of important life activities such as cell survival, proliferation, differentiation, apoptosis, metabolism, etc. The PI3K-AKT signaling pathway is abnormally activated in many tumors. Type I PI3K is divided into four subtypes: PI3K alpha, PI3K beta, PI3K gamma and PI3K delta. Among them, PI3K alpha (PIK3CA gene) and PI3K beta (PIK3CB gene) are widely expressed in many cells, both have similar structures and both have the ability to activate AKT, but the two subtypes have important and potentially different functions. In 2019, the first PI3K alpha inhibitor (also the first PI3K inhibitor drug) was approved by FDA for the clinical treatment of ER-positive advanced metastatic breast cancer. However, the loss of the tumor suppressor gene PTEN has led to drug resistance of tumors to PI3K alpha inhibitors in clinical practice, because PTEN loss induces tumor cells to change from dependence on PI3K alpha subtype to dependence on PI3K beta subtype, thereby leading to tumor resistance to PI3K alpha inhibitors, and the molecular mechanism remains a mystery. In-depth study of the molecular regulation mechanism of PTEN on PI3K-p110beta and exploration of new PI3K-p110beta targets of PTEN-deficient tumors are of great significance for the treatment of various PTEN-deficient tumors and the prevention of canceration of PTEN-deficient cells.

[0003] Post-translational modification of proteins is an important mechanism affecting the mutual combination of proteins. In recent years, it has been found that PTEN has protein phosphatase activity in addition to the classic lipid phosphatase activity. However, the protein substrates dephosphorylated by PTEN as a phosphatase and their functions in tumors are still unknown. SUMMARY

[0004] Based on the above research progress and status, the application provides a rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta and a preparation method and application thereof.

[0005] The application uses phosphorylation modified peptide segments to connect keyhole limpet hemocyanin (KLH) to immunize Japanese white rabbits, and obtain polyclonal antibodies against PI3K beta 962th tyrosine phosphorylation site, which can specifically detect the endogenous PI3K beta 962th site phosphorylation level in cells and tissues, and further become a brand new marker for judging PTEN deletion tumors, and provide necessary detection means for subsequent high-throughput screening of small molecule compounds specifically targeting the PI3K beta 962th phosphorylation site through compound library, thereby having potential to become a new strategy for treating PTEN deletion tumors. It is a high-specificity polyclonal antibody with a brand new phosphorylation modification site of PI3K beta and potential application in tumor marker detection.

[0006] The rabbit polyclonal antibody for PI3K beta 962th tyrosine phosphorylation has specific binding activity with PI3K beta 962th site phosphorylation. The inventors of the present application found that PI3K beta is a new dephosphorylation modification protein substrate of PTEN. PI3K beta phosphorylation modification cannot be detected in PTEN normal cells, but high degree of phosphorylation peptides of two sites (T930, Y962) of PI3K beta protein are detected in PTEN deletion cells. Among them, the phosphorylation of PI3K beta Y962 site can significantly change the mutual combination of PI3K beta and transmembrane kinase to regulate the expression of downstream cancer protein cMyc, thereby driving the occurrence and progression of PTEN deletion tumors. Further exploration found that when PTEN is normal, the phosphorylation level of PI3K beta Y962 site is low, mainly depending on PI3K alpha; when PTEN is deleted, the phosphorylation of PI3K beta Y962 site is significantly up-regulated, and promotes the occurrence and development of tumors by up-regulating the protein level of c-Myc. These results verify that PI3K beta is a new dephosphorylation modification substrate protein of PTEN, and Y962 site is a key phosphorylation site of PI3K beta affecting tumor occurrence and development.

[0007] It is found that PI3K beta Y962 site phosphorylation is crucial for the occurrence and development of PTEN deletion tumors. Therefore, the development of antibodies against PI3K beta Y962 site phosphorylation is of great significance for in-depth study of the molecular mechanism of PTEN deletion-induced tumor survival dependence on PI3K beta subtype, and development of small molecule compounds targeting PI3K beta Y962 site phosphorylation for targeted treatment of PTEN deletion tumors and cancer prevention of PTEN deletion cells.

[0008] The purpose of the present application can be achieved by the following technical solutions:

[0009] One of the technical solutions of the present application: a rabbit polyclonal antibody targeting human PI3K beta tyrosine phosphorylation at position 962, the nucleotide sequence of the rabbit polyclonal antibody targeting human PI3K beta tyrosine phosphorylation at position 962 is shown in SEQ ID NO. 1; the amino acid sequence of the rabbit polyclonal antibody targeting human PI3K beta tyrosine phosphorylation at position 962 is shown in SEQ ID NO. 2.

