Smcp polyclonal antibody, and preparation method and application thereof

Abstract

The application belongs to the technical field of immune detection, and specifically discloses a SMCP polyclonal antibody, a preparation method and application thereof. The preparation method comprises the following steps: using a polypeptide containing an amino acid sequence as shown in SEQ ID NO:1 as an immunogen to immunize animals, collecting whole blood of the immunized animals, and separating to obtain a SMCP polyclonal antibody. The application has the beneficial effect that: the specific SMCP protein antigen epitope is used as the immunogen to immunize animals, which is beneficial to large-scale preparation of the antibody specifically recognizing the SMCP protein. The SMCP antibody prepared by the method has good specificity, can be used for WB, IF and Co-IP experiments, and provides an important research tool for studying the pathogenic mechanism of weak sperm caused by SMCP deletion.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, and specifically discloses an SMCP polyclonal antibody, its preparation method, and its application. Background Technology

[0002] Infertility has become the third leading cause of disease affecting human health, impacting approximately 10% to 15% of couples of reproductive age worldwide, with male infertility accounting for about half of all cases. Therefore, addressing male infertility and enabling couples who wish to have children to have healthy offspring has significant practical and social implications.

[0003] Sperm are highly specialized cells with motility, and normal sperm motility is essential for male fertility. Sperm motility disorders are known as asthenospermia (AZS), characterized by low sperm motility, with less than 40% of sperm exhibiting progressive motility in semen, or less than 32% exhibiting progressive motility. It is the most common cause of male infertility, accounting for approximately 20% to 40% of all cases. Therefore, researching the mechanisms underlying asthenospermia is crucial for maintaining male reproductive health and achieving precise clinical diagnosis and treatment for this condition.

[0004] Asthenospermia often occurs secondary to reproductive tract infections, varicocele, and endocrine disorders. Furthermore, approximately 50% of asthenospermia cases are caused by congenital genetic factors, with single-gene mutations being one of the most prevalent hereditary causes. Examples include primary ciliary motility disorders caused by mutations in genes such as CCDC39, and various morphological abnormalities of sperm flagella caused by mutations in genes such as DNAH1. Currently, identifying the pathogenic genes for asthenospermia and exploring their pathogenic mechanisms has become a research hotspot. Basic research in this field will contribute to the development of targeted treatments, safeguarding male reproductive health and family well-being.

[0005] Mature sperm consists of two main parts: the head and the tail, also known as the flagellum. In most species, the sperm flagellum is a crucial structure for sperm to move along the female reproductive tract to reach the oocyte and fertilize it. In mammals, mitochondria are confined within the mitochondrial sheath of the sperm flagellum, producing ATP through the electron transport chain and oxidative phosphorylation to support flagellar motility. Abnormalities in the structure or function of the flagellum mitochondrial sheath can lead to reduced sperm motility, resulting in asthenospermia (low sperm motility).

[0006] The SMCP (Sperm mitochondria associated cysteine ​​rich protein) gene, also known as MCS or MCSP, is located on chromosome 1 in humans. It contains two exons and encodes a protein of 116 amino acids. In mice, the SMCP gene is located on chromosome 3, also containing two exons, and encodes a protein up to 143 amino acids long. SMCP is a structural protein rich in cysteine ​​and proline, and is associated with the mitochondrial keratinized sac in the sperm mitochondrial sheath. Previous studies have found that silencing SMCP leads to infertility and decreased sperm motility in male mice with a 129 / Sv background, but the pathogenic mechanism is currently unclear, and further research is needed to explore the function of the SMCP protein and its precise molecular mechanism in sperm motility. Specific antibodies against SMCP protein are crucial for studying its functional mechanism. However, commercially available SMCP antibodies currently on the market have poor specificity and cannot effectively perform experiments such as Western blot (WB), immunofluorescence (IF), and co-immunoprecipitation (Co-IP). This has hindered the progress of scientific research in the field of reproduction to some extent and has become a technical problem that urgently needs to be solved. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention provides a polyclonal antibody against SMCP, its preparation method, and its applications. By employing specific antigenic epitopes of the SMCP protein as immunogens to immunize animals, this invention facilitates the large-scale preparation of antibodies that specifically recognize the SMCP protein, thus supporting scientific research in the field of reproduction.

[0008] In a first aspect, the present invention provides a method for preparing SMCP polyclonal antibodies, comprising: immunizing an animal with a polypeptide containing an amino acid sequence as shown in SEQ ID NO:1 as an immunogen, collecting whole blood from the immunized animal, and separating the SMCP polyclonal antibody.

[0009] In some implementations, the C-terminus of the peptide is coupled to a carrier protein.

[0010] In some implementations, the carrier protein is KLH protein (Keyhole LimpetHemocyanin), BSA protein (Bovine Serum Albumin), or OVA protein (Ovalbumin).

[0011] In some implementations, an immune adjuvant is also included, in which the animal is immunized using a composition containing an immunogen and an immune adjuvant.

[0012] In some implementations, the immune adjuvant is Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum salt adjuvant, liposome, monophosphoryl lipid A, or cytokine adjuvant.

[0013] In some implementations, the animal is a mouse or a rabbit.

[0014] In some implementation schemes, animals are immunized 3 to 5 times.

