Separated ubiquitin specific peptidase 30 mutant, nucleic acid, antibody and application

Abstract

The invention belongs to the field of biological medicine, and relates to a separated ubiquitin specific peptidase 30 mutant, nucleic acid, an antibody and application. The ubiquitin specific peptidase 30 mutant is a USP30S104A mutant, and the ubiquitin specific peptidase 30 mutant has an amino acid sequence as shown in SEQ ID NO: 1. The invention discovers a brand new phosphorylation site (104 site) on USP30 protein, and provides a breast cancer gene therapy and chemotherapy drug resistance prediction method based on the mutant and phosphorylation detection by researching a USP30S104A mutant nuclear localization regulation mechanism.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an isolated ubiquitin-specific peptidase 30 mutant, nucleic acid, antibody and application. Background Art

[0002] Ubiquitin-specific peptidase 30 (USP30) is a key member of the deubiquitinating enzyme (DUB) family and belongs to the USP subfamily. Classic studies have shown that USP30 is primarily localized to the outer mitochondrial membrane. By specifically removing ubiquitinated modifications from mitochondrial proteins (such as Parkin and MFN1 / 2), it negatively regulates the PINK1 / Parkin-mediated mitophagy pathway, playing a key role in maintaining mitochondrial homeostasis, energy metabolism, and neuronal survival. Its dysfunction is closely associated with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Furthermore, USP30 is abnormally overexpressed in various tumors (such as breast and lung cancer) and may promote tumor cell survival by stabilizing anti-apoptotic proteins (such as members of the BCL-2 family).

[0003] In current research on the regulation of drug resistance in tumors and pathogens, significant gaps remain in the dynamic regulation of phosphorylation of the deubiquitinase USP30. As a key regulator of the mitochondrial quality control system, USP30, through its deubiquitinase activity, participates in the regulation of important biological processes such as mitochondrial autophagy, apoptosis resistance, and drug efflux. Although studies have shown that multiple kinases (such as PINK1 and AKT) may mediate phosphorylation modifications at specific USP30 sites, effective strategies to predict or reverse drug resistance by regulating USP30 phosphorylation status are currently unavailable. Summary of the Invention

[0004] The purpose of the present invention is to provide a USP30 S104A mutant, and through the study of the nuclear localization regulatory mechanism of the mutant, further provide a breast cancer gene therapy and chemotherapy resistance prediction method based on the mutant and phosphorylation detection.

[0005] To achieve the above objectives, the first aspect of the present invention provides an isolated ubiquitin-specific peptidase 30 mutant, wherein the ubiquitin-specific peptidase 30 mutant is a USP30 S104A mutant having an amino acid sequence as shown in SEQ ID NO: 1. The mutant has enhanced nuclear localization ability compared to wild-type USP30.

[0006] MLSSRAEAAMTAADRAIQRFLRTGAAVRYKVMKNWGVIGGIAAALAAGIYVIWGPITERKKRRKGLVPGLVNLGNTCFMNSLLQGLSACPAFIRWLEEFTSQYARDQKEPPSHQYLSLTLLHLLKALSCQ EVTDDEVLDASCLLDVLRMYRWQISSFEEQDAHELFHVITSSLEDERDRQPRVTHLFDVHSLEQQSEITPKQITCRTRGSPHPTSNHWKSQHPFHGRLTSNMVCKHCEHQSPVRFDTFDSLSLSIPAATW GHPLTLDHCLHHFISSESVRDVVCDNCTKIEAKGTLNGEKVEHQRTTFVKQLKLGKLPQCLCIHLQRLSWSSHGTPLKRHEHVQFNEFLMMDIYKYHLLGHKPSQHNPKLNKNPGPTLELQDGPGAPTPV LNQPGAPKTQIFMNGACSPSLLPTLSAPMPFPLPVVPDYSSSTYLFRLMAVVVHHGDMHSGHFVTYRRSPPSSARNPLSTSNQWLWVSDDTVRKASLQEVLSSSAYLLFYERVLSRMQHQSQECKSEE(SEQ IDNO: 1).

[0007] The USP30 S104A mutant can be obtained by mutating the serine at position 104 of the wild-type USP30 to alanine. The mutation can be obtained by a point mutation method well known to those skilled in the art.

[0008] The second aspect of the present invention provides a nucleic acid encoding the ubiquitin-specific peptidase 30 mutant.

