Anti-human SHLD1 monoclonal antibody and application thereof

Abstract

The present invention relates to an anti-human SHLD1 monoclonal antibody, the anti-human SHLD1 monoclonal antibody comprises a heavy chain and a light chain, the heavy chain comprises three antigen complementary determinants, an H-CDR1 sequence is represented by SEQ ID NO: 1, an H-CDR2 sequence is represented by SEQ ID NO: 2, and an H-CDR3 sequence is represented by SEQ ID NO: 3. The light chain comprises three antigen complementary determinants, an L-CDR1 sequence is as shown in SEQ ID NO: 4, an L-CDR2 sequence is as shown in SEQ ID NO: 5, and an L-CDR3 sequence is as shown in SEQ ID NO: 6. The anti-SHLD1 monoclonal antibody provided by the invention has good recognition specificity on the SHLD1 protein purified in vitro and the SHLD1 protein expressed in cells; and the method can be applied to a plurality of scenes such as western blot experiments and immunohistochemical experiments.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody against human SHLD1 protein and its applications. Background Technology

[0002] Human SHLD1 protein is a key component of the Shieldin complex and plays a central role in DNA damage response (DDR), particularly in protecting DNA ends and maintaining genome stability in the non-homologous end joining (NHEJ) repair pathway.

[0003] Studies have shown that SHLD1 ensures the accuracy of NHEJ repair by inhibiting nuclease-mediated excessive DNA excision and synergistically regulates DNA damage repair with proteins such as 53BP1 and RIF1. Furthermore, SHLD1 expression levels are closely related to tumor therapy sensitivity, especially in BRCA-deficient cancers, where its absence may lead to resistance to PARP inhibitors.

[0004] Given the crucial role of SHLD1 in DNA repair and tumor therapy, the development of anti-SHLD1 monoclonal antibodies is necessary to better understand SHLD1, elucidate its molecular mechanisms in DNA repair and its interaction with the Shieldin complex, its potential role as a biomarker in tumor diagnosis, and its role in assessing patients' DNA repair deficiencies and prognosis. Furthermore, anti-SHLD1 monoclonal antibodies hold promise for providing new strategies for precision medicine, such as predicting the efficacy of PARP inhibitors, developing antibody drugs or combination therapies targeting SHLD1, thereby overcoming tumor drug resistance and improving treatment efficiency.

[0005] Therefore, developing a highly specific and sensitive anti-SHLD1 monoclonal antibody has significant scientific and clinical value. Summary of the Invention

[0006] To address the aforementioned technical problem, the present invention provides a monoclonal antibody against human SHLD1, comprising a heavy chain and a light chain, wherein the heavy chain contains three antigenic complementary determinants, wherein the H-CDR1 sequence is shown in SEQ ID NO:1, the H-CDR2 sequence is shown in SEQ ID NO:2, and the H-CDR3 sequence is shown in SEQ ID NO:3.

[0007] In one specific embodiment, the amino group of the light chain contains three antigenic complementary determinants, wherein the L-CDR1 sequence is shown in SEQ ID NO:4, the L-CDR2 sequence is shown in SEQ ID NO:5, and the L-CDR3 sequence is shown in SEQ ID NO:6.

[0008] The present invention also provides a nucleic acid encoding the above-mentioned monoclonal antibody. Since the antigen complementarity determinant sequence of the monoclonal antibody has been identified, nucleic acids can be designed based on this sequence and commonly used antibody backbone sequences for the biosynthesis of the above-mentioned monoclonal antibody.

[0009] This invention also provides the application of the above-mentioned monoclonal antibody in the preparation of SHLD1 detection reagent.

[0010] The present invention also provides a kit for detecting SHLD1, the kit comprising the above-mentioned monoclonal antibody.

[0011] In one specific implementation, the kit is an immunoblotting kit, an immunohistochemistry kit, or an immunofluorescence kit.