[0015] In some implementation methods, the preparation method includes: Step 1: Immunogen preparation: The polypeptide with the amino acid sequence shown in SEQ ID NO:1 is conjugated with a carrier protein to obtain a conjugated protein, denoted as immunogen A; Freund's complete adjuvant is mixed and emulsified with the conjugated protein to obtain a Freund's complete adjuvant emulsified conjugated protein, denoted as immunogen B; Freund's incomplete adjuvant is mixed and emulsified with the conjugated protein to obtain a Freund's incomplete adjuvant emulsified conjugated protein, denoted as immunogen C; Step 2: Animal immunization: Immunize animals for the first time using immunogen B; immunize animals for the second time using immunogen C 20-22 days after the first immunization; immunize animals for the third time using immunogen C 13-15 days after the second immunization; immunize animals for the fourth time using immunogen A 20-22 days after the third immunization. Step 3: Antibody isolation: On days 13-15 after the fourth immunization of the animals, whole blood was collected from the immunized animals to isolate the SMCP polyclonal antibody.

[0016] In a second aspect, the present invention provides an SMCP polyclonal antibody prepared by the aforementioned method.

[0017] A third aspect of the present invention provides the use of the aforementioned SMCP polyclonal antibody in the preparation of immunoassay reagents or products for detecting SMCP protein.

[0018] In some implementations, immunoassay reagents or products are used for Western blotting, immunofluorescence staining, or immunoprecipitation assays to detect SMCP proteins.

[0019] The beneficial effects of this invention are as follows: This invention utilizes specific SMCP protein epitopes as immunogens to immunize animals, facilitating the large-scale preparation of antibodies that specifically recognize the SMCP protein. The SMCP antibodies prepared by this method exhibit good specificity and can be used in Western blotting, infusion, and co-IP experiments, providing an important research tool for investigating the pathogenic mechanism of asthenospermia caused by SMCP deficiency. Attached Figure Description

[0020] Figure 1 These are results of whole-sequence specificity analysis; Figure 2 The results are from a full sequence conservation analysis. Figure 3 The results are from the analysis of transmembrane structural domains; Figure 4 This is the result of post-translation modification analysis; Figure 5 The results are from the conservative structural domain analysis. Figure 6 This is the result of the three-level structure analysis; Figure 7 The result represents the prediction of the secondary structure; where: e: extended chain; c: random coil; h: α-helix.

[0021] Figure 8A This is the prediction result of the full-sequence antigenic epitopes; Figure 8B The predicted results for the antigenic epitopes in the 128-143AA sequence; Figure 9 The results are from the hydrophilicity test of the 128-143AA sequence; Figure 10 This is the HPLC chromatogram of the crude product; Figure 11 MS analysis chromatogram of crude product; Figure 12 To prepare HPLC chromatograms for purification; Figure 13 For purification and analysis of HPLC chromatograms; Figure 14 MS results after purification; Figure 15 The results are from Western blot experiments; where: A: Western blot was performed in mouse testicular tissue using mouse SMCP antibody at a dilution ratio of 1:1000; B: Western blot was performed in mouse testicular tissue using rabbit SMCP antibody at a dilution ratio of 1:1000; C: Western blot was performed in mouse sperm using mouse SMCP antibody at a dilution ratio of 1:1000. Figure 16 The results of immunofluorescence assay using rabbit SMCP antibody in mouse testes; Figure 17 The results are as follows: A: Co-IP was performed in mouse testes using mouse SMCP antibody. To exclude light chain interference, Western blot (WB) was performed after Co-IP using rabbit SMCP antibody at a dilution ratio of 1:1000. B: Co-IP was performed in mouse testes using rabbit SMCP antibody. To exclude light chain interference, Western blot (WB) was performed after Co-IP using mouse SMCP antibody at a dilution ratio of 1:1000. Figure 18 Results of Western blot experiments using commercially available SMCP monoclonal antibodies; Figure 19 The results are from an immunofluorescence assay using a commercially available SMCP monoclonal antibody. Figure 20 The results of the immunoprecipitation assay using commercially available SMCP monoclonal antibody are shown below; IP: SMCP Antibody (Catalog No.: NBP2-45888, NOVUS) 8 μl; IgG antibody; WB: SMCP Antibody (Catalog No.: NBP2-45888, NOVUS) 1:500. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: SMCP peptide antigen design 1. Background Information

[0024] 2. Antigen Design 2.1 Sequence Information

[0025] 2.2 Analysis of amino acid sequence specificity and conservation 2.2.1 Sequence Specificity Analyzing the specificity of amino acid sequences within the same species helps determine whether antibodies produced by a protein can recognize other proteins. In *Mus musculus*, gene specificity analysis revealed that the gene only aligns to the protein itself, such as... Figure 1 As shown.

[0026] 2.2.2 Sequence Conservation Analyzing the conservation of amino acid sequences across different species facilitates the discovery of whether antibodies produced by the protein can cross-react with other species. For example... Figure 2 As shown, the amino acids conserved in this protein in humans and mice are marked in purple.

[0027] 2.3 Protein expressibility analysis 2.3.1 Transmembrane domain analysis Antigen design should generally avoid transmembrane domains. For example... Figure 3 As shown, this protein has no topological domains that significantly affect its expression.

[0028] 2.3.2 Post-translation embellishment Signal peptides are typically not considered in antigen design because they often exhibit strong hydrophobicity and may limit antigen expression. For example... Figure 4 As shown, this protein does not have any significant post-translational modifications that affect its expression.