[0009] A third aspect of the present invention provides a recombinant vector comprising the aforementioned nucleic acid. Specifically, the vector may be an adenoviral vector, a lentiviral vector, or a plasmid. The recombinant vector may be used as a gene therapy vector.

[0010] The fourth aspect of the present invention provides an antibody that specifically recognizes the phosphorylation state of site 104 of the USP30 protein, that is, specifically recognizes the USP30 protein in which the serine at site 104 is a phosphorylated serine; the antibody can be a monoclonal antibody or a polyclonal antibody.

[0011] Since the antibody can specifically recognize the phosphorylation state of site 104 of the USP30 protein, it can be used to prepare a reagent for detecting the phosphorylation level of USP30 in breast cancer cells.

[0012] The specific antibodies of the present invention can be prepared by immunizing animals. According to a preferred embodiment, the antibodies are obtained by the following preparation method:

[0013] (1) Antigen design and preparation:

[0014] Based on the S104 phosphorylation site of the USP30 protein, a specific phosphorylated peptide was designed and synthesized. The sequence of the phosphorylated peptide was EFTSQYSRDQKEC, in which the serine at position 7 was phosphorylated. The phosphorylated peptide was coupled to a carrier protein by chemical cross-linking to form a complete antigen.

[0015] (2) Animal immunization:

[0016] Japanese big-eared rabbits are immunized with the antigen obtained in step (1), and the immunization method is as follows: first immunization: 0.2-0.8 mg complete antigen and complete Freund's adjuvant, multiple subcutaneous injections; multiple booster immunizations: performed every 14 days, using incomplete Freund's adjuvant, with a dose of 0.2-0.8 mg / time; the titer of the antibody obtained by immunization is detected and the specificity of the antibody is confirmed;

[0017] (3) Antibody purification:

[0018] According to a more specific embodiment, the antibody is obtained by the following preparation method:

[0019] A fifth aspect of the present invention provides a reagent for predicting chemotherapy resistance in breast cancer patients, the reagent comprising: the above-mentioned antibody that specifically recognizes the phosphorylation state of site 104 of the USP30 protein and a total USP30 antibody;

[0020] The antibody that specifically recognizes the phosphorylation state of the USP30 protein site 104 is used to detect the level of USP30 in the patient sample with S104 being phosphorylated.

[0021] The total USP30 antibody is used to detect the level of total USP30 in patient samples. The total USP30 antibody can be commercially obtained, such as from Abcam (ab314749).

[0022] The ratio of the level of USP30 in the phosphorylated state of S104 (P-USP30 S104) to the total USP30 antibody was obtained to determine whether the patient had the risk of chemotherapy resistance.

[0023] The sixth aspect of the present invention provides the use of a ubiquitin-specific peptidase 30 mutant in the preparation of a drug for inhibiting breast cancer stem cell characteristics and / or enhancing the chemotherapy sensitivity of breast cancer cells. The mutant achieves the tumor suppressor function by enhancing the nuclear localization of USP30.

[0024] In gene therapy, delivery of the USP30 S104A mutant can enhance nuclear localization, thereby suppressing tumor stemness and drug resistance. Anti-P-USP30 S104 antibodies can also be used to monitor phosphorylation levels and guide chemotherapy regimen selection.

[0025] The beneficial technical effects of the present invention are:

[0026] The inventors of the present invention discovered a new phosphorylation site (site 104) on the USP30 protein and found that phosphorylation of this site is associated with the nuclear localization of USP30. They further provided a USP30 S104A mutant. Subcellular fractionation and immunoblotting demonstrated that the USP30 S104A mutant was enriched in nuclear components; stem cell sphere formation experiments showed that the USP30 S104A mutant significantly reduced the number and size of spheres and decreased the expression of stemness markers, indicating that the USP30 S104A mutant inhibited tumor stemness; CCK-8 experiments demonstrated that the USP30 S104A mutant reduced the IC of doxorubicin. 50 The value was decreased, indicating that the USP30S104 mutant could enhance chemotherapy sensitivity; immunoblotting of phosphorylated antibody P-USP30 S104 proved that the phosphorylated protein was mainly in the cytoplasm. Since the phosphorylation status of USP30 is related to chemotherapy sensitivity, the ratio of P-USP30 S104 antibody level to total USP30 antibody level can be used to determine whether the patient has the risk of chemotherapy resistance.

[0027] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0029] Figure 1 Shown is the subcellular localization of USP30 in MDA-MB-231 cells.