[0010] The second technical solution of the present application: a preparation method of a rabbit polyclonal antibody targeting human PI3K beta tyrosine phosphorylation at position 962, comprising the following steps:

[0011] S1, synthesize an antigen peptide including a tyrosine phosphorylation site at position 962, the antigen peptide is connected with cysteine at the C-terminal end, and is coupled with a keyhole limpet hemocyanin carrier protein as a complete antigen;

[0012] S2, the complete antigen in S1 is combined with an adjuvant to be applied to an animal for immunization, and then the serum is collected to obtain an antiserum;

[0013] S3, the antiserum in S2 is purified to obtain a PI3K beta Y962 phosphorylation antibody.

[0014] In some embodiments of the present application, in S1, the antigen peptide is ILT(Y-p)DFIH-C, which is a peptide segment including a tyrosine phosphorylation site at position 962 and the site is modified by phosphorylation;

[0015] In some embodiments of the present application, in S2, the adjuvant is Freund's complete adjuvant and Freund's incomplete adjuvant, and the immunized animal is a test-level Japanese white rabbit.

[0016] In some embodiments of the present application, in S3, the purification method of the antiserum is first affinity purification with an affinity chromatography column cross-linked with a phosphorylated polypeptide, then removing the antibody components cross-reacted with the corresponding non-phosphorylated polypeptide with an affinity chromatography column cross-linked with a non-phosphorylated polypeptide, and then removing the antibody components against the keyhole limpet hemocyanin carrier protein with an affinity chromatography column cross-linked with the keyhole limpet hemocyanin; finally, removing the non-antibody protein components by dialysis, concentration and other methods to obtain the purified PI3K beta Y962 phosphorylation antibody.

[0017] Further, the PI3K beta Y962 phosphorylation antibody prepared is subjected to Dot Blot test to verify the titer and specificity of the antibody.

[0018] Further, the PI3K beta Y962 phosphorylation antibody prepared is subjected to Western Blot test to verify the specificity of the antibody.

[0019] The third technical scheme of the present application provides an application of a rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta, which is used as a marker for judging PTEN-deficient tumors.

[0020] Further, the rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta is used for specific detection of the phosphorylation level of an endogenous PI3K beta Y962 site in cells.

[0021] Further, the rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta is used for immunofluorescence detection of Y962 site-phosphorylated PI3K beta protein in PTEN-deficient human triple-negative breast cancer cells BT549.

[0022] Further, the rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta is used for immunofluorescence detection of Y962 site-phosphorylated PI3K beta protein after complementation of PI3K beta in PTEN-deficient human prostate cancer cells PC3.

[0023] Further, the rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta is used for multicolor immunofluorescence detection of Y962 site-phosphorylated PI3K beta protein, PTEN and c-Myc in PTEN-normal and PTEN-deficient human triple-negative breast cancer cells BT549.

[0024] Compared with the prior art, the present application has the following advantages and effects:

[0025] 1. The rabbit polyclonal antibody targeting phosphorylation of a tyrosine at position 962 of human PI3K beta provided by the present application is used for specific detection of the phosphorylation level of an endogenous PI3K beta Y962 site tyrosine in cells and tissues, and is applied to Western Blot and immunofluorescence detection.

[0026] 2. Since PI3K beta Y962 site phosphorylation is crucial for the occurrence and development of PTEN-deficient tumors, the present application plays an important role in in-depth research on the molecular target value of PI3K beta Y962 site phosphorylation in PTEN-deficient tumors and development of new strategies for PTEN-deficient tumor treatment targeting PI3K beta Y962 site phosphorylation. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 2 is a secondary mass spectrum of a PI3K beta tyrosine phosphorylation site at position 962. In HEK293T-PTEN-KO cells, proteins are enriched by immunoprecipitation after overexpression of PI3K beta, and PI3K beta tyrosine phosphorylation at position 962 is identified by phosphorylation mass spectrometry.

[0028] Figure 2 Conservation of PI3K beta Y962 in different species. The conservation of PI3K beta Y962 was compared and found to be very conserved in human, mouse, rat, bovine, dog, chicken and Xenopus, but not conserved in zebrafish.

[0029] Figure 3 Dot blot detection of PI3K beta Y962 phosphorylation antibody. Control peptide containing Y962 site and phosphorylated Y962 site were synthesized and detected by purified PI3K beta Y962 phosphorylation antibody (E19972(P), E19973(P), E19974(P)) respectively. The results showed that the three antibodies had very good specificity and good antibody titer.

[0030] Figure 4 Western blot detection of PI3K beta Y962 phosphorylation antibody. The purified PI3K beta Y962 phosphorylation antibody was used to detect the Y962 site phosphorylation changes of PI3K beta and its mutants T930A, Y962F, T930A / Y962F in PTEN-WT and PTEN-KO cells. It was found that the Y962 site phosphorylation increased significantly after PTEN knockout, and it could not be detected in the Y962F mutant which simulated non-phosphorylated modification. It was proved that the prepared PI3K beta Y962 phosphorylation antibody had good specificity at the protein level.