[0029] 2.3.3 Conservative Structural Domains Antigens should typically contain the entire structural domain. For example... Figure 5 As shown, this protein lacks stable structural domains. Specifically, the first brown region, "Disordered," indicates that this region has not formed a stable structure and is mainly characterized by random coils. The second pink region, "Poly / Rich," represents a low-complexity region, indicating that these regions have a higher proportion of certain amino acids but a lower variety, resulting in insufficient epitope diversity / specificity.

[0030] 2.3.4 Three-level structure analysis Tertiary structure analysis is helpful for identifying B cell epitopes and analyzing protein expression capabilities. The predicted full-length protein structure of AF-P15265-F1, 1-143AA, is shown below. Figure 6 As shown.

[0031] 2.3.5 Secondary Structure Prediction Secondary structure prediction is helpful for the identification of B cell epitopes and the analysis of protein expression capabilities. This protein is predominantly random coiled, such as... Figure 7 As shown.

[0032] 2.4 Protein antigenicity analysis 2.4.1 Antigenic Epitope Prediction Antigenic epitope prediction helps in understanding protein hydrophobicity. The protein has a favorable antigenic epitope, such as... Figure 8A As shown.

[0033] 2.5 Antigen Region Selection

[0034] 2.5.1128-143AA sequence specificity By comparing the 128-143AA sequence of the SMCP protein, 7 / 11 (64%) Identities were found, indicating that the 128-143AA sequence of the SMCP protein has good specificity.

[0035] 2.5.2128-143AA sequence antigenic epitope prediction like Figure 8B As shown, the 128-143AA sequence antigenic epitope has good predictive properties.

[0036] 2.5.3 Hydrophilicity of the 128-143AA sequence like Figure 9 As shown, the amino acids in this sequence have good hydrophilicity (average hydrophilicity value of 0-2; generally speaking, such polypeptides are very soluble in water; good solubility can be achieved without the addition of solubilizers). Example 2: Synthesis of SMCP polypeptide antigen 1. Synthetic Information

[0037] 2. Solid raw materials Fmoc-Gly-OH, Fmoc-Lys(BOC)-OH, Fmoc-Asn(trt)-OH, Fmoc-Gln(trt)-OH, Fmoc-Trp(BOC)-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Ser(tbu)-OH.

[0038] 3. Condensation reagent

[0039] 4. Solvent

[0040] 20% Pip / DMF 6. Resin Information 2-Cl Resin (chlorinated resin) 7. Production instruments Semi-automatic multichannel peptide synthesizer, mechanical shaker, constant temperature water bath, analytical balance, low-speed centrifuge, vacuum drying oven, high-performance liquid chromatograph (HPLC), high-performance liquid chromatograph-mass spectrometer (MS). 8. Production process Step 1. Swelling the resin Add CL-K resin (modified chlorinated resin, i.e. chlorinated resin with Lys attached) to the reactor, add DCM (10ml), and stir to swell for 3 min. Step 2. Resin Deprotection Vacuum dry the swelling reagent, add 6 ml of 20% Pip / DMF and stir for 20 min. Step 3. Remove protective layer and wash. Remove the protective reagent by vacuum drying, wash 6 times with DMF (6 ml), stirring for 1 min each time, and then vacuum dry for 0.5 min. Step 4. Deprotection detection Place 20 resin particles into a test tube, add 1 ml of ninhydrin detection reagent, place the test tube in a water bath at 95°C or above for 2 minutes, remove and observe the resin color. A darker resin color indicates a positive result, signifying successful deprotection. Step 5. Condensate the second amino acid. Add 6 ml of the prepared amino acid (Fmoc-Asn(trt)-OH) / OXYMA / DMF solution, then add 0.95 equivalents of DIC to activate for 1 min, and then start the stirring reaction for 1 h. Step 6. Reaction washing Vacuum dry the condensation reagent, add 6 ml of DMF, stir for 1 min each time, vacuum dry for 0.5 min, and wash 3 times. Step 7. Reaction Detection Place 20 resin grains into a test tube, add 1 ml of ninhydrin test reagent, and place the test tube in a water bath at 95°C or higher for 2 minutes. Remove the tube and observe the resin color. No color change indicates a negative result, signifying successful condensation. Alternatively, remove the tube and test the resin color; no significant color change indicates successful condensation. Step 8. Repeat steps 2 to 7, condensing the amino acids in the sequence from right to left. Step 9. After the last amino acid condensation is completed, perform the deprotection, deprotection washing, and deprotection detection steps in sequence. Then wash the resin twice with methanol (10 ml) and vacuum dry for 10 min.

[0041] 9. Resin pyrolysis 9.1 Information on pyrolysis materials Trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT), purified water, benzyl sulfide, phenol.

[0042] 9.2 Pyrolysis Process After drying, the resin was loaded into a boat-shaped reactor, 10 ml of lysis buffer was added, and the mixture was lysed at room temperature for 3 h. The reaction solution was filtered out and added to 100 ml of ice-cold ether. The mixture was centrifuged to precipitate and washed three times with ice-cold ether to obtain crude polypeptide solid for purification. The crude polypeptide solid was transferred to a clean tray and placed in a vacuum drying oven at 35 °C for 3-5 h. 10. Crude product intermediate control inspection Take a small sample for HPLC and MS mass spectrometry analysis for purification reference. For example... Figure 10 As shown, the HPLC analysis results of the crude product show a single, clean main peak, indicating separability. Figure 11 As shown, the MS analysis results of the crude product were m+2+3+4, which is a good result.

