Use of plk1 protein in preparation of products for identifying, enriching or determining the concentration of x sperm and y sperm of mammals

Abstract

The application relates to application of PLK1 protein in preparation of a product for identifying X sperm and Y sperm of mammals, enriching X sperm or determining the concentration of X sperm, and belongs to the technical field of biological detection. By detecting total proteins of X sperm and Y sperm of a cow, a marker protein PLK1 protein which is specifically expressed in X sperm but not in Y sperm is identified, and the PLK1 protein is further used for identification of X sperm and Y sperm, enrichment of X sperm and determination of the concentration of X sperm. Whether the PLK1 protein is expressed can be used to determine the type of sperm, X sperm can be enriched quickly and simply on the basis of not damaging the sperm, and the concentration of X sperm can be determined according to the expression amount of the PLK1 protein.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to the application of PLK1 protein in the preparation of products for the identification, enrichment or concentration determination of X sperm and Y sperm in mammals. Background Technology

[0002] Sperm are the male reproductive cells in animal sexual reproduction. They undergo three stages: spermatogonial mitosis, spermatocyte meiosis, and spermatogenesis, ultimately forming X sperm and Y sperm carrying either an X or Y chromosome. When an X sperm fertilizes an egg, it produces a female offspring with XX chromosomes, while a Y sperm fertilizing an egg produces a male offspring with XY chromosomes. Animal sex control is a biological technique that artificially intervenes in the normal reproductive process of animals to enable adult females to reproduce offspring of a specific sex. Current sex control technologies utilize differences in sperm DNA, proteins, and charges to separate X and Y sperm. Separation methods include centrifugation, electrophoresis, and flow cytometry; and early embryo identification to screen for the sex of in vitro embryos. However, current sex control technologies are not widely applicable due to high costs and limitations of existing technology.

[0003] Flow cytometry separation of X and Y sperm is currently the most effective method for sex control in livestock. It primarily utilizes the difference in DNA content between X and Y sperm to achieve separation and obtain high concentrations of either. However, flow cytometry requires low-temperature treatment, staining, and laser processing, which can easily damage sperm cells, leading to decreased motility. Furthermore, it involves multiple steps, making the process cumbersome and limiting the processing capacity to only 10-20 million sperm per hour. The equipment is also expensive. Therefore, there is an urgent need for a low-cost, simple, and cell-free method for sex control in mammals to enrich X sperm while simultaneously identifying and determining the concentration of X and Y sperm. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of PLK1 protein in the preparation of products for the identification, enrichment or concentration determination of X sperm and Y sperm in mammals, simplifying the detection process, reducing detection costs, improving detection efficiency, and reducing cell damage during the detection process.

[0005] This invention provides the application of PLK1 protein in the preparation of mammalian X and Y sperm identification products.

[0006] This invention provides the application of PLK1 protein in the preparation of mammalian X sperm concentration detection products.

[0007] This invention provides the application of antibodies against PLK1 protein in the preparation of products for the identification of mammalian X and Y sperm and / or the detection of mammalian X sperm concentration.

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

[0009] This invention provides the application of PLK1 protein in the preparation of mammalian X sperm enrichment products.

[0010] This invention also provides the application of PLK1 protein inhibitors or PLK1 protein agonists in the preparation of mammalian X sperm enrichment products.

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

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

[0013] Preferably, the mammal includes a cow.

[0014] Preferably, the product includes a reagent kit.

[0015] The beneficial effects of this invention are:

[0016] This invention utilizes LC-MS / MS and DIA techniques to perform proteomic analysis on the total protein content of bovine X and Y sperm, and identifies it using Western blot, confirming that PLK1 protein is specifically expressed in X sperm. This invention is the first to identify PLK1 protein in X sperm. This protein can serve as a marker protein for identifying, enriching, and detecting X and Y sperm. The presence or absence of PLK1 protein expression determines whether a sperm is X or Y. PLK1 protein enrichment is used to obtain sperm cells with a higher X sperm content. The concentration of X sperm is determined by the expression level of PLK1 protein in sperm cells. This provides a low-cost, efficient, and simple method for sperm cell identification, enrichment, and detection. Furthermore, the sperm are not subjected to low-temperature treatment, staining, or laser treatment during X sperm enrichment, avoiding damage to sperm cells. This allows for the rapid production of sperm cells with a high X sperm content, providing a large quantity of sex-controlled semen, and has significant application value. Attached Figure Description

[0017] Figure 1 This is a Western blotting result of PLK1 protein used to identify X sperm and 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 level of PLK1 protein in the sperm samples; where blue represents the sperm nucleus after Hoechest staining, green represents the sperm acrosome after PNA staining, and red represents the color development result of PLK1 protein in sperm.