[0031] Figure 5 Immunofluorescence detection of PI3K beta Y962 phosphorylation antibody in PTEN-deficient triple-negative breast cancer cell BT549. Vector, WT and Y962F PIK3CB were stably transfected in BT549 shPIK3CB cell line, and immunofluorescence was performed with purified PI3K beta Y962 phosphorylation antibody. It was found that after the Y962 site mutation F simulated the state of non-phosphorylated modification, PI3K beta Y962 phosphorylation antibody could no longer be recognized, proving that the prepared PI3K beta Y962 phosphorylation antibody had good specificity at the protein level.

[0032] Figure 6 Immunofluorescence detection of PI3K beta Y962 phosphorylation antibody after complementation of PI3K beta in PTEN-deficient prostate cancer cell PC3. Vector, WT and Y962F PIK3CB were stably transfected in PC3 shPIK3CB cell line, and immunofluorescence was performed with purified PI3K beta Y962 phosphorylation antibody. It was found that after the Y962 site mutation F simulated the state of non-phosphorylated modification, PI3K beta Y962 phosphorylation antibody could no longer be recognized, proving that the prepared PI3K beta Y962 phosphorylation antibody had good specificity at the protein level.

[0033] Figure 7 Multicolor immunofluorescence co-detection of PI3Kβ Y962 phosphorylation antibody, PTEN, c-Myc in triple negative breast cancer patient tissue specimens. Multicolor immunofluorescence detection of PI3Kβ Y962 phosphorylation antibody, PTEN, c-Myc in PTEN normal and PTEN deleted triple negative breast cancer patient samples. PI3Kβ Y962 phosphorylation and c-Myc protein expression were found to be significantly enhanced in PTEN deleted tumor cells. It is proved that PI3Kβ Y962 phosphorylation antibody can effectively detect the PI3Kβ Y962 phosphorylation level in human tissue specimens, and is significantly related to PTEN deletion and c-Myc amplification. DETAILED DESCRIPTION

[0034] The present application will be described in detail below with reference to the accompanying drawings and specific examples.

[0035] In the following examples, if there is no special description of reagents or processing techniques, it means that they are all conventional commercially available raw materials or conventional processing techniques in the art.

[0036] Example 1: Identification of PI3Kβ Y962 tyrosine phosphorylation site.

[0037] 1. Construction of plenti-CRISPR-sgPTEN plasmid:

[0038] (1) Find guide RNA (gRNA) sequence: find the CDS sequence of the target protein from NCBI, generally select the region within 200 bp downstream of ATG for screening to improve KO efficiency, find suitable and high-score gRNA sequence at http: / / crispr.mit.edu / website, the selected gRNA sequence is SEQ ID No. 3: CAATTCAGGACCCACACGA.

[0039] (2) Design primers: based on the gRNA sequence found in the previous step, generally 19 bp, synthesize the following primers F primer (SEQ ID No. 4): 5'-CACCG-CAATTCAGGACCCACACGA-3', R primer (SEQ ID No. 5): 5'-AAAC-TCGTGTGGGTCCTGAATTG-C-3'.

[0040] (3) Primer annealing: primer annealing was performed according to the following system

[0041]

[0042] Annealing program: 37℃ 30min, 95℃ 5min, turn off annealing 2h.

[0043] (4) Enzymatic digestion of the vector: the pLentiCRISPR-E vector was digested according to the following system

[0044]

[0045] The mixed system was reacted at 37℃ for 30min.

[0046] (5) Ligation: the digested vector and annealed primer were ligated according to the following system, and the ligation was carried out at room temperature for 1h.

[0047]

[0048] (6) The ligation product was transformed into competent E. coli DH5a, and plated on LB plates with ampicillin resistance. Single colonies were picked, and plasmids were extracted after small shaking. Sequencing was performed to obtain the correct plenti-CRISPR-sgPTEN plasmid.

[0049] 2. Construction of PTEN knockout cell line in HEK293T:

[0050] (1) Preparation of cells: 100cm of 2 dishes of HEK293T cells were prepared, and when the cells grew to 60%-70% density, transfection was prepared.

[0051] (2) Cell transfection: first prepare the transfection system, prepare two eppendorf tubes, and add plasmids according to the ratio of psPAX2:VSVG=5:3:2 μg, pLentiCRISPR-sgPTEN according to psPAX2:VSVG=5:3:2 μg, respectively add 1 mL DMEM, mix the plasmids, and then add 16 μL Lipo8000, gently mix, and then add the mixture to two dishes of HEK293T cells; 16h after transfection, replace 10 mL of fresh complete medium.

[0052] (3) Cell infection: 48h after transfection, collect the supernatant of the transfected cells, filter the virus liquid supernatant with a 45 μm filter, and add it to the prepared HEK293T cells with a density of 60%-70%, and add 10 μg / mL of Poybreen to increase the infection efficiency; 24h after infection, replace the fresh culture medium.