[0043] 11. Crude product purification Purification gradient: 0-20% acetonitrile concentration for 5-35 min, and 0.5% acetic acid aqueous solution for the aqueous phase. The purified preparation results are separable, such as... Figure 12 As shown. The purification results show a single, clean peak shape, indicating good results, as shown. Figure 13 As shown. After purification, the MS results mainly consist of the final product, such as... Figure 14 As shown.

[0044] 12. Freeze-drying and repackaging After purification, the qualified fraction is placed into a freeze-drying tray, covered, and then placed in a freeze dryer for freeze-drying. After lyophilization, remove the lyophilization tray, weigh and aliquot the peptide samples, and store them at -20℃. 13. Quality Control Small samples were taken for quality control testing (endotoxin control: during peptide production, the endotoxin level is ensured to be below 0.01 EU / μg by strictly controlling the production process and environment). After passing the test, the peptide was converted to conjugation.

[0045] 14. Peptide conjugation Step 1: Dissolve 9 mg of polypeptide in pure water to a concentration of 10 mg / ml and set aside. Step 2: Dissolve 9 mg of protein (KLH) in PBS 7.2 to a concentration of 10 mg / ml and set aside. Step 3: Add the peptide (9 mg, 10 mg / ml) to an equal mass of carrier protein (KLH: 9 mg, 10 mg / ml). Step 4: Add an equal volume of EDC (dissolved in PBS 7.2 to 30 mg / ml). Step 5: React on a shaker at room temperature (25℃±2) for 3 hours. Step 6: Dialyze using PBS 7.2 phosphate buffer for 3 hours. Step 7: After completion, change the solution (PBS 7.2) and dialyze overnight. Step 8: Add PBS 7.2 until the final protein concentration is 2 mg / ml. Step 9: Dispense and freeze-dry the product into vials at a rate of 3 × 3.0 mg (1.5 ml / vial). Example 3: Animal immunization with SMCP polypeptide antigen 1. Procedure for mouse immunization experiments 1.1 Ten Charles River CD-1 strain mice weighing 25-30 grams were selected and observed to be in good physical condition for later use.

[0046] 1.2 (Day 1) First Immunization: Take 1 mg of SMCP polypeptide antigen (1 ml) and an equal volume of Freund's complete adjuvant (catalog number: F5881, Sigma) and emulsify until the mixture does not diffuse in water. Inject mice intraperitoneally, with each mouse receiving 0.2 ml of the mixture.

[0047] 1.3 (Day 22) Second immunization: Take 0.5 mg of SMCP polypeptide antigen (0.5 ml) and an equal volume of Freund's incomplete adjuvant (catalog number: F5506, Sigma) and emulsify until the mixture does not diffuse in water. Inject mice intraperitoneally, with each mouse receiving 0.1 ml of the mixture. 1.4 (Day 36) Third immunization: 0.5 mg of SMCP polypeptide antigen (0.5 ml) and an equal volume of Freund's incomplete adjuvant (catalog number: F5506, Sigma) were emulsified until the mixture did not diffuse in water. Mice were immunized by intraperitoneal injection, with each mouse receiving 0.1 ml of the mixture.

[0048] 1.5 (Day 57) Fourth immunization: 1 mg of SMCP polypeptide antigen (1 ml volume) was injected intraperitoneally into mice for immunization. Each mouse was injected with 0.1 ml of SMCP polypeptide antigen.

[0049] 1.6 (Day 71) Whole blood collection: Mice were exsanguinated and bled to death. Approximately 1.2 ml of whole blood was collected from each mouse. The whole blood was incubated at 37°C for 30 minutes, then left at room temperature for 3 hours. The blood was centrifuged at 10,000 rpm for 10 minutes, and the supernatant serum was collected. This was repeated twice, once at 10,000 rpm for 10 minutes, and the supernatant serum was collected again. Approximately 0.6 ml of supernatant serum was collected from each mouse. This is the mouse SMCP antibody.

[0050] Note: 1. The concentration of SMCP polypeptide antigen used in the above steps is 1 mg / ml, dissolved in physiological saline. 2. Freund's complete adjuvant (catalog number: F5881, Sigma) contains cell wall components of Mycobacterium tuberculosis and is suitable for primary immunization to induce a strong immune response. Freund's incomplete adjuvant (catalog number: F5506, Sigma) does not contain these components and is used to enhance immunization to maintain the immune effect while reducing side effects. 3. After aliquoting according to experimental requirements, antibodies should be stored at -80°C, avoiding repeated freeze-thaw cycles as much as possible.

[0051] 2 Rabbit Immunization Experiment Procedure 2.1 Two New Zealand white rabbits weighing 2000-2500 grams were selected and observed in our laboratory for 7-14 days until they were in good health. One day before immunization, 1 ml of blood was collected from each rabbit via the marginal ear vein. The blood was incubated at 37°C for 30 minutes and then left at room temperature for 3 hours. The blood was centrifuged at 10,000 rpm for 10 minutes, and the supernatant serum was collected. This supernatant serum was then centrifuged again at 10,000 rpm for 10 minutes and used as a negative control in subsequent antibody titer testing.