[0030] Figure 2 Shown are the subcellular distribution and expression of endogenous USP30 in breast cancer cell lines (a) and non-breast cancer cell lines (b).

[0031] Figure 3 It was shown that phosphorylation of the S104 site of the USP30 protein regulates the nuclear translocation of USP30.

[0032] Figure 4 Shown that the USP30 S104A mutant suppresses breast cancer stem cell properties.

[0033] Figure 5Shown that the USP30 S104 mutant suppresses drug resistance in breast cancer cells.

[0034] Figure 6 Shown are the results of immunoblotting experiments using the phosphorylation antibody P-USP30 S104. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0036] Example 1 Verification of USP30 Nuclear Localization and S104A Mutation

[0037] 1. Dual localization of USP30 in mitochondria and nucleus

[0038] Subcellular fractionation technology was used to separate the cytoplasmic, nuclear and mitochondrial fractions, and Western blotting experiments were used to detect the distribution and expression of USP30 in different subcellular fractions.

[0039] Cell component separation experimental steps: Use Abcam nuclear, cytoplasmic and mitochondrial isolation kits according to the instructions. The specific steps are as follows:

[0040] (1) Cell collection: Due to the addition of mitochondrial fraction separation, each sample needs to collect at least 10 cm culture dish of cells to meet the needs of subsequent testing. Cells are collected by 0.25% trypsin digestion in the same manner as for nuclear and cytoplasmic separation.

[0041] (2) Cytoplasmic extraction: Resuspend the cell pellet in 1× buffer A and count the cells to adjust the concentration to 6.6×10 6 Cells / mL. Dilute Lysis Buffer I 1000-fold in 1× Buffer A to prepare Lysis Buffer B. Transfer a certain amount of cell suspension to a new test tube. Add an equal volume of Lysis Buffer B to the cell suspension. Mix with a pipette, incubate the sample for 7 minutes, and centrifuge the sample at 5,000 rpm for 1 minute at 4°C. Carefully remove all supernatant and transfer it to a new set of test tubes, and store the pellet on ice. Re-centrifuge the supernatant at 10,000 rpm for 1 minute. The supernatant collected again is the cytoplasmic component.

[0042] (3) Mitochondrial extraction: The "cells" (containing mitochondria and nuclei) from which the cytoplasm has been removed are suspended in 1× buffer A. Lysis solution II is diluted 25-fold in 1× buffer A to prepare lysis solution C. The exact same volume of buffer C is added to the cell suspension. Mix by pipetting. After lysing the sample for 10 minutes, centrifuge the sample at 4°C, 5,000 rpm for 1 minute. Carefully remove all the supernatant and transfer it to a new set of test tubes, and store the precipitate on ice. The supernatant is re-centrifuged at 10,000 rpm for 1 minute to obtain a supernatant containing mitochondrial proteins and a precipitate containing nuclear proteins.

[0043] (4) Preparation of nuclear fraction: Resuspend the cell pellet in 1× buffer A to prepare the nuclear fraction.

[0044] Immunoblotting: Cytoplasmic, mitochondrial and nuclear fractions were separated by SDS-PAGE protein gel electrophoresis, and the cytoplasmic, mitochondrial and nuclear fractions were distinguished by GAPDH, VDAC1 and Lamin B antibodies, respectively.

[0045] (1) Protein extraction: Prepare a lysis buffer containing protease and phosphatase inhibitors in advance, collect and lyse the cells. The lysis process must ensure that the cells are kept at low temperature and fully exposed to the lysis buffer. After 15-30 minutes of lysis, centrifuge at 4°C, 10,000-12,000 rpm for 10-15 minutes. Use a pipette to remove the sedimented gene clusters at the bottom and collect the supernatant.

[0046] (2) Protein quantification: Use lysis buffer to dilute the standard protein in the Bradford protein assay kit into 5 concentration gradients (0.00 μg / μL, 1.25 μg / μL, 2.50 μg / μL, 5.00 μg / μL). Take 1 μL of the sample to be tested or the 5 concentration gradients of standard protein and mix them with 200 μL of Coomassie Brilliant Blue G-250 dye. Use a 96-well plate to measure the absorbance at 590 nm on a microplate reader. Set up 3 replicates for each sample. Draw a concentration-absorbance standard curve based on the absorbance results to calculate the sample concentration.

