Application of PLK1 protein in preparation of products for identifying X sperms and Y sperms of mammals, enriching X sperms or determining concentration of X sperms

Abstract

The invention relates to application of PLK1 protein in preparing products for identifying X sperms and Y sperms of mammals, enriching the X sperms or determining the concentration of the X sperms, and belongs to the technical field of biological detection. By detecting the total protein of the X sperm and the Y sperm of the cattle, the symbolic protein PLK1 which is specifically expressed in the X sperm and is not expressed in the Y sperm is identified and further used for identifying the X sperm and the Y sperm and enriching and measuring the concentration of the X sperm, the sperm variety can be simply, conveniently and quickly judged according to whether the PLK1 protein is expressed or not, and the method has the advantages of high specificity and high specificity. X sperms are quickly and simply enriched on the basis of not damaging the sperms, and the concentration of the X sperms is judged according to the expression quantity of the PLK1 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and in particular relates to the application of PLK1 protein in preparing products for distinguishing mammalian X sperm and Y sperm, and enriching or measuring X sperm concentration. Background Art

[0002] Sperm is the male reproductive cell during sexual reproduction in animals. It undergoes three stages: spermatogonia mitosis, spermatocyte meiosis, and spermatid metamorphosis, ultimately forming X- and Y-sperm, each carrying an X or Y chromosome. When an X-sperm fuses with an egg, it produces a female offspring with XX chromosomes, while when a Y-sperm fuses with an egg, it produces a male offspring with XY chromosomes. Animal sex control involves artificially intervening in the normal reproductive process to produce offspring of a specific sex in adult female animals. Current sex control technologies utilize differences in sperm DNA, protein, and charge to separate X and Y sperm. Separation methods include centrifugation, electrophoresis, and flow cytometry. Other methods include early embryo identification to screen the sex of embryos in vitro. However, current sex control technologies are limited in their widespread application due to high costs and technological limitations.

[0003] Flow cytometry is currently the most effective method for sex control in livestock. It primarily exploits the different DNA content of XY sperm to achieve separation, resulting in high concentrations of either X or Y sperm. However, flow cytometry requires low temperatures, staining, and laser treatment, which can damage sperm cells and reduce their motility. Furthermore, the multi-step process is cumbersome, allowing only 10-20 million sperm to be processed per hour. Furthermore, the equipment is expensive. Therefore, there is an urgent need for a low-cost, simple, and cell-safe method for sex control in mammals that can enrich X sperm and simultaneously identify and measure the concentration of X and Y sperm. Summary of the Invention

[0004] In view of this, the present invention aims to provide a method for using PLK1 protein in the preparation of products for the identification of mammalian X and Y sperm, and for the enrichment or concentration determination of X sperm, thereby simplifying the detection process, reducing detection costs, improving detection efficiency, and reducing cell damage during the detection process.

[0005] The present invention provides the use of PLK1 protein in preparing a product for distinguishing mammalian X sperm and Y sperm.

[0006] The present invention provides the use of PLK1 protein in preparing a product for detecting the concentration of X sperm in mammals.

[0007] The present invention provides the use of an antibody against PLK1 protein in preparing a product for distinguishing mammalian X sperm from Y sperm and / or detecting the concentration of mammalian X sperm.

[0008] The present invention also provides a product for distinguishing mammalian X sperm from Y sperm and / or detecting the concentration of mammalian X sperm, wherein the product comprises an antibody against the PLK1 protein.

[0009] The present invention provides application of PLK1 protein in preparing a mammalian X sperm enrichment product.

[0010] The present invention also provides the use of a PLK1 protein inhibitor or a PLK1 protein agonist in preparing a mammalian X sperm enrichment product.

[0011] The present invention also provides a product for enriching mammalian X sperm, which comprises a PLK1 protein inhibitor or a PLK1 protein agonist.

[0012] Preferably, the PLK1 protein inhibitor includes SBE13.

[0013] Preferably, the mammal comprises cattle.

[0014] Preferably, the product comprises a kit.