[0052] 2.2 (Day 1) First Immunization: Take 2 mg of SMCP polypeptide antigen (1 ml volume) and an equal volume of Freund's complete adjuvant (catalog number: F5881, Sigma) and emulsify until the mixture does not diffuse in water. Administer 8 subcutaneous injections along both sides of the spine on the back of the rabbits, plus subcutaneous injection into both hind legs. The total volume of the mixture injected into each rabbit is 1 ml.

[0053] 2.3 (Day 22) Second immunization: Emulsify 1 mg of SMCP polypeptide antigen (0.5 ml) and an equal volume of Freund's incomplete adjuvant (catalog number: F5506, Sigma) until the mixture does not diffuse in water. Administer 0.5 ml of the mixture subcutaneously at 6 points along both sides of the spine on the back of the rabbits.

[0054] 2.4 (Day 36) Third immunization: Emulsify 1 mg of SMCP polypeptide antigen (0.5 ml) and an equal volume of Freund's incomplete adjuvant (catalog number: F5506, Sigma) until the mixture does not diffuse in water. Administer 0.5 ml of the mixture subcutaneously at 6 points along both sides of the spine on the back of the rabbits.

[0055] 2.5 (Day 50): 1 ml of blood was collected from the marginal ear vein of each rabbit and incubated at 37°C for 30 minutes, then left at room temperature for 3 hours. The blood was then centrifuged at 10,000 rpm for 10 minutes, and the supernatant serum was collected. This supernatant serum was then centrifuged again at 10,000 rpm for 10 minutes for further testing to determine the antiserum titer.

[0056] 2.6 (Day 57) Fourth immunization: Take 2mg of SMCP polypeptide antigen (1ml volume) and inject it subcutaneously at 6 points along both sides of the spine on the back of the rabbit. Each rabbit is injected with 0.5ml of SMCP polypeptide antigen.

[0057] 2.7 (Day 71) Whole blood collection: Rabbits were bleeded to death by cannulation of the carotid artery. The whole blood was incubated at 37°C for 30 minutes, then left at room temperature for 3 hours. The blood was centrifuged at 10,000 rpm for 10 minutes, and the supernatant serum was collected. This was repeated twice, once at 10,000 rpm for 10 minutes, and the supernatant serum was collected again. Approximately 50 ml of supernatant serum was collected from each rabbit. This is the rabbit SMCP antibody.

[0058] Note: 1. The concentration of SMCP antigen used in the above steps is 2 mg / ml, dissolved in physiological saline. 2. Freund's complete adjuvant (catalog number: F5881, Sigma) contains cell wall components of Mycobacterium tuberculosis and is suitable for primary immunization to induce a strong immune response. Freund's incomplete adjuvant (catalog number: F5506, Sigma) does not contain these components and is used to enhance immunization, maintaining the immune effect while reducing side effects. 3. After aliquoting according to experimental requirements, antibodies should be stored at -80°C, avoiding repeated freeze-thaw cycles as much as possible.

[0059] Example 4: Specific Detection and Application of SMCP Polyclonal Antibody The applicant utilized a 129 / Sv background constructed using CRISPR / Cas9 technology. Smcp The specificity of the prepared SMCP antibodies (including rabbit SMCP antibodies and mouse SMCP antibodies) was tested using a whole-body homozygous knockout mouse model.

[0060] 1. Western blot (WB) Experimental steps: (1) Prepare protein lysis solution according to the ratio of protease inhibitor (100× cocktail): protein lysis buffer (RIPA potent lysis buffer) = 1:100. Add 1 ml of protein lysis solution to every 100 mg of testicular tissue and 100 µl of protein lysis solution to every unilateral epididymal tail sperm. In wild-type control male mice ( Smcp + / + Testicular samples, sperm samples and Smcp Systemically homozygous knockout male mice ( Smcp - / - Protein lysis solution was added to the testicular and sperm samples, and the testicular tissue was cut into pieces with scissors. (2) Ultrasonic fragmentation of testicular and sperm samples: 25%, 50J, 4 degrees, 3s. After each ultrasonic fragmentation, place the sample on ice for 10s before performing the next ultrasonic fragmentation until the sample is completely fragmented. (3) At 4℃, the sample was pyrolyzed on a rotating shaker for 40 min at a speed of 10 rpm; (4) Centrifuge at 13000 rpm / 4℃ / 40 min, and take the supernatant into a new 1.5 ml EP tube, which is testicular or sperm protein; (5) Protein concentration determination. Protein concentration was determined using the BCA method. The required protein standard curve is as follows:

