Immunogens for the production of testis-specific glyceraldehyde-3-phosphate dehydrogenase lysine malonylation site-specific antibodies

Abstract

The application discloses an immunogen for preparing testis-specific glyceraldehyde-3-phosphate dehydrogenase lysine malonylation site-specific antibody, and belongs to the technical field of biotechnology.The immunogen is a malonylation modified polypeptide, and the amino acid sequence is IIPASTGAAKAVT, corresponding to the 278th-290th amino acid of a human GAPDHS protein, wherein a lysine at the 287th position (the 10th position in the sequence) is subjected to malonylation modification.A specific antibody designed according to the immunogen has the characteristics of high titer and high specificity. By screening a human sperm sample by using the antibody, it is found that the antibody can not only specifically recognize the GAPDHS with the lysine at the 287th position subjected to malonylation modification in vitro and in human sperm, but also is closely related to sperm forward movement and asthenozoospermia. The application has great significance in the field of preparation of reagents for precision diagnosis and treatment of idiopathic male infertility.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an immunogen for preparing an antibody specific to the 287th lysine malonylation site of testis-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHS). This immunogen can be used to produce antibodies that specifically recognize the 287th lysine malonylation modification of GAPDHS, and can be applied to the development of diagnostic reagents for idiopathic asthenospermia. Background Technology

[0002] Male infertility, a widespread health problem, now affects 10%-15% of couples of reproductive age in my country. Half of male infertile patients exhibit insufficient progressive motility of sperm, and clinically, a progressive motility (PR) sperm percentage below 32% is defined as asthenospermia (World Health Organization, Laboratory Manual of Human Semen Examination and Processing (Fifth Edition)). Due to the complex and diverse etiologies of asthenospermia, its pathogenesis is still far from being fully understood.

[0003] Sperm are highly specialized cells with both transcription and translation highly silenced, and their functional regulation is highly dependent on post-translational modifications (PTMs). Post-lysine modifications (PLMs) have become a research hotspot in recent years, affecting not only the cellular protein translation process by regulating nuclear histone function but also cytoplasmic proteins and regulating various cellular physiological functions. Lysine malonylation (Kmal) is a novel PLM discovered in recent years. By adding a malonyl group, the charge of lysine is changed from +1 to -1, ultimately affecting the three-dimensional structure and function of proteins. Previous studies have found that compared with men who have abstained from ejaculation for a long period, men who have abstained from ejaculation for a short period have significantly increased sperm concentration, motility, viability, normal morphology rate, acrosome reaction capacity, and total antioxidant capacity, while the overall level of sperm Kmal is decreased. Other studies have found that Kmal is mainly located in the tail of human sperm, suggesting that the level of lysine malonylation modification is likely closely related to the regulation of human sperm motility.

[0004] Glycolysis is the main pathway for ATP production in mammalian sperm, and the testis-specific glycolytic enzyme GAPDHS plays a crucial regulatory role in it. GAPDHS is responsible for catalyzing the oxidation of glyceraldehyde-3-phosphate to generate ATP and NADH. Studies have shown that the sperm of GAPDHS gene knockout mice exhibit complete blockage of glycolysis, leading to severe sperm motility dysfunction and infertility. This finding fully confirms the indispensable role of GAPDHS in maintaining normal sperm motility. Meanwhile, the inventors found that compared with normal fertile individuals, the level of GAPDHS-K287 malonylation modification in the sperm of patients with asthenospermia was significantly increased and negatively correlated with sperm forward motility. These findings suggest that GAPDHS-K287 malonylation is likely a key biomarker for asthenospermia.

[0005] However, at present, there is still a lack of antibodies that specifically detect GAPDHS K287 malonylation, which limits the application of this modification in clinical diagnosis. Finding a suitable immunogen for preparing specific antibodies is the key to solving the above dilemma. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides an immunogen for preparing a specific antibody against the 287th lysine malonylation site of GAPDHS. The antibody prepared by this immunogen has the characteristics of high titer and good specificity, and can specifically recognize malonylated GAPDHS in vitro and in sperm, and can be used to prepare diagnostic reagents for idiopathic asthenospermia.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution:

[0008] Firstly, the present invention provides a malonylated modified polypeptide immunogen, which is obtained by malonylating the 10th lysine residue based on the amino acid sequence shown in SEQ ID NO.1.