[0015] Beneficial effects of the present invention:

[0016] The present invention utilizes LC-MS / MS and DIA techniques to perform proteomic analysis of total proteins from bovine X and Y sperm, followed by Western blot analysis, identifying PLK1 as a protein specifically expressed in X sperm. This invention, for the first time, identifies PLK1 in X sperm. This protein can be used as a marker protein for distinguishing, enriching, and detecting X and Y sperm. The presence of PLK1 protein expression determines whether a sperm is X or Y sperm. X sperm are enriched using PLK1 to obtain sperm cells with a higher X sperm content. The concentration of X sperm can be determined by the expression of PLK1 protein in sperm cells. This provides a low-cost, efficient, and simple method for sperm cell identification, enrichment, and detection. Furthermore, during the X sperm enrichment process, sperm are not subjected to treatments such as low temperatures, staining, and laser treatment, thus avoiding damage to sperm cells. This allows for the rapid acquisition of sperm cells with a high X sperm content, providing a large quantity of sexed semen, which has significant application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a Western blotting test result of PLK1 protein used to distinguish X sperm from Y sperm. Sample 1 represents X sperm, and sample 2 represents Y sperm.

[0018] Figure 2To detect the concentration of X sperm in sperm samples, laser confocal microscopy was used to observe the expression of PLK1 protein in sperm samples; blue represents sperm nuclei stained with Hoechst, green represents sperm acrosomes stained with PNA, and red represents the color development results of PLK1 protein in sperm.

[0019] Figure 3 This is the result of Western blotting analysis of X sperm in the upper layer of semen after treatment with PLK1 inhibitor.

[0020] Figure 4 This is the grayscale analysis result of X sperm in the upper layer of semen after the PLK1 inhibitor acts on semen.

[0021] Figure 5 This is the Western blotting analysis result of X sperm in the lower layer of semen after the treatment of semen with PLK1 inhibitor.

[0022] Figure 6 This is the grayscale analysis result of X sperm in the lower layer of semen after the PLK1 inhibitor acts on the semen. DETAILED DESCRIPTION

[0023] The present invention provides an application of PLK1 protein in preparing a product for distinguishing mammalian X sperm from Y sperm or detecting the concentration of mammalian X sperm.

[0024] In the present invention, the gene sequence of the PLK1 protein is shown as SEQ ID NO.1, and the amino acid sequence of the PLK1 protein is shown as SEQ ID NO.2.

[0025] The present invention preferably determines whether a mammalian sperm cell is an X sperm or a Y sperm cell by detecting whether the mammalian sperm cell expresses PLK1 protein. If PLK1 protein expression is detected in the mammalian sperm cell, the mammalian sperm cell is an X sperm cell; if PLK1 protein expression is not detected in the mammalian sperm cell, the mammalian sperm cell is a Y sperm cell. The detection of whether PLK1 protein expression in the mammalian sperm cell is preferably performed using an antibody against PLK1 protein. The present invention preferably detects the concentration of X sperm in the sperm cell by detecting the expression level of PLK1 protein in the mammalian sperm cell. The detection of PLK1 protein expression in the mammalian sperm cell is preferably performed using an antibody against PLK1 protein.

[0026] The present invention also provides the use of an antibody against PLK1 protein in preparing a product for distinguishing mammalian X sperm from Y sperm and / or detecting the concentration of mammalian X sperm.

[0027] The present invention also provides a product for distinguishing mammalian X sperm from Y sperm and / or detecting the concentration of mammalian X sperm, wherein the product comprises an antibody against the PLK1 protein.

[0028] The present invention does not specifically limit the source or preparation method of the PLK1 protein antibody. In one embodiment, conventional commercially available products in the art or antibodies prepared using the PLK1 protein as an immunogen can be selected. The present invention does not specifically limit the specific method for preparing antibodies using the PLK1 protein as an immunogen; conventional methods for preparing antibodies using immunogens in the art can be used. As a specific embodiment, the PLK1 protein antibody can be prepared by immunizing mammals (such as mice, rats, rabbits, sheep, and humans) with the PLK1 protein as an immunogen to produce polyclonal antibodies, or by immunizing mammals (such as mice, rats, rabbits, sheep, and humans) with the PLK1 protein as an immunogen to produce monoclonal antibodies using hybridoma or DNA recombination technology. The monoclonal antibodies preferably include humanized monoclonal antibodies. In one embodiment, the PLK1 protein antibody includes, but is not limited to, the PLK1 protein antibody commercially available under the product number ab189139 (from Abcam).

[0029] The present invention does not specifically limit the specific method for distinguishing X sperm from Y sperm; methods commonly used in the art for substance identification using marker proteins can be employed. In one embodiment, the method for distinguishing X sperm from Y sperm can be an immunological method based on the principle of antigen-antibody reaction, preferably including immunoblotting. The present invention does not specifically limit the method for detecting X sperm concentration; methods commonly used in the art for detecting substance concentration using marker proteins can be employed; in one embodiment, immunofluorescence can be employed.