[0047] (3) Protein denaturation: The protein solution in (1) was mixed with 5× Loading Buffer (4:1), the EP tube cap was fixed with an EP tube explosion-proof clamp, and then placed in a metal bath and heated at 100°C for 7 minutes to denature the protein.

[0048] (4) Gel preparation: After the above steps are successfully completed, 10%, 12.5%, and 7.5% SDS-PAGE gels are prepared using a PAGE gel rapid preparation kit.

[0049] (5) Sample loading: Calculate the required sample volume for 40 μg of protein based on the concentration measured in step (2) and add the corresponding protein sample solution to the sample wells of the SDS-PAGE gel. Select two sample wells for each gel and add 3 μL of Protein Maker.

[0050] (6) Electrophoresis: The electrophoresis was performed at a constant voltage of 150 V. The end time of electrophoresis was determined based on the protein separation results.

[0051] (7) Transfer: Transfer the membrane at a constant current of 220 mA for about 2 hours. During the transfer process, the temperature must be continuously lowered to prevent the transfer device from being too hot, until proteins of different molecular weights are transferred to the nitrocellulose membrane.

[0052] (8) Blocking: Blocking solution (5% skim milk powder) at room temperature; 1 h.

[0053] (9) Incubation with primary antibodies: 2 h at room temperature or overnight at 4°C. The antibodies used include: anti-USP30 (#ab314749, Abcam), anti-Lamin A / C (#4777, CST), anti-Tubulin (#2125, CST), anti-VDAC1 (#4866, CST), and the antibody dilution factor is 1000.

[0054] (10) Incubation with secondary antibodies: 1 h at room temperature, with HRP-goat anti-rabbit IgG (H+L) (#5220-0336, Seracare-KPL) and HRP-goat anti-mouse IgG (H+L) (#5220-0341, Seracare-KPL), diluted 5000.

[0055] (11) Imaging: Using luminescent liquid, developer, and fixer, darkroom imaging or iBright TM Protein expression was detected using the CL750 imaging system.

[0056] Figure 1 The subcellular localization of USP30 in MDA-MB-231 cells is shown: (a) Endogenous USP30 expression in the cytoplasm (C), mitochondria (M), and nucleus (N) at different exposure times; (b) Endogenous USP30 expression at different nuclear protein loading levels. The results show that USP30 is primarily localized in the mitochondrial fraction, but when the exposure time of the immunoblot band is prolonged or the amount of nuclear protein loaded is increased, USP30 protein bands can also be detected in the nuclear fraction.

[0057] Figure 2The subcellular distribution and expression of endogenous USP30 in breast cancer cell lines (a) and non-breast cancer cell lines (b) are shown; Cyto, cytoplasm, Nuc, nucleus. The results show that USP30 is nuclear-localized in various breast cancer cell types.

[0058] Figure 3 Figure 2 shows that phosphorylation of the S104 site of the USP30 protein regulates USP30 nuclear translocation: USP30-His (WT), USP30-S104A-His, and USP30-S104E-His were overexpressed in MDA-MB-231 (a) and HEK293 cells (b), and the cytoplasmic and nuclear fractions were separated to examine the subcellular distribution of USP30. The results showed that the S104A mutant was enriched in the nuclear fraction.

[0059] 2. Phosphorylation of serine 104 of USP30 protein regulates nuclear translocation of USP30

[0060] Ser104 of wild-type USP30 was mutated to alanine A and glutamic acid E to mimic the dephosphorylation and phosphorylation states of this site.

[0061] Construction of mutant plasmids: The USP30 S104A mutation mutates serine at position 104 to alanine (A), and its sequence is shown in SEQ ID NO: 1; the S104E mutation mutates serine at position 104 to glutamic acid (E). Both mutant plasmids were constructed using pcDNA3.1-USP30 as a template by single-site mutagenesis. USP30 WT (wild type), S104A, and S104E were overexpressed in MDA-MB-231 and HEK293 cells, respectively, and nuclear fractions were isolated and analyzed by immunoblotting. The method included:

[0062] 48 hours after cells were transfected with wild-type USP30, S104A, or S104E mutant plasmids, nuclear and cytoplasmic fractions were separated according to the protocol provided by the Biyuntian Nuclear / Cytoplasmic Fractionation Kit as follows:

[0063] (1) Cell collection: Isolation of nuclear and cytoplasmic proteins requires a larger number of cells than conventional protein extraction. Generally, the amount of cells in a 6 cm culture dish is appropriate. Remove the old cell culture medium and rinse the cells 2-3 times with PBS before digesting them. Terminate digestion when the cells detach from the cell wall and collect the cell pellet by centrifugation (800 rpm, 3 min).