[0012] The monoclonal antibody against human SHLD1 provided by this invention has good recognition specificity for human SHLD1 protein expressed in vitro, cell lysates expressing human SHLD1 protein, or tissues expressing SHLD1 protein; compared with polyclonal antibodies, this monoclonal antibody has stronger recognition specificity. Attached Figure Description

[0013] Figure 1 Western blot images of SHLD1 protein in 293T cells and HTR8 / SVneo cells detected using the monoclonal antibody of this invention;

[0014] Figure 2 IHC image of SHLD1 protein in human placental tissue detected using the monoclonal antibody of the present invention;

[0015] Figure 3 Immunofluorescence micrographs of SHLD1 protein in 293T cells and HTR8 / SVneo cells detected using the monoclonal antibody of this invention. Detailed Implementation

[0016] The following will describe in detail the technical solution, implementation steps, and effects of the present invention with reference to embodiments and experimental data. The embodiments described are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can adjust or expand the specific implementation conditions through conventional experiments based on the content disclosed in this invention, without departing from the core ideas of the invention.

[0017] Terms and definitions:

[0018] Unless otherwise expressly stated, the technical terms used in this specification (such as "SHLD1" and "NHEJ") shall be understood by those skilled in the art to which this invention pertains. In case of any ambiguity, the definitions in this specification shall prevail.

[0019] Unless otherwise specified, the raw materials, reagents and equipment involved in this invention (such as ELISA detectors, hybridoma cell lines, etc.) are all commercially available products or can be prepared by standard methods (e.g., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 1992).

[0020] Example 1: Monoclonal Antibody and its Preparation Method

[0021] 1. Construction of recombinant protein vectors and prokaryotic expression of proteins

[0022] (1) The human SHLD1 gene CDS sequence was cloned into the pET42b vector to obtain the recombinant vector.

[0023] The primer pairs used were: forward primer (5'-aagaaggagatatacatatggagagcgacgagagcggcctgccc-3'); reverse primer (5'-tggtggtggtggtgctcgaggcgagatccggtggagccgggtcc-3'), cloned into the NdeI-XhoⅠ restriction site of the pET42b vector.

[0024] (2) Protein expression and purification:

[0025] ① Plasmid transformation:

[0026] The constructed recombinant plasmid was transformed into BL21 competent bacteria and plated on LB agar plates containing kanamycin resistance. After single colonies grew, single colonies were picked and cultured in 5 mL of culture medium.

[0027] ② Protein-induced expression:

[0028] After incubating the bacterial culture overnight (approximately 12 hours), transfer it to a large volume (1 L) of culture medium and continue culturing until OD (outlet capacity) is reached. 600 =0.4-0.6, add IPTG (0.1-0.3 mM) to induce protein expression, and continue induction culture at 16-25 ℃ for 24 hours.

[0029] ③Preparation before protein purification:

[0030] Collect the induced bacterial culture at low temperature (4 ℃ unless otherwise specified) using a centrifuge (4000 rpm, 20 minutes), then wash with PBS to remove residual culture medium; add an appropriate amount of pre-cooled (stand on ice unless otherwise specified) bacterial lysis buffer (30 mL / 1 L bacteria, formula below) and working concentration of proteasome inhibitor (such as PMSF) and lysozyme, place on ice for 10 minutes, then lyse the bacteria using sonication; after thorough lysis, centrifuge at low temperature and high speed (e.g., 12000 rpm, 20 minutes), and collect the supernatant for subsequent purification.

[0031] ④ Protein purification:

[0032] Wash the agarose beads coupled with the affinity ligands (such as Ni NTA beads for histidine tags) three times with lysis buffer, then add them to the supernatant recovered in step ③ and bind at low temperature for 1 hour; centrifuge to remove the supernatant, wash the agarose beads twice with lysis buffer for 10 minutes each time, and then wash them three times with washing buffer (formula below) for 10 minutes each time; thoroughly aspirate the washing buffer, and elute the protein with elution buffer (formula below).