[0053] (4) Positive cell screening: 48h after infection, 1 μg / mL puromycin was added for screening, and the screened cells were counted and inoculated in 96-well plates at a density of 1 cell per well; after growth, they were successively subcultured into 24-well plates, 12-well plates, 6-well plates and 60-mm culture dishes. Part of the cells were taken for PCR verification when subcultured into 12-well plates to determine the success of knockout.

[0054] 3. Phosphorylation mass spectrometry to identify new phosphorylation modification sites of PI3Kβ;

[0055] (1) Cell preparation: the above successfully constructed HEK293T pLentiCRISPR-sgCON and HEK293T pLentiCRISPR-sgPTEN cell lines were each prepared with 4 dishes of 150-mm culture dish cells, and PIK3CB-Flag plasmid (15 μg per dish) was transfected with transfection reagent Lipo8000 when the cells grew to about 60% density. The medium was changed after 16h, and the cells were collected after 48h;

[0056] (2) Immunoprecipitation: collect cells, discard culture medium, wash cells with pre-cooled PBS three times, add 0.5% NP40 (IP) lysis buffer containing protease inhibitors and phosphatase inhibitors, 3 mL 0.5% NP40 lysis buffer per dish, add 5 mM iodoacetamide to protect the sulfhydryl group on the cysteine of the protein during cell lysis to prevent oxidation, add 5 mM DTT to neutralize the excess iodoacetamide after lysis for 30 min, and react for 30 min;

[0057] Transfer the reacted lysis buffer to an EP tube, centrifuge at 12000 rpm at 4°C for 15 min, transfer the supernatant to a new EP tube, and obtain the cell lysate. Take out 40 μL of supernatant, add 10 μL of 5×SDS loading buffer, and boil at 100°C for 10 min to obtain the input sample.

[0058] (3) Wash Beads: Add 160 μL of Flag beads to 2 mL of pre-cooled 0.1% NP40 lysis buffer, place on a magnetic stand for 2 min, discard the supernatant, and repeat 3 times. Add the washed Flag beads to 1 mL of 0.1% NP40 lysis buffer, mix well, and evenly distribute them into 2 samples (500 μL each, cut off the front part of the gun when sucking the Flag beads). 4°C drum lysis overnight.

[0059] Place the overnight lysed mixture on a magnetic rack for 2 min and discard the cell lysis buffer. Wash the Flag beads with 1 mL of pre-cooled 0.5% NP40, place them on a magnetic rack for 2 min and discard the supernatant. Repeat 3 times to wash away non-specifically bound proteins and precipitate Flag beads enriched with the target protein. 1×SDS loading buffer can be added for subsequent detection.

[0060] (4) LC-MS detection: The lysed Flag beads were added to 100 μL of 1×SDS loading buffer and subjected to SDS-PAGE electrophoresis. The protein gel after electrophoresis was stained with Coomassie Brilliant Blue. The corresponding band of PI3Kβ protein in the Coomassie Brilliant Blue stained gel was cut off with a blade and placed in an EP tube. This is the prepared sample, which was sent for LC-MS / MS detection. This detection was completed by the public platform of the School of Life Sciences, Fudan University.

[0061] (5) Identification of phosphorylation modification sites: The RAW file generated by LC-MS / MS is searched using Mascot software. The modification parameters are T / S / Y of the three amino acids, which are increased by 80 to obtain the phosphorylation modification sites and secondary mass spectra of the target protein.

[0062] (6) Results Analysis: Two phosphorylation modification sites, T930 and Y962, were identified in the PTEN-KO cell line, but not in the PTEN-WT cells; furthermore, the secondary mass spectra showed a high number of b and y ions, indicating high reliability. Figure 1 The image shown is a secondary mass spectrum of phosphorylation at the PI3KβY962 site.

[0063] Example 2: Preparation and validation of PI3Kβ962 phosphorylated antibody.

[0064] 1. Antigen preparation: This invention uses the 8 amino acid sequences at positions 959-966 of human PI3Kβ to synthesize the phosphorylated modified polypeptide ILT(Yp)DFIH-C, and simultaneously synthesize the control peptide ILTYDFIH-C.

[0065] (1) Analysis of the homology of this site across different species revealed that this peptide and the Y962 site are highly conserved in humans, mice, rats, cattle, dogs, chickens, and Xenopus laevis, but this site is not conserved in zebrafish. Figure 2 );

[0066] (2) Since the peptides are small and not enough to produce immunogenicity in animals, the phosphorylated modified peptides and control peptides are dissolved in 8M urea-PBS buffer solution and coupled to keyhole hemocyanin as antigens for subsequent immunization processes. This increases the immunogenicity of the antigen peptides and avoids the degradation of the peptides.

[0067] 2. Antibody preparation:

[0068] (1) Three experimental Japanese white rabbits were immunized with phosphorylated modified peptide antigen and control peptide antigen, respectively. On day 1, 0.7 mg peptide fragment plus Freund's complete adjuvant was immunized. On days 7, 19, 40, 61 and 82, 0.35 mg peptide fragment plus Freund's incomplete adjuvant was immunized. The rabbits were immunized by subcutaneous multi-point injection.