[0064] (2) Cytoplasmic protein extraction: Add protease inhibitors to pre-cooled cytoplasmic protein extraction reagent A and take 200 μL to resuspend and lyse the cells. After fully lysing on ice for 10 minutes, add 10 μL of phase separation promoter B and vortex to mix. After the lysate system stands on ice for 1 minute, centrifuge at 4°C (12,000 rpm, 5 minutes). Collect the supernatant and mark it as the cytoplasmic protein fraction.

[0065] (3) Nuclear protein extraction: The remaining precipitate was lysed with nuclear lysis buffer containing protease inhibitors, and centrifuged again at 4°C and 12,000 rpm to obtain the supernatant to obtain the nuclear fraction.

[0066] (4) Immunoblotting: The cytoplasmic and nuclear fraction proteins were quantified, and after adding Loading Buffer (5×), the tube cap was fixed with an EP tube explosion-proof clamp and placed in a 100°C metal bath for 7 min. Protein samples were separated by SDS-PAGE protein gel electrophoresis, and nuclear and cytoplasmic proteins were distinguished using Lamin A / C antibodies (#4777, CST) and GAPDH (#10494-1-AP, Proteintech), respectively.

[0067] The results showed that the expression level of S104A in the cell nucleus was significantly higher than that of WT and S104E mutation ( Figure 3 ).

[0068] The above experiments proved that USP30 was enriched in the nuclei of breast cancer cells, and its nuclear localization was directly related to dephosphorylation of the S104 site (S104A mutation).

[0069] Example 2 USP30 S104A mutant inhibits breast cancer stem cell characteristics

[0070] The USP30 S104A mutant was used to further explore the relationship between USP30 nuclear localization and its regulation of breast cancer stem cell-like behavior, including:

[0071] MDA-MB-231 cells stably transfected with empty vector, wild-type USP30 or S104A mutation were serum starved for 48 h using serum-free DMEM / F12 1:1 medium. After the cell density reached 80%, the cells were trypsinized and digested with DMEM / F12 1:1 complete medium to terminate the digestion. The cells were resuspended in serum-free DMEM / F12 1:1 medium. DMEM / F12 1:1 medium was added with bFGF (20 ng / mL), EGF (20 ng / mL) and b27 (2%) to prepare a special medium for inducing stem cells. The cells were diluted to 10 4 / mL (cell number / medium volume) density and transfer to a non-adhesive 6cm culture dish or 6-well plate. Depending on the growth status of the stem cell spheres, change the culture medium every 72 hours. It takes about 14 days to successfully induce stem cell spheres. Randomly select 5 fields of view and take pictures under a microscope to calculate the number of cell spheres with a diameter greater than 60μm and the sphere formation rate. Collect the cell stem cell spheres, lyse the cells, and detect the expression of stem cell markers by protein immunoblotting. The antibodies used include: anti-P-gp (#22336-1-AP, Proteintech), anti-ALDH1A1 (#15910-1-AP, Proteintech), anti-OCT4 (#11263-1-AP, Proteintech), anti-CD44 (#15675-1-AP, Proteintech), anti-OCT4 (#11263-1-AP, Proteintech), anti-USP30 (#ab314749, Abcam), and anti-β-actin (#60008-1-lg, Proteintech).

[0072] Figure 4 Figure 3 shows the results of the USP30 S104 site affecting the characteristics of breast cancer stem cells: (a) After stably overexpressing Vector, USP30, and USP30-S104A in MDA-MB-231 cells, a stem cell sphere formation experiment and the number of stem cell spheres were counted; (b) After stably overexpressing Vector, USP30, and USP30-S104A in MDA-MB-231 cells, stem cells were induced into spheres, and the stem cell spheres were collected to extract protein components, and the differences in the expression of stem cell stemness marker proteins were detected by immunoblotting.

[0073] The results showed that the USP30 S104A mutant significantly reduced the number and size of stem cell spheres and decreased the expression of stemness markers. Compared with wild-type USP30, the USP30 S104A mutant had a more significant inhibitory effect on breast cancer stem cell sphere formation and stemness gene expression.

[0074] Example 3 USP30 S104 mutant enhances chemotherapy sensitivity

[0075] The USP30-S104A mutant was used to explore the relationship between the nuclear localization of USP30 and breast cancer resistance.