[0009] Furthermore, the amino acid sequence shown in SEQ ID NO.1 corresponds to amino acids 278-290 of the human GAPDHS protein (UniProtKB: O14556), and the lysine at position 10 is located at position 287 of the human GAPDHS protein.

[0010] Secondly, this invention provides a method for preparing the above-described malonyl-modified polypeptide immunogen, comprising the following steps:

[0011] (1) A malonylated modified polypeptide was synthesized and cross-linked with keyhole hemocyanin to obtain a keyhole hemocyanin cross-linked polypeptide;

[0012] (2) Dissolve the keyhole hemocyanin crosslinked polypeptide and emulsify it with Freund's complete adjuvant by shaking to obtain the malonylated modified polypeptide immunogen.

[0013] Thirdly, this invention provides the application of the malonylated modified polypeptide immunogen described above in the preparation of a specific antibody for detecting GAPDHS-K287mal.

[0014] Fourthly, this invention provides a method for preparing a specific antibody for detecting GAPDHS-K287mal, comprising the following steps: immunizing immunized animals by subcutaneous injection at multiple points on the back using the malonylated modified polypeptide immunogen; immunizing a total of 3-5 times, once every 2-3 weeks, at a dose of 350 μg / injection to 450 μg / injection; collecting blood 6-8 days after the last injection, separating and purifying to obtain a specific antibody with a titer greater than 1:600,000.

[0015] Furthermore, the immunized animals include New Zealand white rabbits.

[0016] The specific preparation steps of the antibody for detecting GAPDHS-K287mal in this invention are as follows:

[0017] (1) Peptide synthesis: The GAPDHS peptide-287 lysine site malonylated and unmodified GAPDHS peptide was synthesized and crosslinked with keyhole hemocyanin (KLH);

[0018] (2) Immunogen preparation and immunization animals: Two New Zealand white rabbits (approximately 2-2.5 kg) were used as immunization animals. KLH cross-linked peptides were dissolved in 400 μL of 0.01 mol / L phosphate buffer and mixed with Freund's complete adjuvant in equal proportion. The mixture was thoroughly shaken and emulsified. The emulsified immunogen was used for subcutaneous immunization by injecting it into multiple sites on the back of the rabbits. A total of 4 immunizations were performed, with 400 μg administered every 2-3 weeks. Seven days after the last injection, ear artery blood was collected, and the antibody titer was determined using an indirect ELISA method. When the titer was greater than 1:600,000, blood was collected to prepare and purify the antibody.

[0019] (3) Antibody purification: The antibody was purified by antigen affinity purification method, and the titer and specificity of the purified antibody were detected by ELISA and dot blot hybridization.

[0020] Fifth aspect: The present invention provides a specific antibody for detecting GAPDHS-K287mal, which is prepared by the above preparation method.

[0021] The specific antibody provided in this invention for detecting GAPDHS-K287mal peptide has a detection sensitivity of 0.5 ng / mL and can detect GAPDHS-K287mal peptide as low as 1 ng.

[0022] Sixth aspect: The present invention provides the use of the specific antibody in the preparation of reagents for detecting GAPDHS-K287mal.

[0023] Furthermore, the reagent is used for the in vitro or in vivo detection of GAPDHS-K287mal.