[0019] Figure 3 The results of Western blotting analysis of X sperm in the upper layer of semen after the PLK1 inhibitor has been applied to the semen.

[0020] Figure 4 The results are grayscale analysis of X sperm in the upper layer of semen after the PLK1 inhibitor has been applied to the semen.

[0021] Figure 5 The results of Western blotting analysis of X sperm in the lower layer of semen after the PLK1 inhibitor has acted on the semen.

[0022] Figure 6 The results are grayscale analysis of X sperm in the lower layer of semen after the PLK1 inhibitor has been applied. Detailed Implementation

[0023] This invention provides the application of PLK1 protein in the preparation of products for identifying mammalian X and Y sperm or detecting mammalian X sperm concentration.

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

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

[0026] The present invention also provides the application of antibodies against PLK1 protein in the preparation of products for the identification of mammalian X sperm and Y sperm and / or the detection of mammalian X sperm concentration.

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

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

[0029] This invention does not specifically limit the method for identifying X and Y sperm; any method conventional in the art that uses marker proteins for substance identification can be used. In one embodiment, the method for identifying X and Y sperm can be an immunological method based on an antigen-antibody reaction, preferably including Western blotting. This invention does not specifically limit the method for detecting X sperm concentration; any method conventional in the art that uses marker proteins to detect substance concentration can be used. In one embodiment, immunofluorescence can be selected.

[0030] Using the PLK1 protein, a marker protein of X sperm in this invention, to identify mammalian X sperm and Y sperm or to determine the concentration of mammalian X sperm, can be a simple, quick, and low-cost method for identifying sperm types and detecting the concentration of X sperm in sperm cells.

[0031] This invention provides the application of PLK1 protein in the preparation of mammalian X sperm enrichment products.

[0032] This invention also provides the application of PLK1 protein inhibitors or PLK1 protein agonists in the preparation of mammalian X sperm enrichment products.

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

[0034] This invention does not impose any particular limitation on the source and type of the PLK1 protein inhibitor or PLK1 protein agonist; conventional commercially available products in the art can be used. Preferably, the PLK1 protein inhibitor includes SBE13.

[0035] This invention preferably uses a PLK1 protein inhibitor to enrich X sperm. When a PLK1 protein inhibitor is added to the sperm cell mixture, X sperm aggregate in the upper layer of the sperm cell solution, increasing their concentration and facilitating their separation from Y sperm, resulting in sperm cells with a higher X sperm content. Compared to flow cytometry, this method avoids cumbersome experimental procedures, simplifies the process, avoids damage to sperm cells caused by staining and lasers, and can process large volumes of semen simultaneously, improving processing efficiency.

[0036] In this invention, the mammal preferably includes a cow. The product preferably includes a kit.

[0037] The technical solutions provided by the present invention will be 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] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1

[0041] Specific expression of PLK1 in X sperm

[0042] I. Obtaining Total Sperm Protein

[0043] 1. Sperm samples A and B from Holstein cattle were obtained using flow cytometry. Neither sample A nor sample B contained animal-derived proteins. Sperm sample A contained 150 million X sperm cells; sperm sample B contained 150 million Y sperm cells.

[0044] 2. After completing step 1, total sperm protein was extracted from samples A and B using Lysis buffer (Lysis buffer consisted of RIPA lysis buffer, 1% PMSF, 1% phosphatase inhibitor 2, and 1% phosphatase inhibitor 3). The specific steps are as follows:

[0045] Take a 15 mL centrifuge tube, add the sperm sample, centrifuge at 2000 rpm for 20 min, and discard the supernatant. Add Lysis buffer to the centrifuge tube and boil in a boiling water bath for 10 min. Then centrifuge at 12000 rpm for 10 min, collect the supernatant, and the resulting solution is the total sperm protein. Store the protein solution at -80℃.

[0046] II. Proteomics Analysis

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

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

[0049] (2) After completing step (1), the microplate reader detects the wavelength of each sample at 595nm to generate a standard curve;

[0050] (3) Dilute the total sperm protein 10 times, take 20 μL of the protein dilution solution and add it to 180 μL of working solution. After standing in the dark for 20 min, use an ELISA reader to detect the absorbance of the protein sample at 595 nm and use it to calculate the concentration of sperm protein in the standard curve.

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

[0052] (1) Mix total sperm protein with 6× protein loading buffer to obtain a mixture;

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

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

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

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

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

[0058] (7) After decolorization, add dithiothreitol (DTT) and treat at 56℃ for 45 min;

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

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

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

[0062] (11) After the gel block absorbs the trypsin solution, add 25mM NH4HCO3 and place it at 37℃ overnight for digestion;

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

[0064] (13) After completing step (12), add a solution containing 50% acetonitrile and 5% TFA, sonicate for 10 minutes, and aspirate the solution.