[0033] ⑤ Concentrated and dialysis proteins

[0034] If the obtained protein concentration is low, it needs to be concentrated using an ultrafiltration tube. During the concentration process, the elution buffer can be replaced with PBS buffer; if the protein concentration is high, it can be directly dialyzed with a large amount of PBS buffer.

[0035] ⑥ Protein storage

[0036] After the protein is dialyzed, its concentration is determined by the BCA method, then 10-20% glycerol is added, the mixture is flash-frozen in liquid nitrogen, and stored at -80 °C.

[0037] (3) Protein purification reagent formulation

[0038] Lysis buffer: Tris-HCl: 100 mM, NaCl: 300 mM, NP-40: 0.05%, pH=8.0;

[0039] Washing buffer: Tris-HCl: 100 mM, NaCl: 300 mM, Imidazole: 20-50 mM, NP-40: 0.05%, pH=8.0;

[0040] Elution buffer: Tris-HCl: 100 mM, NaCl: 300 mM, Imidazole: 250 mM, NP-40: 0.05%, pH=8.0.

[0041] 2. Animal immunization

[0042] Purified human SHLD1 protein was used as the antigen to immunize mice. Three 6-8 week old Balb / C mice were selected. The first master injection used Freund's complete adjuvant to emulsify the antigen, and subsequent booster injections used Freund's incomplete adjuvant to emulsify the antigen. Immunization was performed via subcutaneous injection at four points in the abdomen. Immunization dosage: 0.1 mg antigen / mouse for the initial immunization, followed by three booster immunizations, with an interval of two weeks between each immunization. The first two booster immunizations were 0.1 mg antigen / mouse, and the third booster immunization was 0.05 mg antigen / mouse.

[0043] 3. Antiserum titer detection

[0044] (1) A small amount of blood was taken from the outer canthus vein of the mouse and the serum was collected by centrifugation.

[0045] (2) ELISA method is used to detect the antiserum titer to determine whether the titer reaches 1:10000.

[0046] 4. Cell fusion and hybridoma subclonal screening

[0047] (1) Preparation of myeloma cells (SP2 / 0)

[0048] One week before fusion, SP2 / 0 cells were revived and cultured normally to the logarithmic growth phase.

[0049] (2) Spleen cell preparation

[0050] Select mice for fusion, euthanize them by cervical dislocation on the day of fusion, harvest spleens, and collect and count spleen cells according to standard procedures.

[0051] (3) Cell fusion

[0052] Myeloma cells and spleen cells were mixed at a ratio of 1:3 to 1:10, and cell fusion was performed using standard procedures. The cells were then cultured in HAT DMEM complete medium. Hybridoma cells were visible 3 days after fusion. On day 7, the medium was replaced with half HAT complete medium, and on day 8, it was replaced with half HT medium. Screening and testing began approximately 10 days after fusion.

[0053] Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope. Multiple growing hybridoma cells were observed, proving that the fusion operation was successful.

[0054] (4) Screening of fusion cells

[0055] 100 μL of cell supernatant was aspirated per well for indirect ELISA detection. Positive wells were identified based on the ELISA results. A single-channel pipette was used to pick up any positive wells detected on the entire plate for a second test to further confirm their positive status.

[0056] (5) Subclonal cell culture

[0057] Two rounds of subcloning were performed on the positive well cells from the second screening. Because the positive well cell lines obtained from the first subcloning are not yet stable and may contain multiple hybridoma cells, it is generally believed that the hybridoma cells after the second subcloning are single cell lines and are confirmed as positive.

[0058] The cells in the positive wells were first subcloned and diluted into multiple wells. They were then cultured in HT DMEM medium and observed under a microscope after about 7 days. Wells with clonal growth were detected by indirect ELISA, and wells with high OD values ​​were selected as positive wells. Cells from the positive wells were picked for a second subcloning to detect stable positive hybridoma cell lines, which were then used as cells for the final preparation of monoclonal antibodies. The cells were then expanded to obtain hybridoma cell lines.