[0024] Seventh aspect: The present invention provides the use of the specific antibody in the preparation of a diagnostic reagent for idiopathic asthenospermia.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The GAPDHS-K287mal site-specific antibody designed using a polypeptide with a specific amino acid sequence as an antigen in this invention exhibits high titer and high specificity. Screening human sperm samples with this antibody revealed that it can specifically recognize GAPDHS modified with malonylation at lysine position 287 in vitro and in human sperm, and that the level of GAPDHS-K287mal in sperm is closely related to sperm forward motility and asthenospermia. The GAPDHS-K287mal site-specific antibody designed, developed, and prepared in this invention can be used to prepare a precision diagnostic reagent for idiopathic male infertility. Attached Figure Description

[0027] Figure 1 This is a mass spectrometry analysis of the GAPDHS-K287mal site-specific antibody immunogen in Example 1 of the present invention.

[0028] Figure 2 This is a mass spectrometry analysis chromatogram of the unmodified GAPDHS peptide in Example 1 of the present invention.

[0029] Figure 3 This figure shows the purity identification results of the GAPDHS-K287mal site-specific antibody in Example 1 of this invention. M represents the protein molecular weight standard, and the molecular weight values ​​corresponding to different bands are marked on the left side of the figure. Lane 1 shows the results of SDS-PAGE electrophoresis and Coomassie brilliant blue staining of the antibody obtained after purification using the GAPDHS-K287 malonylation site-specific antibody immunogen. Lane 2 shows the electrophoretic detection results of the product obtained after further reverse screening and purification using unmodified peptides based on the purified antibody product of lane 1. The protein bands with a molecular weight of 50 kDa shown in lanes 1 and 2 are both heavy chain bands of the target antibody.

[0030] Figure 4 This is the specific detection result of the GAPDHS-K287mal site-specific antibody in Example 2 of the present invention.

[0031] Figure 5 This document presents the verification results of the GAPDHS-K287mal site-specific antibody used in Example 1 of this invention for recognizing malonylated GAPDHS in vitro and in sperm. A: An immunofluorescence assay was performed using the GAPDHS-K287mal site-specific antibody to detect the malonylation level at the GAPDHS-K287 site in normal human sperm samples; the scale bar in the figure is 5 μm. B: An immunoblotting assay was performed using the GAPDHS-K287mal site-specific antibody to detect the difference in GAPDHS-K287mal levels in total sperm protein samples from normal individuals and patients with asthenospermia in vitro.

[0032] Figure 6 This presents the experimental results of the correlation between GAPDHS-K287mal levels and sperm progressive motility and asthenospermia in Application Example 2 of this invention. A: An immunoblotting experiment was performed using a GAPDHS-K287mal site-specific antibody to detect the relative content of GAPDHS-K287mal in sperm samples from normal individuals and patients with asthenospermia; experimental data are presented in the form of "mean ± standard error", with each data point in the figure corresponding to the detection result of a single sample; statistical analysis of inter-group differences was performed using Welch's t-test, where... express P <0.001. B: The correlation between sperm progressive motility and the relative content of GAPDHS-K287mal was evaluated using Pearson correlation coefficient analysis; in the statistical analysis, when P A value <0.05 is considered statistically significant. Detailed Implementation

[0033] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0034] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0035] Example 1: Design and preparation of GAPDHS-K287mal site-specific antibody

[0036] 1. Analysis and design of peptide sequences

[0037] The amino acids of GAPDHS were analyzed using DNAstar software to determine their epitope composition and to evaluate various indices, including hydrophilicity and antigenicity. Further consideration was given to amino acid type, distribution, and structural complexity. Based on the amino acid sequences before and after lysine at position 287 of the malonylated GAPDHS, the 13 amino acids from positions 278 to 290 were ultimately determined as the sequence for the synthesized polypeptide, corresponding to the sequence 278-IIPASTGAAKAVT-290 (SEQ ID NO. 1). The malonylated GAPDHS polypeptide at position 287 (278-IIPASTGAAKmalAVT-290, molecular weight 1284.67, Kmal being the malonylated lysine) and the unmodified GAPDHS polypeptide (278-IIPASTGAAKAVT-290, molecular weight 1199.40) were synthesized. Mass spectrometry analysis results of the polypeptides are shown below. Figure 1 and Figure 2 As shown.