[0030] The use of the X sperm marker protein PLK1 protein of the present invention to distinguish mammalian X sperm from Y sperm or to measure the concentration of mammalian X sperm can simply, quickly and at low cost identify the type of sperm and detect the concentration of X sperm in sperm cells.

[0031] The present invention provides application of PLK1 protein in preparing a mammalian X sperm enrichment product.

[0032] The present invention also provides the use of a PLK1 protein inhibitor or a PLK1 protein agonist in preparing a mammalian X sperm enrichment product.

[0033] The present invention also provides a product for enriching mammalian X sperm, which comprises a PLK1 protein inhibitor or a PLK1 protein agonist.

[0034] The present invention has no particular limitation on the source and type of the PLK1 protein inhibitor or PLK1 protein agonist, and conventional commercially available products in the art can be used. The PLK1 protein inhibitor preferably includes SBE13.

[0035] The present invention preferentially enriches X sperm using a PLK1 protein inhibitor. Adding the PLK1 protein inhibitor to the sperm cell mixture causes X sperm to aggregate in the upper layer of the sperm cell solution, increasing the concentration of X sperm in the upper layer. This enrichment facilitates their separation from Y sperm, resulting in sperm cells with a higher X sperm content. Compared to flow cytometry-based separation and enrichment methods for obtaining sperm cells with a higher X sperm content, this method avoids cumbersome experimental procedures, simplifies the process, and prevents damage to sperm cells caused by staining and laser treatment. It can also process large amounts of semen simultaneously, improving processing efficiency.

[0036] In the present invention, the mammal preferably includes cattle. The product preferably includes a kit.

[0037] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] In the following examples, unless otherwise specified, all methods are conventional.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] PLK1 is specifically expressed in X sperm

[0042] 1. Obtaining total sperm protein

[0043] 1. Sperm Samples A and B were obtained from Holstein cattle using flow cytometry. Both Sperm Samples A and B were free of animal-derived proteins. Sperm Sample A contained 150 million X sperm; Sperm Sample B contained 150 million Y sperm.

[0044] 2. After completing step 1, use lysis buffer (lysis buffer is RIPA lysis buffer, 1% PMSF, 1% phosphatase inhibitor 2 and 1% phosphatase inhibitor 3) to extract total sperm protein from sample A and sample B respectively. The specific steps are as follows:

[0045] Add the sperm sample to a 15 mL centrifuge tube and centrifuge at 2000 rpm for 20 minutes. Discard the supernatant. Add Lysis Buffer to the tube and boil in a boiling water bath for 10 minutes. Centrifuge at 12000 rpm for 10 minutes and remove the supernatant. This solution is the total sperm protein. Store the protein solution at -80°C.

[0046] 2. Proteomic Analysis

[0047] 1. Take the total sperm protein solution and determine the concentration using the Bradford method. The specific steps are as follows:

[0048] (1) Prepare bovine serum albumin (BSA) standards of different concentrations. Add 20 μL of BSA standard to 180 μL of working solution and store in the dark for 20 min.

[0049] (2) After completing step (1), the wavelength of each sample is detected by an enzyme-labeled instrument at 595 nm to prepare a standard curve;

[0050] (3) Dilute the total sperm protein 10-fold, take 20 μL of protein dilution solution and add it to 180 μL of working solution. After placing it in the dark for 20 minutes, use a microplate reader to detect the absorbance of the protein sample at 595 nm and use it to calculate the concentration of sperm protein using the standard curve.

[0051] 2. After completing step 1, take the total sperm protein and perform SDS-PAGE electrophoresis. The specific steps are as follows:

[0052] (1) Mixing total sperm protein with 6× protein electrophoresis loading buffer to obtain a mixed solution;

[0053] (2) The mixed solution was used for spotting, and the sample loading volume was 40 μg;

[0054] (3) Connect the power supply, set the voltage to 150 V, and turn off the power supply when bromophenol blue migrates to the bottom;

[0055] (4) Remove the gelatin plate, stain with Coomassie Brilliant Blue for 1 hour, and then decolorize with distilled water;

[0056] (5) Take the strip after the previous step of decolorization, add acetonitrile and continue to shake and decolorize for 15 minutes;

[0057] (6) Repeat step (5) until the decolorization is completed;

[0058] (7) After decolorization, dithiothreitol (DTT) was added and the mixture was incubated at 56°C for 45 min.

[0059] (8) Discard the waste liquid, add iodoacetamide (IAA), and place in the dark for 30 minutes;

[0060] (9) Discard the waste liquid, dehydrate with acetonitrile and dry for 10 min;

[0061] (10) After completing step (9), add trypsin at a final concentration of 0.1 μg / μL and digest at 4°C for 30 min;

[0062] (11) After the trypsin solution is completely removed from the gel, 25 mM NH4HCO3 is added and the gel is incubated at 37°C overnight for digestion.