[0059] 5. Ascites preparation and antibody purification

[0060] (1) Preparation of ascites

[0061] The positive cells were cultured and injected into the peritoneal cavity of Balb / C mice (sensitized with Freund's incomplete adjuvant). Abdominal distension was observed in the mice within 7-10 days, indicating the presence of ascites. The ascites was promptly aspirated when significant ascites was observed.

[0062] (2) Purification of ascites

[0063] The ascites fluid from the above cells was purified (using ammonium octanoate sulfate + DEAE ion column purification method), and the antibody purity after purification was greater than 90%.

[0064] (3) Detection of antibody concentration and purity. The antibody concentration was measured to be 1.5 mg / mL. The purity of the purified antibody was detected by Coomassie brilliant blue staining assay.

[0065] 6. Antibody sequence analysis

[0066] (1) Culture of hybridoma cells

[0067] After reviving the hybridoma cell line, culture it until the cell number expands to approximately 1 × 10⁻⁶. 7 Centrifuge at 1000 rpm for 5 minutes at room temperature to collect cells.

[0068] (2) Extracting cellular RNA

[0069] In a clean bench environment, add 1 mL of Trizol reagent to the collected cells, let stand for 5 minutes, add 200 μL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 5 minutes, and centrifuge at 12000 rpm for 15 minutes at low temperature; transfer the upper aqueous layer to a new EP tube, add 0.5 mL of isopropanol, let stand at -20°C for 10 minutes, and centrifuge at 12000 rpm for 10 minutes; discard the supernatant, add 1 mL of 75% ethanol, centrifuge at 7500 rpm for 5 minutes, repeat the washing once to remove as much ethanol as possible from the precipitate, let stand at room temperature for 2 minutes, and add 50 μL of RNase-free double-distilled water to dissolve the precipitate; after concentration determination, the RNA can be stored at -80°C for later use or used directly.

[0070] (3) Preparation of cDNA by reverse transcription

[0071] 1 μL total cellular RNA, 6 μL RNase-free ddH2O, 0.5 μL oligo dT Primer, 0.5 μL PrimeScript RT Enzyme Mix I, and 2 μL 5×Prime Script Buffer were mixed and incubated at 37 °C for 15 minutes followed by 85 °C for 5 seconds.

[0072] (4) Amplify cDNA

[0073] Mouse IgG V H V L Primer library was used to amplify the above cDNA separately. A 2×PCR mix was prepared with 20 μL of cDNA, 2 μL of upstream primer, 2 μL of downstream primer, and water added to a final volume of 40 μL. PCR was performed under the following conditions: 98 °C for 5 minutes, 98 °C for 30 seconds of denaturation, 63 °C for 20 seconds of annealing, 72 °C for 90 seconds of extension, and 30 cycles followed by a 5-minute extension at 72 °C.

[0074] (5) Agarose gel electrophoresis and gel recovery

[0075] The PCR products were subjected to agarose gel electrophoresis. The electrophoresis results were observed. The amplification products with molecular weights of 700-800bp (light chain) and 1400-1600bp (heavy chain) were sent for sequencing.

[0076] (6) Antigenic determinant analysis

[0077] The nucleic acid sequences in the sequencing results can be translated into protein sequences using software (such as Snapgene) and submitted to the corresponding CDR prediction website to obtain the corresponding CDR region information (as shown in Table 1).

[0078] Table 1. CDR sequences of antigens and monoclonal antibodies

[0079]

[0080] Example 2: Monoclonal antibodies used for immunoblotting detection

[0081] (1) HTR8 / SVneo or 293T cells overexpressing 3*FLAG-PCMV-SHLD1 plasmid cultured in 6-well plates: discard the culture medium, wash twice with PBS, add an appropriate amount of lysis buffer, lyse on ice for 30 minutes, scrape off the cells, centrifuge at 4°C (12,000×g, 15 minutes), and take the supernatant.

[0082] (2) Protein quantification: Protein concentration was determined by BCA method. The sample concentration was adjusted to be consistent. 5× Loading Buffer was added, and the protein was boiled for 5-10 minutes to denature it. The protein was stored at -20℃ for later use.