[0038] 2. Immunization regimen

[0039] New Zealand white rabbits weighing 2.0-2.3 kg (6-7 months old, Hunan Taiping Biotechnology Co., Ltd.) were selected as immunization subjects. 400 μg of the KLH carrier protein-conjugated GAPDHS-K287mal immunogen was dissolved in 400 μL of 0.01 M phosphate-buffered saline (PBS, pH 7.4) and thoroughly emulsified with an equal volume of Freund's complete adjuvant to form a water-in-oil emulsion. The initial immunization was performed via subcutaneous injection at multiple sites on the back. Subsequent booster immunizations were administered every 3 weeks, for a total of 4 immunizations. The initial immunization used complete adjuvant, while subsequent booster immunizations used Freund's incomplete adjuvant. Seven days after the last immunization, auricular artery blood was collected, and serum antibody titers were monitored using indirect enzyme-linked immunosorbent assay (ELISA). When the titer against the target GAPDHS-K287mal peptide consistently exceeded 1:600,000, carotid artery puncture was performed to collect highly immune serum for purification.

[0040] 3. Antibody purification

[0041] An antigen affinity purification chromatography column was prepared by coupling malonylated peptide GAPDHS with a mercapto gel. The resulting antiserum was mixed with phosphate-buffered saline (PBS) in equal volumes to prepare an antibody sample. This sample was loaded into the pre-equilibrated affinity chromatography medium at a suitable flow rate to ensure sufficient binding of the antibody components to the ligands. Subsequently, a low-pH glycine-hydrochloric acid buffer was used as the elution buffer to dissociate the specifically bound antibody from the medium and collect it. The collected antibody solution was immediately transferred to a dialysis bag with an appropriate molecular weight cutoff and dialyzed against PBS at 4°C for at least 12 hours to replace the buffer system and remove small molecule impurities. Finally, the purified antibody solution was analyzed for key parameters including purity, concentration, and titer. Indirect ELISA showed that the purified antibody exhibited extremely high affinity for the GAPDHS-K287mal target peptide, with a stable titer of approximately 512 K. To accurately assess its specificity, we simultaneously tested the cross-reactivity of the antibody to the unmodified peptide, and its titer was no higher than 64K (Table 1). Calculations showed that the antibody's specific affinity for the target modified epitope was approximately eight times that for the unmodified form, fully demonstrating its potential as a highly specific diagnostic reagent. Identification by SDS-PAGE combined with Coomassie brilliant blue staining showed that the antibody purity was higher than 85%, and the concentration, as determined by the BCA method, was 0.56 mg / mL. Figure 3 Lane 1).

[0042] Table 1. Results of purified antibody assay (using a non-malonylated peptide column).

[0043]

[0044] To obtain a highly specific anti-malonylated GAPDHS antibody, we employed affinity chromatography based on non-malonylated GAPDHS peptides as negative ligands for antibody purification. The peptide was immobilized on a thiol-gel medium to prepare an adsorption column. The initially obtained malonylated antibody solution was passed through the column at a controlled flow rate to adsorb and remove antibody components that could cross-react with the non-malonylated form. The permeate was collected to obtain the highly specific malonylated antibody. After equilibration by dialysis with PBS at 4°C, key parameters, including purity, concentration, and titer, were measured in the purified antibody solution. Indirect ELISA showed that the antibody had a titer of at least 1,024 K against the malonylated GAPDHS peptide, while the titer against the non-malonylated form was only about 16 K, a ratio exceeding 60-fold, demonstrating excellent modification specificity (Table 2). SDS-PAGE and Coomassie Brilliant Blue staining showed a purity higher than 90%. Figure 3The concentration was determined to be 0.63 mg / mL using the BCA method (lane 2). Based on the above parameters, the detection sensitivity of this antibody for the target peptide GAPDHS-K287mal was calculated to be 0.61 ng / mL.