[0069] (2) On day 94, blood was collected from the carotid artery of the immunized animals. The animals were injected intraperitoneally with 10% chloral hydrate (3 mL / kg). After the carotid artery was dissected, it was obliquely cut, and about 100 mL of whole blood was collected. The blood was allowed to stand at room temperature for 1 hour, then transferred to 4°C and allowed to settle overnight. After centrifugation at 3000 rpm and 4°C for 10 min, the supernatant was transferred to obtain antiserum.

[0070] 3. Antibody purification: Using modified peptides and control peptides as antigens, the antiserum is coupled to magnetic beads for affinity purification to obtain concentrated antibodies. P represents the modified peptide, and C represents the control peptide. The peptide numbers and concentrations are as follows:

[0071] E19972(P) concentration: 2.39 mg / mL; E19972(C) concentration: 1.02 mg / mL;

[0072] E19973(P) concentration: 2.58 mg / mL; E19973(C) concentration: 1.31 mg / mL;

[0073] E19974(P) concentration: 1.74 mg / mL; E19974(C) concentration: 0.86 mg / mL.

[0074] 4. Antibody validation: The titer and specificity of the phosphorylated modified peptide ILT(Yp)DFIH-C and the control peptide ILTYDFIH-C were validated by Dot-blot method.

[0075] (1) The synthesized 0.1 mg phosphorylated modified peptide ILT(Yp)DFIH-C and control peptide ILTYDFIH-C were dissolved in 1 mL ddH2O to obtain a peptide fragment of 100 μg / mL, which was further diluted tenfold to 10, 1, 0.1, 0.01 and 0.001 μg / mL;

[0076] (2) Prepare the NC membrane, and point 1 μL of the six ten-fold gradient diluted phosphorylated modified and control peptides on the membrane in order, dry, block with 3% BSA prepared in TBST at room temperature for 1 h, wash once with PBST for 5 min;

[0077] (3) Prepare the obtained purified antibodies E19972(P), E19973(P) and E19974(P) in the primary antibody diluent at a ratio of 1:100, and add the antibodies to the blocked membrane, and incubate on a shaking table at 4°C overnight;

[0078] (4) Collect the primary antibody, and wash with TBST on a shaking table for 10 min, repeat three times;

[0079] (5) Prepare 5% skimmed milk powder in TBST, and add goat anti-rabbit (G-R) at a ratio of 1:5000, and incubate on a shaking table at room temperature for 1 h;

[0080] (6) Collect the secondary antibody, and wash with TBST on a shaking table for 10 min, repeat three times;

[0081] (7) Color development, slightly drain the liquid on the membrane, select Pierce ECL Plus Western blotting substrate kit to prepare ECL Plus color developing liquid (A:B=1:50 in the kit), evenly pour on the membrane, count for about 3 min, develop on Typhoon, first start the machine, then start the software, set the parameters as ECL Plus / 250v and select the region;

[0082] (8) According to the experimental results as shown in Figure 3 , the three antibodies have very good specificity, recognize the phosphorylated modified polypeptide but not the control polypeptide, and the three antibodies can recognize at least 1 ng of peptide amount, the antibody titers of E19972(P) and E19974(P) are slightly stronger, and the antibody titer of E19973(P) is slightly weaker, and the overall titer is very good.

[0083] Example 3: Western Blot identification of PI3K beta 962 phosphorylation antibody.

[0084] 1. Construct a mutant simulating a non-modified state:

[0085] (1) Primer design: Construct mutant PI3KCB-T930A-Flag, PI3KCB-Y962F-Flag, PI3KCB-T930A / Y962F-Flag plasmids simulating non-modified state, design primers based on Novagen site-directed mutagenesis kit instructions, the principle is that the 5' end of the forward and reverse primers contains a 15-21 bp reverse complementary region (GC content 40-60%); each primer is mutated at the site to the 3' end region Tm value higher than 60°C is best, the primer sequences of the above two sites PI3KCB-Flag point mutation plasmid are as follows:

[0086]

[0087] (2) PCR amplification reaction: Wild-type PI3KCB-Flag plasmid as template for amplification, the program is 95°C for 5 min; 95°C for 15 s, 50°C-60°C for 15 s, 72°C for 30 s / kb, 30-35 cycles; 72°C for 10 min, 4°C for 5 min;

[0088] (3) Remove the methylated template plasmid: add 1 μL Dpn1 enzyme to the reaction system of (2), 37°C for 1 h, agarose gel electrophoresis to identify the PCR product band, and conduct gel recovery;

[0089] (4) Product recombination: recombine as follows, 37°C for 30 min, then immediately put on ice;

[0090]

[0091] Transformation, plating, plasmid extraction, sequencing, and correct PI3KCB-T930A-Flag, PI3KCB-Y962F-Flag plasmids are obtained;

[0092] (5) PI3KCB-T930A / Y962F-Flag plasmid is a template for sequencing successful PI3KCB-T930A-Flag plasmid, using Y962F primers to repeat the above mutation steps, and finally obtaining a double-site mutant plasmid with successful sequencing.