[0083] (3) SDS-PAGE electrophoresis: Prepare a 12% separating gel and a 5% stacking gel, insert a 10-well comb, and wait for the stacking gel to solidify (about 20 minutes).

[0084] (4) Add the sample to the well in sequence (add the marker at the same time), constant voltage electrophoresis: 80V for the stacking gel stage, and adjust to 120V after the sample enters the separating gel, until the bromophenol blue is close to the bottom of the gel (about 1.5-2 hours).

[0085] (5) Transfer (wet transfer): Soak the PVDF membrane in methanol for 10 seconds beforehand, then equilibrate it with transfer buffer; soak the filter paper and sponge pad in buffer. Place the membrane into the transfer holder in the order of "sponge → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge", ensuring that there are no air bubbles between each layer (air bubbles can be removed by rolling with a roller). Place the transfer holder into the transfer tank (membrane facing the positive electrode), add pre-cooled transfer buffer, and transfer at constant current for 2 hours under ice bath conditions.

[0086] (6) Blocking and antibody incubation: After the transfer is completed, the PVDF membrane is placed in 5% skim milk blocking solution and blocked on a shaker at room temperature for 1-2 hours. Primary antibody incubation: Discard the blocking solution and wash the membrane 3 times with TBST for 10 minutes each time.

[0087] Dilute the primary antibody (SHLD1; 1:2000) proportionally, add it to the membrane, and incubate overnight at 4°C. Secondary antibody incubation: After primary antibody incubation, wash the membrane 5 times with TBST for 10 minutes each time (to thoroughly wash away unbound antibody). Add HRP-labeled secondary antibody (1:10000 dilution) and incubate on a shaker at room temperature for 2 hours. Repeat the TBST washing 5 times for 10 minutes each time, and finally wash once with TBS for 5 minutes.

[0088] (7) Chemiluminescence and development: ECL luminescence solution A and B are mixed in a 1:1 ratio and evenly dropped onto the membrane. The reaction is carried out in the dark for 1-2 minutes. Protein expression is detected using an ECL chemiluminescence analyzer.

[0089] The results are as follows Figure 1 As shown, the anti-SHLD1 monoclonal antibody can specifically detect SHLD1 protein expression.

[0090] Example 3: Monoclonal antibodies used for immunohistochemical detection

[0091] (1) Placental tissue paraffin sections were placed in a 65℃ oven and dried for 2 hours. The sections were then dewaxed to water (the sections were placed in xylene I for 20 min, xylene II for 20 min, anhydrous ethanol I for 10 min, anhydrous ethanol II for 10 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, and 70% ethanol for 5 min). The sections were washed three times with PBS for 5 min each time.

[0092] (2) Perform antigen repair on the slices, heat for 8 minutes on medium heat, cool down for 8 minutes, then heat on medium-low heat for 7 minutes and then turn off the power.

[0093] (3) After natural cooling, wash with PBS 3 times, 5 min each time.

[0094] (4) Wash with PBS 3 times, 5 min each time, spin dry and then block with 3% BSA for 30 min.

[0095] (5) Remove the BSA solution, add about 50 μl of diluted SHDL1 monoclonal antibody to each slice to cover the tissue, and incubate overnight at 4°C.

[0096] (6) Wash with PBS 3 times, 5 min each time.

[0097] (7) Remove the PBS solution, add 50-100 μl of secondary antibody to each slice, and incubate at room temperature for 50 min.

[0098] (8) Wash with PBS 3 times, 5 min each time.

[0099] (9) Remove the PBS solution, add 50-100 μl of freshly prepared DAB solution to each slice, and control the color development under a microscope.

[0100] (10) After the color development is complete, rinse with distilled water or tap water, counterstain with hematoxylin, differentiate with 1% hydrochloric acid alcohol (about 1 second), rinse with tap water, return to blue with ammonia water, and rinse with running water.

[0101] (11) Dehydrate with anhydrous ethanol and clear with xylene. Mount with neutral resin. Detect antibody specificity.