[0045] Table 2 Results of secondary purified antibody detection

[0046]

[0047] Example 2: Evaluation of the specificity of the GAPDHS-K287mal antibody

[0048] To accurately evaluate the recognition sensitivity and cross-reactivity of the antibody of this invention, we designed a peptide array-dot hybridization experiment: the immunogen of this invention (SEQ ID NO.1, K287mal) and its unmodified control peptide were serially diluted and precisely spotted onto a nitrocellulose membrane using a spotter. After blocking, the membrane was incubated overnight at 4°C with the antibody of this invention (working concentration: 0.5 μg / mL). After washing, it was incubated with HRP-labeled secondary antibody, and chemiluminescence detection was performed using a high-sensitivity ECL substrate, followed by quantitative analysis of optical density using an imaging system. Figure 4 As shown, the antibody of this invention can specifically recognize the GAPDHS-K287mal target peptide at concentrations as low as 1 ng, and its detection signal exhibits a good dose-response relationship with peptide concentration. Crucially, even at the highest concentration (25 ng), no cross-reactivity was detected between the antibody of this invention and the unmodified peptide. Figure 4 This demonstrates the high affinity of the antibody of this invention for the specific epitope K287mal.

[0049] Application Example 1: The GAPDHS-K287mal antibody recognizes malonylated GAPDHS in vitro and in sperm.

[0050] 1. Immunofluorescence analysis:

[0051] (1) Preparation, fixation and attachment of sperm samples

[0052] Normal human sperm precipitate purified by Percoll density gradient centrifugation was resuspended in PBS solution containing 4% paraformaldehyde and fixed at room temperature for 15 minutes. After fixation, sperm were collected by centrifugation at 500×g for 5 minutes and washed twice with PBS buffer to completely remove the fixative. The washed sperm precipitate was resuspended in an appropriate amount of PBS and added to the bottom of a confocal culture dish pretreated with 0.01% poly-L-lysine. The dish was incubated at 37°C for 30 minutes to allow the sperm to adhere to the bottom. Excess suspension was aspirated, and the sample was then gently washed three times with PBS for 2-3 minutes each time.

[0053] (2) Cell permeation and non-specific site blockade

[0054] Discard the PBS and add 0.3% Triton X-100 in PBS solution. Permeabilize at room temperature for 12 minutes to enhance cell membrane permeability. After permeabilization, gently rinse the sample three times with PBS. Then, add blocking buffer containing 5% bovine serum albumin in PBS and incubate at room temperature for 90 minutes to effectively block non-specific binding sites.

[0055] (3) Antibody incubation and washing

[0056] Remove the blocking buffer and directly add the GAPDHS-K287mal specific primary antibody of this invention, diluted 1:150 with the blocking buffer, ensuring the solution completely covers the sample. Incubate overnight (approximately 16 hours) in a humidified chamber at 4°C. The next day, recover the primary antibody and wash the sample four times with PBS wash buffer containing 0.1% Tween-20 on a shaker for 8 minutes each time to thoroughly remove unbound primary antibody. Then, add DyLight 594-labeled goat anti-rabbit secondary antibody, diluted 1:500 with the blocking buffer, and incubate at room temperature in the dark for 60 minutes.

[0057] (4) Nuclear staining and mounting observation

[0058] After secondary antibody incubation, the samples were washed four times with the aforementioned PBST buffer, 8 minutes each time, in the dark. Subsequently, the samples were stained with 1 μM DAPI solution and incubated at room temperature in the dark for 10 minutes. After staining, the samples were rinsed three times with PBS to remove excess dye. Finally, a small amount of anti-fluorescence quenching mounting medium was added to the culture dish, and observation and image acquisition were immediately performed using a laser scanning confocal microscope. Figure 5 As shown in Figure A, in normal human sperm, the GAPDHS-K287mal signal (red fluorescence) is specifically located in the principal and terminal segments of the sperm flagellum (FL: fluorescence field; BF: bright field), which is consistent with the biological functional location of GAPDHS, further confirming the high specificity of the antibody.