[0093] 2. Expression and purification of mutant proteins:

[0094] (1) Cell transfection: the above constructed HEK293T-WT and HEK293T-PTEN KO cell lines are subcultured into 5 60 cm dishes; respectively transfect 2 μL Vector, PI3KCB-T930A-Flag, PI3KCB-Y962F-Flag, PI3KCB-T930A / Y962F-Flag plasmids, change the liquid 16 h after transfection, and collect the cells 48 h after transfection.

[0095] (2) Immunoprecipitation: collect cells, discard the culture medium, wash the cells with pre-cooled PBS three times, add 0.5% NP40 (IP) lysis solution containing protease inhibitors, 500 mL of lysis solution per dish, 4°C refrigerator lysis for 30 min;

[0096] (3) Transfer the lysis solution to an EP tube, centrifuge at 12000 rpm at 4°C for 15 min, transfer the supernatant to a new EP tube, and obtain the cell lysate. Take out 40 μL of the supernatant, add 10 μL of 5x SDS loading buffer, and boil at 100°C for 10 min to obtain the input sample.

[0097] (4) Wash the beads: add 80 μL of Flag beads to 1 mL of pre-cooled 0.5% NP40, place on a magnetic stand for 2 min, discard the supernatant, and repeat 3 times. Add the washed Flag beads to 1 mL of lysis solution, mix well, and evenly distribute into ten samples (100 μL each, cut off the front part of the gun when sucking the Flag beads). 4°C drum IP overnight.

[0098] (5) Place the IP overnight mixture on a magnetic stand for 2 min, discard the cell lysate; wash the Flag beads with 1 mL of pre-cooled 0.1% NP-40, place on a magnetic stand for 2 min, discard the supernatant, repeat 3 times, and wash away the non-specifically bound proteins. The precipitate is the Flag beads enriched with the target protein, which can be added with 1x SDS loading buffer for subsequent WB detection.

[0099] 3. Western Blot detection:

[0100] According to Figure 4The results show that in HEK293T-WT cells, no phosphorylation of PI3K beta WT, T930A, Y962F, T930A / Y962F is detected. In HEK293T-PTEN-KO cell lines, PI3K beta WT and T930A are detected to have a significant increase of PI3K beta Y962 phosphorylation antibody (E19974(P)), and in PI3K beta Y962F, T930A / Y962F mutants simulating non-modified state mutations, no PI3K beta Y962 phosphorylation antibody is detected. It is indicated that after the Y962 site is mutated F, the PI3K beta Y962 phosphorylation antibody cannot be recognized, which represents that no phosphorylation modification can occur. It is proved that the prepared PI3K beta Y962 phosphorylation antibody has good specificity at the protein level, and can be well applied to Western Blot experiment to quantitatively and semi-quantitatively analyze and compare the levels of PI3K beta Y962 phosphorylation antibody under different conditions.

[0101] Example 4: Immunofluorescence identification of anti-PI3K beta 962 tyrosine phosphorylation antibody.

[0102] 1. Construction of shPIK3CB stable knockdown cell lines of BT549 and PC3:

[0103] (1) Construction of pLKO-shPIK3CB plasmid: In the MERCK MISSION shRNA library, a verified sequence targeting 5'UTR was searched, and the primers were designed and synthesized as follows, F primer (SEQ ID No. 10): 5'-CCGGCGACAAGACTGCCGAGAGATTCTCGAGAATCTCTCGGCAGTCTTGTCGTTTTTG-3',

[0104] R primer (SEQ ID No. 11):

[0105] 5'-AATTCAAAAACGACAAGACTGCCGAGAGATTCTCGAGAATCTCTCGGCAGTCTTGTCG-3', the primer annealing, vector digestion and ligation were carried out according to the system and steps in Example 1-1-(3);

[0106] The pLKO vector was digested according to the following system:

[0107]

[0108] The mixed system was digested at 16°C overnight.

[0109] According to the system and steps in Example 1-1 (5), the ligation was performed at room temperature for 1 h, and then the ligation product was converted, plated, and single colonies were picked. After a small shake, plasmid was extracted, sequenced, and the pLKO-shPIK3CB plasmid with correct sequencing was obtained.

[0110] (2) Construction of stable knockdown cell lines: According to the method and steps of Example 1-2, first transfection was performed in HEK293T cells, in which shCON was added according to the ratio of psPAX2:VSVG = 5:3:2 μg, and shPIK3CB was added according to the ratio of psPAX2:VSVG = 5:3:2 μg; then 48 h after the supernatant was collected, it was infected into BT549 and PC3 cell lines, and then puromycin was added for screening, and stable knockdown shPIK3CB cell strains were obtained.