[0065] (14) Mix the solutions obtained in steps (12) and (13) and freeze-dry them;

[0066] (15) Add 10 μL of 0.1% TFA solution to the sample obtained in step (14) and perform instrument detection;

[0067] (16) After completing step (15), the raw file obtained by mass spectrometry was analyzed using MaxQuant. The database used was the bovine Uniprot protein sequence library (https: / / www.uniprot.org / proteomes / UP000009136, downloaded on 2024-07-01, with 57591 proteins).

[0068] (17) Some analysis results are shown in Table 1. The results show that PLK1 protein is expressed in X sperm samples but not detected in Y sperm samples, indicating that PLK1 protein is specifically expressed in X sperm samples. 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. Partial analysis results of total sperm protein in sample A (X sperm) and sample B (Y sperm).

[0070] Uniprot search 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 in X sperm samples 14.89162499 Average abundance in Y sperm samples NA

[0071] Example 2

[0072] X sperm and Y sperm samples were identified by detecting PLK1 expression levels using Western blotting.

[0073] (1) Fresh sperm were collected from three Holstein cattle. After mixing the samples, high-purity (≥90%) X sperm and Y sperm were obtained by flow cytometry and used 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) Extract total protein from sample 1 and sample 2 respectively;

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

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

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

[0078] (6) Seal the PVDF membrane obtained in step (5) with 5% skim milk powder at 37°C for 1 hour.

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

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

[0081] (9) Place the PVDF film 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 indicates that sample 1 contained X sperm and sample 2 contained Y sperm, consistent with actual findings. This demonstrates that PLK1 protein can be used to distinguish between X and Y sperm.

[0083] Example 3

[0084] The concentration of X sperm in sperm samples was determined by immunofluorescence detection of PLK1 expression in routine sperm samples.

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

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

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

[0088] (4) Transfer the sperm sample from step (3) to PLK1 primary antibody (PLK1 protein antibody, catalog number ab189139, Abcam) blocking solution and incubate overnight at 4°C.

[0089] (5) Transfer the sperm sample from step (4) to the secondary antibody (ALEXAFLUOR 568DONKEY ANTI-RA 0.5ML, catalog number A10042, Thermo) blocking solution and incubate at 37°C for 1 h;

[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 hour;

[0091] (7) The expression of PLK1 in the sperm samples from step (6) was observed using laser confocal microscopy.

[0092] (8) Immunofluorescence such as Figure 2 As shown, PLK1 protein is expressed in the midpiece of sperm, and PLK1 was observed in 50% of the sperm sample. The concentration of X sperm in untreated fresh sperm was also 50%, indicating that the measured results are consistent with actual results. Therefore, the concentration of X sperm in sperm cells can be detected using PLK1 protein.

[0093] The above results further confirm that PLK1 is highly expressed in X sperm but not in Y sperm, indicating that PLK1 protein is specifically expressed in X sperm and can be used to distinguish between X and Y sperm.

[0094] Example 4

[0095] Enrichment of X sperm

[0096] (1) Wash the fresh Holstein bovine semen solution three times with physiological saline.

[0097] (2) Prepare a 3 mL sperm solution with a concentration of 100 million / mL using sperm diluent;

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

[0099] (4) Place the sperm suspension in a 37°C cell culture incubator. After one hour, obtain different semen samples by aspirating 1 mL of the top (upstream) and 1 mL of the bottom (downstream) sperm.

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

[0101] The above results demonstrate that using PLK1 protein inhibitors can enrich X sperm in the upper layer of mixed semen, thereby obtaining semen with an X sperm content of 70%. Compared to flow cytometry, this method significantly simplifies the detection process and avoids damage to sperm cells. By simply adding an appropriate concentration of PLK1 protein inhibitor to the mixed semen based on its volume, a large quantity of sex-controlled semen with a higher X sperm content can be obtained in a short time. Furthermore, this experiment can process over 100 million sperm cells per hour, compared to the 10-20 million sperm cells processed per hour by flow cytometry. Therefore, the method for enriching and separating X sperm in this invention is far more efficient.

[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of antibodies against PLK1 protein in the preparation of products for the identification of mammalian X and Y sperm and / or the detection of mammalian X sperm concentration, wherein the mammal is Holstein cattle.

2. Application of PLK1 protein inhibitor in the preparation of mammalian X sperm enrichment products, wherein the PLK1 protein inhibitor is SBE13, and the mammal is Holstein cattle.

3. The application according to any one of claims 1 to 2, characterized in that, The product includes a reagent kit.

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