[0059] 2. Extraction of sperm protein

[0060] Semen samples from normal individuals and patients with asthenospermia were liquefied for 30-60 minutes and then purified using Percoll density gradient centrifugation. The specific steps were as follows: 1.5 mL of 80% Percoll solution was spread on top of 1.5 mL of 40% Percoll solution, 2 mL of liquefied semen was slowly added, and the mixture was centrifuged at 300 × g for 20 minutes at room temperature. Remove the supernatant, collect the sperm pellet, and wash twice with preheated HEPES buffer (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM glucose, 1 mM Na-pyruvate, 10 mM lactic acid, 20 mM HEPES, pH 7.4) to remove residual Percoll solution. Finally, lyse the somatic cells with 200 μL of somatic cell lysis buffer (0.2% SDS, 0.5% Triton X-100), centrifuge at 800×g for 10 minutes, remove the supernatant, and resuspend the purified sperm pellet in RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) containing 1× protease inhibitor cocktail and 1× deacylate inhibitor (containing NAM and TSA). The samples were vortexed on ice for 2 hours to ensure complete lysis. Subsequently, they were centrifuged at 12,000 × g for 15 minutes at 4 °C, and the supernatant was collected. Total protein concentration was determined using a BCA protein quantification kit. All samples were uniformly diluted to 2 μg / μL with RIPA lysis buffer, aliquoted, and stored at -80 °C.

[0061] 3. Western blot analysis:

[0062] 30 μg of total sperm protein was separated by 10% SDS-PAGE gel electrophoresis. The protein was transferred to a PVDF membrane using a wet transfer method at 4°C and a constant current of 250 mA for 2 hours. After transfer, the membrane was blocked for 1 hour at room temperature on a rocker at 60 rpm using TBST solution (blocking buffer) containing 5% bovine serum albumin (BSA). After blocking, the membrane was incubated overnight at 4°C with the GAPDHS-K287mal specific primary antibody prepared according to this invention (diluted 1:1000 in blocking buffer). The next day, the membrane co-incubated with the primary antibody was removed from 4°C and incubated at room temperature for 1 hour. The primary antibody dilution was recovered from the incubation chamber, and the PVDF membrane was washed 8 times with TBST for 5 minutes each time. After washing, the TBST was removed, and HRP-labeled goat anti-rabbit secondary antibody (diluted 1:10000 in blocking buffer) was added and incubated at room temperature for 1 hour. The secondary antibody dilution solution in the incubation chamber was recovered, and the PVDF membrane was washed 8 times with TBST for 5 minutes each time. Finally, TBST was added to the incubation chamber to completely cover the PVDF membrane. Equal volumes of solution A and solution B from the ECL chemiluminescence kit were mixed and evenly applied to the PVDF membrane, then developed and exposed using a chemiluminescence imaging system. Figure 5 As shown in B, the antibody of the present invention can specifically pull down and detect a specific band corresponding to the molecular weight of GAPDHS (approximately 66 kDa) in total sperm protein, demonstrating the specificity of antibody binding, and the GAPDHS-K287mal level in asthenospermia sperm is significantly higher than that in normal sperm.

[0063] Application Example 2: Assessing the correlation between GAPDHS-K287mal levels and sperm progressive motility and asthenospermia.

[0064] 1. Establishment and grouping of clinical sample cohorts

[0065] A rigorously selected clinical sample cohort was established based on the World Health Organization's Laboratory Manual for the Examination and Processing of Human Semen (Fifth Edition). Forty healthy men with normal semen parameters and a history of fertility within the past year were included as the normal control group, and 40 patients diagnosed with idiopathic asthenospermia were included as the case group. Among these, asthenospermia patients had normal sperm concentration and the proportion of normally morphological sperm in routine semen analysis, a sperm progressive motility rate of less than 32%, and a history of infertility for more than 12 months.