[0076] MDA-MB-231 cells stably transfected with an empty vector, wild-type USP30, or the S104A mutant were seeded in 96-well plates at 5,000 cells per well, with six replicates per group. A cell-free control group was set up as a blank control. After cell attachment, the old culture medium was discarded, and 100 μL of fresh complete culture medium containing varying concentrations of doxorubicin (0, 0.005, 0.01, 0.02, 0.05, 0.10, 0.20, 0.50, 1, 2, and 10 μg / mL) or cisplatin (0, 0.005, 0.01, 0.05, 0.10, 0.50, 1, 2, 5, and 10 μg / mL) was added to each well and culture continued. After 72 hours of drug treatment, discard the old culture medium, wash with PBS to remove dead cells to reduce errors, add 100 μL of fresh serum-free culture medium containing 10% CCK-8 to each culture well, return to the incubator and incubate for 1-1.5 hours. Place the well on a microplate reader to measure OD450nm. Calculate the IC value for each group using the absorbance. 50 value.

[0077] Figure 5 The results of USP30 S104 mutant inhibiting breast cancer cell drug resistance are shown: (a) Wild-type USP30 and USP30 S104A phosphorylation site mutant were stably overexpressed in MDA-MB-231 cells. CCK-8 assay was used to detect the effect of USP30 S104A mutant on doxorubicin and cisplatin resistance under different concentrations of doxorubicin and cisplatin. Drug concentration-cell survival rate curves were plotted and IC 50 value.

[0078] The results showed that the IC of wild-type USP30 to doxorubicin 50 The value is 0.20 μg / mL, USP30 S104A is the IC of doxorubicin 50 The IC value of wild-type USP30 for cisplatin is 0.130 μg / mL; 50 The value is 1.02 μg / mL, USP30 S104A is the IC of cisplatin 50 The value was 0.78 μg / mL. It can be seen that compared with wild-type USP30, the USP30 S104A mutant significantly enhanced the sensitivity of breast cancer cells to doxorubicin and cisplatin.

[0079] The above experiments demonstrated that the USP30 S104A mutant significantly inhibited the ability of tumor stem cells to form spheres and reduced the IC value of chemotherapy drugs. 50 Furthermore, the phosphorylation level of USP30 S104 is positively correlated with chemotherapy resistance and can be used as a predictive marker for drug resistance.

[0080] Example 4 Preparation of phosphorylated antibodies and Western blotting experiments

[0081] The antibody that specifically recognizes USP30 in the phosphorylated state of S104 (P-USP30 S104) was prepared by the following method:

[0082] (1) Antigen design and preparation:

[0083] Based on the S104 phosphorylation site of USP30 protein, a specific phosphorylated peptide sequence (EFTSQY S RDQKEC (SEQ ID NO: 2), where the underlined serine S indicates phosphorylation, was synthesized simultaneously with a non-phosphorylated control peptide. The phosphorylated peptides were conjugated to carrier proteins (BSA and OVA) via chemical cross-linking to form complete antigens. SDS-PAGE was used to verify the conjugation efficiency and confirm that the antigen concentration was 1 mg / mL, meeting immunization requirements.

[0084] (2) Animal immunization:

[0085] Two Japanese big-eared rabbits were selected, and the immunization regimen was as follows: first immunization: 0.5 mg phosphorylated antigen (BSA conjugate) + complete Freund's adjuvant, subcutaneous multiple injection; booster immunization: every 14 days (a total of 4 times), using incomplete Freund's adjuvant, dose of 0.5 mg / time; blood was collected on the 35th day after the third immunization and the 49th day after the fourth immunization for serum titer detection.

[0086] (3) Serum titer test (ELISA):

[0087] Phosphorylated peptides (10 μg / mL) and non-phosphorylated peptides were coated on ELISA plates separately. Serum was serially diluted (1:2000 to 1:32000), primary antibody was added (100 μL / well), and incubation was performed at 37°C for 1 hour. After washing, HRP-labeled secondary antibody (1:10000 dilution) was added and incubated at 37°C for 45 minutes. TMB was used for color development for 20 minutes, and the OD450 value was measured. The results showed that the titer of the antiserum of both rabbits exceeded 1:50K, and the OD value of the phosphorylated peptide group was significantly higher than that of the non-phosphorylated group, indicating that the antibody has high specificity for the phosphorylation site.