[0061] (6) Take 1 μl of protein sample, add 19 μl of protein lysis buffer to make up to 20 μl of system, and then add 200 μl of BCA staining solution; (7) After adding the staining solution, incubate in a 37°C oven in the dark for 30 min, and then detect it using an ELISA reader; (8) Add 6× Loading buffer to the protein sample until the final concentration is 1×; (9) Boil egg whites in a metal bath at 95°C for 10 minutes, then store at -20°C for later use. (10) Clean the WB glass plate and rinse it with double-distilled water (ddH2O), then dry it in an oven; (11) Install the glue-making frame and add ddH2O to check for leaks; (12) Prepare 12% separating adhesive according to the separating adhesive formula in Table 1, pour the separating adhesive into the glass plate, and slowly add isopropanol sealant from left to right; (13) Let stand for 30-40 minutes until the separated gel solidifies; (14) Discard the isopropanol and wash with ddH2O 2-3 times; (15) Prepare 5% concentrated glue according to the formula in Table 2. Pour the concentrated glue into the glass plate and insert the comb at an angle to avoid generating air bubbles. (16) Let stand for 20-30 minutes until the concentrated gel solidifies; (17) Install WB glue in the electrophoresis tank, insert the glass plate into the glue tank, prepare the electrophoresis buffer according to the electrophoresis buffer formula in Table 3, and pour the electrophoresis buffer into the electrophoresis tank to check for leaks. (18) Remove the comb, add the sample, and add 50 μg of protein; (19) Add electrophoresis buffer, perform electrophoresis at a constant voltage of 90V. When the protein sample reaches the separating gel, change the voltage to a constant voltage of 120V and continue electrophoresis. Stop electrophoresis when the bromophenol blue reaches the bottom of the WB gel; (20) Transfer membrane: Prepare transfer buffer according to the transfer buffer formula in Table 4. Put the filter paper into the pre-cooled transfer buffer to equilibrate in advance, and soak the PVDF membrane in methanol to activate it. After electrophoresis, take out the gel plate, wash the surface of the electrolytic buffer with ddH2O, pry open the glass plate, put the filter paper, gel block and PVDF membrane into the transfer sandwich clamp in sequence and clamp them tightly. Install them into the transfer frame, pour in the transfer buffer, add two ice blocks, and transfer membrane at 90V for 2 hours. (21) Seal: Use tweezers to remove the PVDF membrane from the sandwich clamp, place it in the incubation box, pour in 5% skim milk powder (5g skim milk powder + 100ml 1×TBS) to immerse the membrane, shake at 50rpm, and seal at room temperature for 2h. (22) Discard the blocking solution, wash repeatedly with 1×TBS 3-5 times to remove the residual blocking solution, discard the TBS, add the primary antibody (rabbit SMCP antibody dilution ratio is 1:1000, mouse SMCP antibody dilution ratio is 1:1000), and incubate at 4 degrees Celsius at 50 rpm for 12 h. (23) After 12 hours, discard the primary antibody, wash the membrane with TBST (2L 1×TBS + 1 ml Tween-20), and shake on a shaker at 150 rpm for 10 min × 3 times. (24) Discard the TBST, incubate the secondary antibody, and dilute the secondary antibody with 5% skim milk powder. Shake at 50 rpm and incubate at room temperature for 2 hours; (25) Discard the secondary antibody, wash the membrane with TBST, and shake on a shaker at 150 rpm for 10 min × 3 times; (26) The ECL exposure solution is prepared and used immediately, and the WB film is exposed using the gel imaging system.

[0062] Table 1 12% Separating Gel Formulation / Block

[0063] Table 2 5% Stacking Gum Formulation / Block

[0064] Table 3 Electrophoresis buffer formulation (1×)

[0065] Table 4 Transfer buffer formulation (1×)

[0066] Experimental results: Western blotting results showed that both rabbit and mouse SMCP antibodies could detect it. Smcp + / + SMCP protein in male rat testes, and in Smcp - / - SMCP protein was not detected in the testes of male rats. Figure 15 (A~15B). Detectable using mouse SMCP antibody. Smcp + / + SMCP protein in male mouse sperm, and in Smcp - / - SMCP protein was not detected in the sperm of male mice. Figure 15 C). This indicates that the SMCP antibody prepared in this invention has good specificity and can be used for Western blotting experiments. Smcp + / + Wild-type control male rats Smcp - / - : Smcp Systemically homozygous knockout male mice (β-tubulin: internal reference) 2. Immunofluorescence (IF) method Experimental steps: (1) Material selection: Smcp + / + and Smcp- / - Complete testicular tissue (with intact capsule) of a male rat; (2) Fixation: Prepare MDF fixative according to Table 5. Place one testis in 1 ml of MDF fixative and fix for 6 hours. Then cut the testis in half vertically along the longitudinal axis with a blade and continue fixing for 48 hours. (3) Dehydration: Soak in 70% ethanol for 24 hours, soak in 80% ethanol for 2 hours, soak in 90% ethanol for 2 hours, soak in 100% ethanol for 30 minutes, and then soak in 100% ethanol for 30 minutes. (4) Permeation: Soak in a solution of 100% ethanol: xylene = 1:1 for 25 min, then soak in xylene for 25 min; (5) Wax impregnation: Immerse in paraffin at 65℃ for 45 minutes, then immerse in paraffin at 65℃ for another 45 minutes; (6) Embedding: The tissue was embedded in a paraffin embedding machine, cooled into blocks, and stored at room temperature; (7) Sectioning: The paraffin tissue block was sectioned to a thickness of 5 μm, and baked in an oven at 65℃ for 12 h and then in an oven at 37℃ for 2 h. (8) Dewaxing: Place the slices in xylene in a 37℃ oven and immerse them twice for 15 min each time; (9) Hydration: Soak in 100% ethanol for 2 min, then soak in 100% ethanol for 2 min, 90% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and ddH2O for 10 min. (10) Antigen retrieval: Prepare the acidic antigen retrieval solution according to Table 6. Immerse the slide in the acidic antigen retrieval solution and heat in a microwave oven on high for 3 minutes and then on low for 7 minutes. Remove and cool to room temperature for 1 hour.