[0102] The results are as follows Figure 2 As shown, the monoclonal SHLD1 antibody has excellent recognition specificity for SHLD1.

[0103] Example 4: Monoclonal antibodies used for immunofluorescence detection

[0104] (1) In a clean bench, open a 6-well plate (HTR8 / SVneo or 293T cells overexpressing the 3*FLAG-PCMV-SHLD1 plasmid) and place a sterile coverslip. Add the cell suspension to the coverslip and incubate at 37°C in a 5% CO2 incubator until the cells are immobilized (about 2 hours). Add 2 ml of cell culture medium and continue culturing for about 24 hours. Transfect the 3*FLAG-PCMV-SHLD1 overexpression plasmid. After 48 hours, collect the cells. Discard the culture medium and wash the cells three times with PBS for 5 minutes each time.

[0105] (2) Take out the 6-well plate with the cell-adhering side facing up, aspirate the culture medium, add sterile PBS buffer to rinse 3 times, aspirate the PBS buffer, add 4% PFA to fix for 20 min.

[0106] (3) Aspirate the PFA and add 0.3% Triton X-100 to break the membrane at room temperature for 8 min. Wash 3 times with sterile PBS buffer, 5 min each time. Add sterile PBS buffer immediately after each well to prevent drying.

[0107] (4) Aspirate the sterile PBS buffer, add 5% BSA / PBS blocking solution and block at room temperature for 1 h, adding 800 μl to each well.

[0108] (5) Remove the blocking solution, dilute the primary antibody with PBS (1:100, operate on ice), add SHLD1 antibody, 200 μl per well, and incubate overnight at 4°C. Mix thoroughly before antibody dilution and addition, and ensure that the antibody completely covers the coverslip in each well.

[0109] (6) On the second day, take the 6-well plate out of the refrigerator, warm it to room temperature for 5 min, aspirate the primary antibody, and wash it 3 times with sterile PBS buffer for 5 min each time.

[0110] (7) Aspirate sterile PBS buffer, dilute the secondary antibody with PBS (operate on ice), add 200 μL of secondary antibody to each well, and incubate at room temperature in the dark for 2 hours or at 37°C in the dark for 1 hour.

[0111] (8) Aspirate the secondary antibody and wash with PBS 3 times, 5 min each time.

[0112] (9) Add DAPI to stain the nucleus for 10 min, aspirate the staining reagent, and wash 3 times with sterile PBS buffer for 5 min each time.

[0113] (10) Add an appropriate amount of anti-fluorescence quencher to the cells, cover with a coverslip, and observe under a fluorescence microscope. Take a picture at 400x magnification.

[0114] The results are as follows Figure 3 As shown, the monoclonal SHLD1 antibody can specifically detect the expression of SHLD1 (red fluorescence).

[0115] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against human SHLD1, characterized in that, It comprises a heavy chain and a light chain, wherein the heavy chain contains three antigen complementarity determinants, wherein the H-CDR1 sequence is shown in SEQ ID NO:1, the H-CDR2 sequence is shown in SEQ ID NO:2, and the H-CDR3 sequence is shown in SEQ ID NO:

3.

2. The monoclonal antibody against human SHLD1 according to claim 1, characterized in that, The light chain contains three antigen complementarity determinants, wherein the L-CDR1 sequence is shown in SEQ ID NO:4, the L-CDR2 sequence is shown in SEQ ID NO:5, and the L-CDR3 sequence is shown in SEQ ID NO:

6.

3. A nucleic acid, characterized in that, Encoding the monoclonal antibody as shown in claim 1 or 2.

4. The use of the antibody according to claim 1 or 2 in the preparation of SHLD1 detection reagent.

5. A kit for detecting SHLD1, characterized in that, A monoclonal antibody comprising the anti-human SHLD1 as described in claim 1 or 2.

6. The reagent kit according to claim 5, characterized in that, The kit is an immunoblotting kit, an immunohistochemistry kit, or an immunofluorescence kit.

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