[0111] 2. Construction of stable PIK3CB WT and Y962 mutant cell lines in BT549 shPIK3CB and PC3 shPIK3CB cell lines:

[0112] According to the method and steps of Example 1-2, the PIK3CB-Flag and PIK3CB-Y962-Flag constructed in Example 3-1 and the virus packaging plasmid psPAX2, VSVG were transfected together in HEK293T cells to produce the corresponding virus; 48 h after transfection, the supernatant was collected and infected into the constructed BT549 shPIK3CB and PC3 shPIK3CB cell lines, and 48 h later, puromycin was added for screening to obtain BT549 shPIK3CB and PC3 shPIK3CB stable PIK3CB WT and Y962 mutant cell lines.

[0113] 3. Immunofluorescence detection:

[0114] (1) Preparation of samples: Add cell culture slides to 12-well plates, and inoculate BT549 shPIK3CB (Vector, PIK3CB-Flag, and PIK3CB-Y962-Flag) three cell lines into each well, 10 5 cells per well. When the cells are digested, try to blow them apart to avoid the presence of cell clumps that interfere with the shooting of immunofluorescence photos. Culture for 24 h to make them fully adhere;

[0115] (2) Fixing of samples: Remove the culture medium from the fully adherent cells, wash with PBS twice, and add 4% paraformaldehyde for room temperature fixation for 15 min;

[0116] (3) Permeabilization: aspirate the paraformaldehyde, wash 3 times with PBS for 3 min each, add 0.2% Triton X-100 (PBST prepared) to permeabilize the cell membrane for 5 min; aspirate the Triton X-100, wash 3 times with PBS for 3 min each;

[0117] (4) Blocking: add 5% BSA or goat serum (PBST prepared) to block for 1 hr at room temperature, aspirate the blocking solution, wash 3 times with PBS for 3 min each;

[0118] (5) Incubate the primary antibody: prepare the PI3K beta Y962 phosphorylation antibody (E19974(P) antibody) at a ratio of 1: 100 in 5% goat serum, add 50 μL per well on the glass slides, cover with the round cover pieces cut from the parafilm, to prevent the antibody from evaporating, incubate overnight at 4°C;

[0119] (6) Incubate the secondary antibody: aspirate the primary antibody, wash 3 times with PBS for 3 min each, add the Goat-anti-Rabbit Alexa Fluor 488 fluorescent secondary antibody prepared in 5% goat serum, incubate for 1 hr at room temperature in the dark;

[0120] (7) Nuclei staining: aspirate the secondary antibody, wash 3 times with PBS for 3 min each; dilute DAPI in ddH2O at a ratio of 1: 1000, incubate for 10 min at room temperature;

[0121] (8) Mounting: aspirate the DAPI, wash 3 times with PBS for 3 min each; gently pick out the cell climbing sheets in the 12-well plate with tweezers, invert on the glass slides with water-soluble mounting medium, add nail polish around to fix, which is the successful mounting, can be stored in the dark or used for fluorescence microscope photography;

[0122] (10) Result analysis: in the BT549 shPIK3CB-Vector, the PI3K beta Y962 phosphorylation antibody staining is light, in the shPIK3CB-WT cell line (such as Figure 5 ), the fluorescence intensity of the PI3K beta Y962 phosphorylation antibody staining is significantly increased, in the shPIK3CB-Y962F cell line, the fluorescence intensity of the PI3K beta Y962 phosphorylation antibody staining is reduced to a similar level as the shPIK3CB-Vector. After the Y962 site mutation F simulates the non-phosphorylated modification state, the PI3K beta Y962 phosphorylation antibody can no longer be recognized, proving that the prepared PI3K beta Y962 phosphorylation antibody has good specificity at the protein level and can be well applied to the immunofluorescence experiment to compare the level of PI3K beta Y962 phosphorylation antibody under different conditions. The results in the PC3 shPIK3CB cell line are similar to those in the BT549 (such as Figure 6 ).

[0123] Example 5: Multicolor immunofluorescence identification of PI3K beta Y962 phosphorylation antibody.

[0124] The experiment was performed using a four-color multiplex fluorescence multiplex staining kit (absin #abs50012).

[0125] 1. Paraffin section dewaxing:

[0126] (1) Select PTEN wild type and mutant breast cancer tissue sections, immerse in fresh xylene for 10 min, repeat 3 times;

[0127] (2) Immerse in different concentrations of ethanol: the selected ethanol concentration and immersion time are 100% ethanol for 5 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min. Wash with sterile water for 1 min, repeat 3 times;

[0128] (3) Immerse in 10% neutral formalin for 10 min, wash with sterile water for 1 min, repeat 3 times, to obtain dewaxed hydrated glass slides.

[0129] 2. Microwave antigen retrieval:

[0130] (1) Place the dewaxed and hydrated glass slides from step 1 in the repair cup, immerse in the antigen retrieval solution (EDTA pH 9.0), place the repair cup in the microwave oven, boil at high heat, and maintain at low heat for 15 min;

[0131] (2) Remove the repair cup and cool it to room temperature naturally.