[0066] 2. Sperm protein extraction and target modification detection

[0067] Total sperm protein was extracted from sperm samples from both normal and asthenospermia patients using an optimized RIPA lysis buffer (supplemented with protease inhibitors and deacylase inhibitors). Western blot experiments were performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and wet transfer (see Application Example 1). The expression level of the target modified protein was detected using the GAPDHS-K287mal specific antibody prepared in this invention (Example 1) as the primary antibody.

[0068] 3. Quantification and analysis of protein modification levels

[0069] ImageJ image analysis software was used to scan the grayscale values ​​of the Western blot bands. For accurate quantification, the ratio of the grayscale value of the GAPDHS-K287mal specific band to the grayscale value of the internal reference protein β-ACTIN in the same lane was calculated to obtain the relative quantitative level of GAPDHS-K287mal in each sperm sample.

[0070] 4. Statistical processing and correlation studies

[0071] All statistical analyses were performed using GraphPad Prism 10.0 software. Comparisons between two independent samples were performed using the Shapiro-Wilk test to verify that the data conformed to a normal distribution, followed by Welch's t-test for significance analysis. The correlation between the relative quantification of GAPDHS-K287mal and the percentage of sperm progressive motility was tested using Pearson linear correlation analysis. All statistical tests were expressed as follows: P <0.05 indicates statistical significance.

[0072] 5. Research Results and Conclusions

[0073] like Figure 6 As shown, the relative quantitative analysis results clearly indicate that the relative quantification of GAPDHS-K287mal in the sperm of patients with asthenospermia (1.53 ± 0.20) was significantly higher than that in the normal control group (1.07 ± 0.28), and the difference was statistically significant. P <0.001). Correlation analysis further revealed a significant negative correlation between GAPDHS-K287mal levels and sperm forward motility (r <0.001). 2 =0.504, P <0.0001).

[0074] Conclusion: The above experimental data collectively confirm that GAPDHS-K287mal is closely related to impaired forward sperm motility in humans, and its upregulation is an important molecular characteristic of asthenospermia, providing solid clinical data support for the diagnostic reagent described in this invention.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A malonylated modified polypeptide immunogen, characterized in that, The malonylated modified polypeptide is obtained by malonylating the lysine at position 10 based on the amino acid sequence shown in SEQ ID NO. 1; the immunogen of the malonylated modified polypeptide is obtained by crosslinking the malonylated modified polypeptide with keyhole hemocyanin.

2. The method for preparing the malonyl-modified polypeptide immunogen according to claim 1, characterized in that, Includes the following steps: (1) A malonylated modified polypeptide was synthesized and cross-linked with keyhole hemocyanin to obtain a keyhole hemocyanin cross-linked polypeptide; (2) Dissolve the keyhole hemocyanin crosslinked polypeptide and emulsify it with Freund's complete adjuvant by shaking to obtain the malonylated modified polypeptide immunogen.

3. The use of the malonylated modified polypeptide immunogen of claim 1 in the preparation of a specific antibody for detecting GAPDHS-K287mal.

4. A method for preparing a specific antibody for detecting GAPDHS-K287mal, characterized in that, The procedure includes the following steps: immunizing immunized animals by subcutaneous injection at multiple points on the back using the malonylated modified polypeptide immunogen described in claim 1; immunizing 3-5 times in total, once every 2-3 weeks, at a dose of 350 μg / time to 450 μg / time; collecting blood 6-8 days after the last injection, separating and purifying to obtain a specific antibody with a titer greater than 1:600,000.

5. The preparation method according to claim 4, characterized in that, The immunized animals include New Zealand white rabbits.

6. A specific antibody for detecting GAPDHS-K287mal, characterized in that, The specific antibody is prepared by any one of the preparation methods described in claims 4-5.

7. Use of the specific antibody according to claim 6 in the preparation of a reagent for detecting GAPDHS-K287mal.

8. The application according to claim 7, characterized in that, The reagent is used for the in vitro or in vivo detection of GAPDHS-K287mal.

9. The use of the specific antibody according to claim 6 in the preparation of a diagnostic reagent for idiopathic asthenospermia.

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