[0067] (11) Wash the slides 3 times with 1×PBS, 5 min each time, on a shaker at 50 rpm; (12) Blocking: Add 1 / 1000 of Triton-100X to 1% BSA (1g BSA + 100ml 1×PBS) and block at room temperature for 2h; (13) Primary antibody incubation: Discard the blocking solution, add primary antibody (1 μl rabbit SMCP antibody + 200 μl 1% BSA), and incubate at 4℃ for 12 h.

[0068] (14) Discard the primary antibody incubation solution, wash the primary antibody with 1×PBS, 5 min × 3 times, shake at 50 rpm; (15) Add fluorescent secondary antibody and Hoechst (1 μl fluorescent secondary antibody + 1 μl Hoechst + 1 ml 1% BSA), and incubate at room temperature in the dark for 2 h; (16) Discard the liquid, wash with 1×PBS, 5 min × 3 times, shake at 50 rpm; (17) Mount the slide with glycerin and take a picture.

[0069] Table 5 MDF fixative (100ml)

[0070] Table 6 Antigen Acidic Retrieval Solution (200ml)

[0071] Experimental results: Testicular IF results are as follows Figure 16 As shown, rabbit SMCP antibodies can be used to detect... Smcp + / + Fluorescent signal of SMCP protein in male mouse testes. SMCP protein is located in the cytoplasm of elongated sperm cells in wild-type mouse testes, and... Smcp - / - No fluorescent signal of SMCP protein was detected in the testes of male mice. This indicates that the SMCP antibody prepared in this invention has good specificity and can be used for IF experiments. 3. Co-immunoprecipitation (Co-IP) (1) Take Smcp + / + and Smcp - / - Bilateral testicular tissue from male rats was removed from its capsule. Protein lysis solution was prepared according to a ratio of 1:100:1 protease inhibitor (100×cocktail) to protein lysis buffer (RIPA potent lysis buffer). Protein lysis solution was added to both sides of the testicular tissue at a standard ratio of 1 ml of protein lysis solution per 100 mg of testicular tissue. The testicular tissue was then cut into small pieces with scissors. (2) Ultrasonic disruption: 25%, 50J, 4℃, 3s. After each ultrasonic treatment, let it stand on ice for 10s before performing the next ultrasonic treatment until the sample is completely disrupted. (3) Lyse the protein on a rotating shaker for 40 min at 4℃ and 10 rpm; (4) Centrifuge at 13000 rpm / 4℃ / 40 min and retain the protein supernatant; (5) Wash the beads during centrifugation, take 30µl of Protein A / G beads into a 1.5ml EP tube as the pretreatment group, and remove the magnetic bead protection solution from the magnetic rack; (6) Add 1 ml of RIPA high-efficiency lysis buffer, shake on a rotating shaker for 5 min at 10 rpm, and remove RIPA from the magnetic rack; (7) Repeat step (6), and wash the beads 3 times with RIPA; (8) The protein supernatant from step (4) was added to the pretreated beads and shaken at 4°C for 1 hour at 10 rpm. (9) Aspirate the supernatant onto a new 1.5ml EP tube using a magnetic rack, leaving 50µl as the Input group sample, and add 5µl of SMCP antibody to the remaining supernatant protein; (10) Shake on a rotating shaker at 4℃ for 12 hours at 10 rpm. The purpose of this step is to allow the antigen and antibody to bind. (11) Take 60 µl of Protein A / G beads into a 1.5 ml EP tube as the IP group, and wash the beads three times with RIPA as above; (12) After 12 hours of antigen-antibody binding, all the liquid was transferred to the beads of the IP group; (13) Shake on a rotating shaker at 4℃ for 3 hours at 10 rpm. The purpose of this step is to allow the antigen, antibody and beads to bind. (14) Discard the supernatant on the magnetic rack and keep the beads; (15) Wash the beads three times on a rotating shaker, RIPA wash, 5 minutes each time, 10 rpm; (16) Boiling beads: Add 60 μl of 2× Loading buffer, boil in a 95℃ metal bath for 10 min, centrifuge after boiling, and transfer the supernatant to a new 1.5 ml EP tube on a magnetic rack. This is the IP group sample. (17) Conduct WB experiments.

[0072] Experimental results: Testicular Co-IP results are as follows Figure 17 As shown in A~17B, rabbit SMCP antibody and mouse SMCP antibody were used in... Smcp + / + SMCP protein was detected in the testes of male mice, and in Smcp - / - SMCP protein was not detected in the testes of male rats. This indicates that the SMCP antibody prepared in this invention has good specificity and can be used for Co-IP experiments.

[0073] Specificity assay of commercially available SMCP polyclonal antibody (catalog number: NBP2-45888, NOVUS) The applicant utilized a 129 / Sv background constructed using CRISPR / Cas9 technology. Smcp The specificity of a commercially available SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS) was tested in a whole-body homozygous knockout mouse model.