[0063] (12) After completing step (11), add a solution containing 30% acetonitrile and 5% TFA, sonicate for 10 min, and aspirate the solution;

[0064] (13) After completing step (12), a solution containing 50% acetonitrile and 5% TFA was added, ultrasonicated for 10 min, and the solution was aspirated;

[0065] (14) mixing the solutions obtained in step (12) and step (13) and freeze-drying the mixture;

[0066] (15) Add 10 μL of 0.1% TFA solution to the sample obtained in step (14) and test on a microscope;

[0067] (16) After completing step (15), the raw files obtained by mass spectrometry were analyzed using MaxQuant, and the database used was the bovine Uniprot protein sequence library (URL: https: / / www.uniprot.org / proteomes / UP 000009136, download date: 2024-07-01, number of proteins: 57591).

[0068] (17) Some of the analysis results are shown in Table 1. The results showed that PLK1 protein was expressed in the sperm sample of X, but not detected in the sperm sample of Y, indicating that PLK1 protein was specifically expressed in the sperm sample of X. The gene sequence of PLK1 protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0069] Table 1 Analysis results of total sperm protein fraction of sample A (X sperm) and sample B (Y sperm)

[0070] Uniprot accession number Q2TA25 Protein name Serine / threonine-proteinkinasePLK1 Gene name PLK1 species Bostauros (Bovine) Protein length 602aa Number of matching peptides 2 Theoretical molecular weight 68kDa Average abundance of X in sperm samples 14.89162499 Average abundance of Y sperm in sperm samples NA

[0071] Example 2

[0072] Western blotting was used to detect the expression level of PLK1 to distinguish between X sperm samples and Y sperm samples.

[0073] (1) Fresh sperm was collected from three Holstein cows. After mixing the samples, high-purity (purity ≥ 90%) X sperm and Y sperm were obtained by flow cytometry as test samples for blind testing. The X sperm sample was named sample 1, and the Y sperm sample was named sample 2.

[0074] (2) Total protein of sample 1 and sample 2 were extracted respectively;

[0075] (3) Western blotting was performed on the total proteins of sample 1 and sample 2 respectively;

[0076] (4) The loading amount of sample 1 and sample 2 was 40 μg, and the electrophoresis program was 80 V for 15 min and 150 V for 1 h;

[0077] (5) After electrophoresis, transfer the protein from the SDS-PAGE gel to a PVDF membrane using a semi-dry transfer tank at 20 V for 30 min.

[0078] (6) The PVDF membrane obtained in step (5) was blocked with 5% skim milk powder at 37°C for 1 h;

[0079] (7) Transfer the PVDF membrane obtained in step (6) to the blocking solution of PLK1 primary antibody (PLK1 protein antibody, catalog number ab189139, Abcam) and incubate at 4°C overnight;

[0080] (8) Transfer the PVDF membrane obtained in step (7) to the dilution solution of the secondary antibody (peroxidase-labeled goat anti-rabbit IgG (H+L), product number 33101ES60, Yisheng Biotechnology (Shanghai) Co., Ltd.) and incubate at room temperature for 1 h;

[0081] (9) placing the PVDF membrane obtained in step (8) under an electronic tablet press for observation;

[0082] (10) Western blotting results are as follows Figure 1 As shown, PLK1 protein was highly expressed in sample 1 but not in sample 2. This result indicates that sample 1 is X sperm and sample 2 is Y sperm, which is consistent with the actual situation. This suggests that PLK1 protein can be used to distinguish X sperm from Y sperm.

[0083] Example 3

[0084] Determine the concentration of X sperm in normal sperm samples by immunofluorescence detection of PLK1 expression in sperm samples

[0085] (1) Collect fresh semen from three Holstein cows and mix the semen samples from the three cows;

[0086] (2) Dilute the fresh sperm solution with 4% paraformaldehyde to a suspension of 1 million sperm / mL and drop it onto a glass slide for fixation;

[0087] (3) After fixation, the sperm samples were blocked with 1% BSA blocking solution at 37°C for 1 h;

[0088] (4) The sperm sample from step (3) was transferred to the PLK1 primary antibody (PLK1 protein antibody, catalog number ab189139, Abcam) blocking solution and incubated at 4°C overnight;

[0089] (5) The sperm sample from step (4) was transferred to the secondary antibody (ALEXAFLUOR 568DONKEY ANTI-RA 0.5mL, Catalog No. A10042, Thermo) blocking solution and incubated at 37°C for 1 hour;