[0088] (4) Antibody purification (affinity chromatography):

[0089] A phosphorylated antigen affinity column (for specific purification of phosphorylated antibodies) and a non-phosphorylated antigen affinity column (for removal of non-specific antibodies) were used. Key steps include: (i) Column pretreatment: After rinsing with deionized water, equilibrate with 0.02M PB + 0.3M NaCl. (ii) Sample loading: 8mL of antiserum was filtered through 0.22μm and loaded onto the column at a flow rate of 5-7 seconds / drop. (iii) Elution: Elution was performed with 0.1M glycine (pH 3.0), and the elution peak was collected and neutralized to pH 7.4. (iv) Concentration and dialysis: Concentrate to 1-3mL using a 10kDa ultrafiltration tube and dialyze overnight against 0.01M PBS. (v) Purity verification: SDS-PAGE showed an antibody purity of >90%. Storage conditions: The final antibody concentration was 0.5mg / mL, and the product was stored at -80°C with 0.02% NaN3 as a preservative.

[0090] The nuclear fraction was isolated and immunoblotted according to the method of Example 1. The antibodies used included P-USP30 S104, anti-USP30 (#ab314749, Abcam), GAPDH (#10494-1-AP, Proteintech), and Lamin A / C antibody (#4777, CST) prepared in this example. Figure 6 As shown in the figure, the phosphorylated protein is mainly located in the cytoplasm. Since the phosphorylation state of USP30 is associated with chemotherapy sensitivity, the ratio of P-USP30 S104 level to total USP30 antibody can be used to determine whether a patient is at risk of chemotherapy resistance.

[0091] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An isolated ubiquitin-specific peptidase 30 mutant, characterized in that The ubiquitin-specific peptidase 30 mutant is a USP30 S104A mutant, which has an amino acid sequence as shown in SEQ ID NO:

1.

2. The ubiquitin-specific peptidase 30 mutant according to claim 1, characterized in that The USP30 S104A mutant is obtained by mutating the serine at position 104 of wild-type USP30 to alanine.

3. A nucleic acid, characterized in that The nucleic acid encodes the ubiquitin-specific peptidase 30 mutant according to claim 1 or 2.

4. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid according to claim 3.

5. The recombinant vector according to claim 4, characterized in that The vector is an adenoviral vector, a lentiviral vector or a plasmid.

6. An antibody, characterized in that The antibody specifically recognizes the phosphorylation state of site 104 of the USP30 protein, and the antibody is a monoclonal antibody or a polyclonal antibody.

7. The antibody according to claim 6, characterized in that The antibody is obtained by the following preparation method: (1) Antigen design and preparation: Based on the S104 phosphorylation site of the USP30 protein, a specific phosphorylated peptide was designed and synthesized. The sequence of the phosphorylated peptide was EFTSQYSRDQKEC, in which the serine at position 7 was phosphorylated. The phosphorylated peptide was coupled to a carrier protein by chemical cross-linking to form a complete antigen. (2) Animal immunization: Japanese big-eared rabbits are immunized with the antigen obtained in step (1), and the immunization method is as follows: first immunization: 0.2-0.8 mg complete antigen and complete Freund's adjuvant, multiple subcutaneous injections; multiple booster immunizations: performed every 14 days, using incomplete Freund's adjuvant, with a dose of 0.2-0.8 mg / time; the titer of the antibody obtained by immunization is detected and the specificity of the antibody is confirmed; (3) Antibody purification: The antibody obtained in step (2) is purified using a phosphorylated antigen affinity column and a non-phosphorylated antigen affinity column.

8. Use of the antibody according to claim 6 or 7 in the preparation of a reagent for detecting the phosphorylation level of USP30 in breast cancer cells.

9. A reagent for predicting chemotherapy resistance in breast cancer patients, characterized in that The reagents include: the antibody that specifically recognizes the phosphorylation state of site 104 of the USP30 protein according to claim 6 and a total USP30 antibody; The antibody that specifically recognizes the phosphorylation state of the USP30 protein site 104 is used to detect the level of USP30 in the patient sample with S104 being phosphorylated. The total USP30 antibody is used to detect the level of total USP30 in patient samples.

10. Use of the ubiquitin-specific peptidase 30 mutant according to claim 1 or 2 in the preparation of a drug for inhibiting breast cancer stem cell characteristics and / or a drug for enhancing the chemotherapy sensitivity of breast cancer cells.

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