[0074] 1. The experimental procedure for Western blotting differs from that of Example 4 only in the following aspects: Step (1) is as follows: Prepare protein lysis solution according to the ratio of protease inhibitor (100× cocktail): protein lysis buffer (RIPA potent lysis buffer) = 1:100. Add 1 ml of protein lysis solution to every 100 mg of testicular tissue. In wild-type control male mice ( Smcp + / + Testicular samples, Smcp Heterozygous knockout male mice ( Smcp + / - Testicular samples and Smcp Systemically homozygous knockout male mice ( Smcp - / - Protein lysis solution was added to the testicular sample, and the testicular tissue was cut into pieces with scissors; Step (22) is as follows: Discard the blocking solution, wash repeatedly with 1×TBS 3-5 times to remove the residual blocking solution, discard the TBS, add the primary antibody (commercial SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS) diluted at 1:500), and incubate at 4 degrees Celsius for 50 rpm for 12 h. Experimental results: WB results are as follows Figure 18 As shown, a commercially available SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS) was used in... Smcp + / + male rat ,Smcp + / - male rats and Smcp - / - No difference was detected in the protein bands at the target location in the testes of male mice. This indicates that the commercially available SMCP antibody has poor specificity and cannot be used for Western blotting experiments. Smcp + / + Wild-type control male rats Smcp + / - : Smcp Gene-integrated heterozygous knockout male mice Smcp - / - : Smcp Systemically homozygous knockout male mice (β-tubulin: internal reference) 2. Immunofluorescence assay: The experimental steps differ from those in Example 4 only in that: Step (13) is as follows: Primary antibody incubation: Discard the blocking solution, add primary antibody (1 μl commercial SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS) + 100 μl 1% BSA), and incubate at 4℃ for 12 h.

[0075] Experimental results: Testicular IF results are as follows Figure 19 As shown, a commercially available SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS) was used in... Smcp + / + male rat testicles and Smcp - / - The fluorescence signal of the target protein detected in the testes of male mice showed no difference. This indicates that the commercially available SMCP antibody has poor specificity and cannot be used for IF experiments. Smcp + / + Wild-type control male rats Smcp - / - : Smcp (Hypozygous knockout male mice) 3. Immunoprecipitation, the only difference between this and the immunofluorescence method in Example 4 is: Step (1) is: Take two Smcp + / + Bilateral testicular tissue from male rats was used, one as the SMCP group and the other as the IgG group. The capsule was removed, and a protein lysis solution was prepared according to a ratio of 1:100:1 for protease inhibitor (100× cocktail) and protein lysis buffer (RIPA potent lysis buffer). The protein lysis solution was added to both testicular tissues at a rate of 1 ml per 100 mg of testicular tissue, and the testicular tissue was then minced with scissors. Step (9) is as follows: Aspirate the supernatant from the magnetic rack into a new 1.5ml EP tube. 50µl of the SMCP group and IgG group are reserved as Input group samples. 8µl of commercial SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS) is added to the remaining supernatant protein of the SMCP group, and 8µl of IgG antibody is added to the remaining supernatant protein of the IgG group. Experimental results: Testicular Co-IP results are as follows Figure 20 As shown, IgG antibodies were used in Smcp + / + The amount of the target site protein drawn from the testes of male mice was greater than that drawn using a commercial SMCP monoclonal antibody (catalog number: NBP2-45888, NOVUS), indicating that the commercial SMCP antibody has poor specificity and cannot be used for Co-IP experiments.

[0076] Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing an SMCP polyclonal antibody, characterized in that, The preparation method includes: immunizing animals with a polypeptide whose amino acid sequence is shown in SEQ ID NO:1 as an immunogen, collecting whole blood from the immunized animals, and separating the SMCP polyclonal antibody.

2. The preparation method according to claim 1, characterized in that, The polypeptide is coupled with a carrier protein.

3. The preparation method according to claim 2, characterized in that, The carrier protein is KLH protein, BSA protein, or OVA protein.

4. The preparation method according to any one of claims 1 to 3, characterized in that, It also includes immune adjuvants, which involve immunizing animals with a composition containing an immunogen and an immune adjuvant.

5. The preparation method according to claim 4, characterized in that, The immune adjuvant is Freund's complete adjuvant, Freund's incomplete adjuvant, aluminum salt adjuvant, liposome, monophosphoryl lipid A, or cytokine adjuvant.

6. The preparation method according to claim 1, characterized in that, The animal in question is either a mouse or a rabbit.

7. The preparation method according to claim 1, characterized in that, The animals were immunized 3 to 5 times.

8. The preparation method according to claim 1, characterized in that, The preparation method includes: The polypeptide with the amino acid sequence shown in SEQ ID NO:1 was coupled to a carrier protein to obtain a coupled protein, denoted as immunogen A; Freund's complete adjuvant was mixed and emulsified with the coupled protein to obtain a coupled protein emulsified with Freund's complete adjuvant, denoted as immunogen B; Freund's incomplete adjuvant was mixed and emulsified with the coupled protein to obtain a coupled protein emulsified with Freund's incomplete adjuvant, denoted as immunogen C. Animals were immunized for the first time using immunogen B; 20-22 days after the first immunization, animals were immunized for the second time using immunogen C; 13-15 days after the second immunization, animals were immunized for the third time using immunogen C; 20-22 days after the third immunization, animals were immunized for the fourth time using immunogen A. On days 13-15 after the fourth immunization, whole blood was collected from the immunized animals, and the SMCP polyclonal antibody was isolated.

9. The SMCP polyclonal antibody prepared by the method according to any one of claims 1 to 8.

10. The use of the SMCP polyclonal antibody according to claim 9 in the preparation of immunoassay reagents or products for detecting SMCP protein.

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