[0132] 3. Blocking: Remove the residual wash solution on the glass slide, circle the sample area on the glass slide with the histo pen, add 3% hydrogen peroxide blocking solution, cover the sample area, and incubate at room temperature for 10 min.

[0133] 4. Primary antibody incubation: Remove the blocking solution on the glass slide, add 10% goat serum diluted primary antibody, shake and incubate overnight; wash the glass slide with 1x TBST buffer for 3 min, repeat 1 time.

[0134] 5. Secondary antibody incubation: Remove the residual wash solution on the glass slide, directly add HRP secondary antibody working solution, immerse the sample area, incubate at room temperature for 30 min; wash the glass slide with 1x TBST buffer for 3 min, repeat 1 time.

[0135] 6. Fluorescent dye amplification signal: Remove the residual washing solution on the slide, and use a pipette to add 100 μL of 1 x dye working solution (diluted by 1:100 using signal amplification solution) to the sample area of the slide, immerse the sample area, and incubate at room temperature for 10 min. Wash the slide with 1 x TBST buffer, and immerse the slide at room temperature for 3 min. Repeat 3 times.

[0136] 7. Microwave the slide, and naturally cool to room temperature. Wash the slide with sterilized water once, and immerse the slide with 1 x TBST buffer for 2 min. End the single staining, and seal the slide for observation or add subsequent staining. Start the subsequent staining from step 3 after the blocking. Each round of primary antibody and secondary antibody staining corresponds to: (1) PTEN stained with TSA520; (2) c-Myc stained with TSA570; (3) P-P110β Y962 stained with TSA650. After the end of each round of staining, the staining condition can be confirmed by a fluorescent microscope.

[0137] 8. Stain the nucleus and seal the slide: Add 1 x DAPI staining solution to the sample, immerse the sample area, and incubate at room temperature for 5 min. Wash the slide with 1 x TBST buffer 3 times, each for 2 min. Then add an anti-fluorescence quenching sealing agent, and seal the slide with a cover glass. Scan the stained tissue slice by a fluorescent scanner.

[0138] 9. Result analysis: As shown in Figure 7 , the levels of c-Myc and P-p110β Y962 are low in the triple-negative breast cancer samples with wild-type PTEN, but the levels of c-Myc and P-p110β Y962 significantly increase in the triple-negative breast cancer samples with mutant PTEN.

[0139] SEQ ID NO. 1:

[0140]

[0141] SEQ ID NO. 2:

[0142]

Claims

1. A method for preparing a polyclonal antibody targeting the phosphorylation of the tyrosine at position 962 of human PI3Kβ, characterized in that, The method comprises the following steps: S1. Synthesizing an antigen peptide of a tyrosine phosphorylation site at position 962 of an amino acid sequence shown in SEQ ID NO. 2, and coupling a keyhole limpet hemocyanin carrier protein as a complete antigen; the antigen peptide is ILT(Y-p)DFIH-C; S2. Collecting serum after applying the complete antigen in S1 and an adjuvant to an immunized animal to obtain antiserum; S3. Purifying the antiserum in S2 to obtain a PI3Kβ Y962 phosphorylation antibody.

2. The method for preparing a polyclonal antibody targeting the phosphorylation of the tyrosine at position 962 of human PI3Kβ according to claim 1, characterized in that, In S2, the adjuvant is Freund's complete adjuvant and Freund's incomplete adjuvant, and the immunized animal is a test-level Japanese white rabbit.

3. The method for preparing a polyclonal antibody targeting the phosphorylation of the tyrosine at position 962 of human PI3Kβ according to claim 1, characterized in that, In S3, the antiserum purification method is as follows: first, affinity purification is performed by using an affinity chromatography column cross-linked with a phosphorylated polypeptide, then, an antibody component cross-reacting with a corresponding non-phosphorylated polypeptide is removed by using an affinity chromatography column cross-linked with a non-phosphorylated polypeptide, then, an antibody component against the keyhole limpet hemocyanin carrier protein is removed by using an affinity chromatography column cross-linked with the keyhole limpet hemocyanin, finally, non-antibody protein components are removed by dialysis and concentration, and a purified PI3Kβ Y962 phosphorylation antibody is finally obtained.

4. A polyclonal antibody targeting the phosphorylation of the tyrosine at position 962 of human PI3K beta, characterized in that, The method is obtained by any one of claims 1-3.

5. Use of a polyclonal antibody targeting human PI3Kβ tyrosine phosphorylation at position 962 in the preparation of a detection reagent for judging PTEN-deficient tumors, characterized in that, the detection reagent is used for detecting the phosphorylation level of PI3Kβ at position Y962 in human breast cancer with PTEN deficiency; and / or the detection reagent is used for detecting the phosphorylation level of PI3Kβ at position Y962 in human prostate cancer with PTEN deficiency.

Images