[0090] (6) Transfer the sperm sample from step (5) to a mixed staining solution of Hoechest 33343 and peanut agglutinin (PNA) and incubate at 37°C for 1 h;

[0091] (7) observing the expression of PLK1 in the sperm sample obtained in step (6) using laser confocal microscopy;

[0092] (8) Immunofluorescence Figure 2 As shown, PLK1 protein is expressed in the sperm midpiece and is observed in 50% of sperm samples. The concentration of X sperm in fresh, untreated sperm is also 50%, indicating that the measurement results are consistent with actual results and that the PLK1 protein can be used to detect the concentration of X sperm in spermatids.

[0093] At the same time, the above results further verified that PLK1 is highly expressed in X sperm and not expressed in Y sperm. PLK1 protein is specifically expressed in X sperm and can be used to distinguish X sperm from Y sperm.

[0094] Example 4

[0095] X sperm enrichment

[0096] (1) Wash the fresh Holstein semen solution with normal saline three times;

[0097] (2) using sperm diluent to prepare 3 mL of a 100 million / mL sperm solution;

[0098] (3) A certain proportion of PLK1 inhibitor SBE13 (PLK1 inhibitor 1) with different final concentrations (0 μM, 0.1 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 2 μM) was added to the prepared sperm solution. It was purchased from MCE (MedChemExpress).

[0099] (4) The sperm suspension was placed in a cell culture incubator at 37°C. One hour later, 1 mL of sperm from the uppermost (upstream) layer and 1 mL from the lowermost (downstream) layer were aspirated to obtain different semen samples;

[0100] (5) The expression of X sperm-specific protein TLR7 in the above-separated semen samples was detected by Western blotting. The results of Western blotting analysis of 1 mL of sperm in the upper layer after treatment with different concentrations of PLK1 inhibitors are shown in Figure 3 The results of the grayscale analysis of the upper 1mLX sperm before and after treatment with 0.8μM PLK1 inhibitor are shown in Figure 4 (The normal sperm in the figure is the proportion of X sperm before treatment with PLK1 inhibitors.) The results show that after treatment with 0.8 μM PLK1 inhibitors, the proportion of X sperm in the upper 1 mL layer increased to 70%. The results of Western blotting analysis of the lower 1 mL sperm after treatment with different concentrations of PLK1 inhibitors are shown in Figure 5 The results of the grayscale analysis of the lower layer 1mLX sperm before and after treatment with 0.8μM PLK1 inhibitor are shown in Figure 6 (The normal sperm in the figure is the proportion of X sperm before treatment with PLK1 inhibitors). The results showed that under treatment with 0.8 μM PLK1 inhibitors, the proportion of X sperm in the lower 1 mL layer dropped to 35%.

[0101] These results demonstrate that using a PLK1 protein inhibitor can enrich X sperm in the upper layer of mixed semen, thereby isolating semen containing 70% X sperm. Compared to flow cytometry, this method significantly simplifies the detection process and avoids damage to sperm cells. Simply adding a PLK1 protein inhibitor at a concentration appropriate for the volume of mixed semen can quickly yield large quantities of sexed semen with a higher X sperm content. Furthermore, this assay can process over 100 million sperm cells within an hour, compared to flow cytometry, which can only process 10-20 million sperm cells per hour. This method for enriching and isolating X sperm is more efficient.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of PLK1 protein in the preparation of products for the identification of mammalian X sperm and Y sperm.

2. Application of PLK1 protein in the preparation of mammalian X sperm concentration detection products.

3. Use of antibodies against PLK1 protein in the preparation of products for distinguishing mammalian X sperm from Y sperm and / or detecting the concentration of mammalian X sperm.

4. A product for distinguishing mammalian X sperm from Y sperm and / or detecting the concentration of mammalian X sperm, characterized in that: The product includes antibodies to the PLK1 protein.

5. Application of PLK1 protein in the preparation of mammalian X sperm enrichment products.

6. Use of a PLK1 protein inhibitor or PLK1 protein agonist in the preparation of a mammalian X sperm enrichment product.

7. A product for enriching mammalian sperm, characterized in that: The product includes a PLK1 protein inhibitor or a PLK1 protein agonist.

8. The use according to claim 6 or the product according to claim 7, characterized in that The PLK1 protein inhibitor includes SBE13.

9. The use according to any one of claims 1 to 8, characterized in that The mammals include cattle.

10. The use according to any one of claims 1 to 9, characterized in that: The products include kits.

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