Application of porcine membrane protein EPG5 in identifying and distinguishing porcine Y sperms

Abstract

The invention discloses application of porcine membrane protein EPG5 in identifying and distinguishing porcine Y sperms. According to the new application of the porcine membrane protein EPG5 provided by the invention, the porcine X and Y sperm membrane proteins are subjected to proteomics analysis through a DIA proteomics technology, the porcine membrane protein EPG5 differentially expressed in the Y sperm membrane protein is screened out, the expression of the protein on the Y sperm is obviously higher than that of the X sperm, and the protein can be used as a molecular marker to identify and distinguish the porcine X and Y sperms. Research shows that the X and Y sperms of the pig can be accurately identified and distinguished through differential expression analysis of the porcine membrane protein EPG5, and the result is consistent with the sorting result of a flow cytometry. The invention provides new application of the porcine membrane protein EPG5 in identifying and distinguishing the porcine Y sperms, protein resources are expanded, more and better methods and selections are provided for identifying and distinguishing the porcine Y sperms, sperm sex sorting is performed through differential expression of the porcine Y sperm membrane protein EPG5, and the method has the advantages of low cost, small damage, high efficiency, simplicity and convenience and good application prospect.

Description

Technical Field

[0001] This invention relates to the fields of biology and breeding technology, and more specifically, to the application of porcine membrane protein EPG5 in the identification and differentiation of porcine Y sperm. Background Technology

[0002] Sex control, or artificial intervention, allows offspring to be bred to the desired sex of piglets. This has revolutionary applications in modern pig farming. Differentiating and identifying X and Y sperm in pigs maximizes economic benefits: boars grow faster, have higher feed conversion rates, and typically have higher lean meat percentages. It improves breeding stock reproduction: expanding sow herds requires a large number of female offspring. Producing all-female herds can rapidly optimize the core population and accelerate genetic progress. It optimizes production management: herds of the same sex are more consistent in growth rate and behavior, reducing fighting due to sex differences (especially in boars), facilitating unified feeding management and batch production, and improving herd uniformity and health.

[0003] The existing publicly available methods for distinguishing and identifying pig X and Y sperm are: (1) Flow cytometry sorting: This is currently the only commercially available and most reliable method. Because the Y chromosome is smaller than the X chromosome, after DNA is combined with fluorescent dyes for staining, the fluorescence intensity emitted by X sperm is slightly higher than that of Y sperm, and sorting is performed. However, this method has a high accuracy rate for X sperm but a low accuracy rate for Y sperm. Moreover, the equipment is expensive, the operation technique is highly demanding, and the processing may cause certain physical and chemical damage to the sperm, affecting the subsequent conception rate. (2) Immunological methods: Based on sex-specific membrane proteins (HY antigen, etc.) on the surface of X and Y sperm. By preparing specific antibodies, the antigen-antibody reaction is used to identify and separate sperm. This has been a research hotspot for many years, but no commercially available, stable and reliable antibodies have been developed on a large scale. The challenge lies in finding sex-specific surface markers with high specificity and high affinity. (3) Identification methods based on polymerase chain reaction: These methods cannot directly separate live sperm, but are used to verify the purity of sorting or to identify the sex of the embryo.

[0004] Because the pig Y chromosome is smaller than the X chromosome, the identification and differentiation of Y sperm is much more difficult than that of X sperm. Currently available sorting methods are largely unsuitable for identifying and differentiating Y sperm. Furthermore, current gene markers are primarily used for PCR identification, not direct isolation. For example, the SRY gene, located on the Y chromosome, encodes the testis-determining factor and is a male-specific genetic marker; while the presence or absence of the SRY gene can accurately identify the sex of sperm or embryos, it cannot be used to identify and differentiate Y sperm. Our team has only researched and disclosed that the porcine membrane proteins PPP4C and CSNK2A2 can be used for the identification and differentiation of pig Y sperm. Currently, there is a lack of more gene resources and release methods specifically related to pig Y sperm that can be used to separate pig X and Y sperm. Therefore, it is necessary to develop more products that can be used to identify and differentiate pig Y sperm. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing gene resources and methods that can identify and distinguish specific pig Y sperm. The present invention provides the application of porcine membrane protein EPG5 in the identification and differentiation of pig Y sperm.

[0006] The first objective of this invention is to provide a novel application of porcine membrane protein EPG5 in the identification and differentiation of porcine Y sperm.

[0007] A second objective of this invention is to provide a reagent for detecting the expression level of the porcine membrane protein EPG5 in the preparation of products for identification and / or differentiation of porcine Y sperm.

[0008] A third objective of this invention is to provide a method for identifying and / or distinguishing porcine Y sperm.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides the application of porcine membrane protein EPG5 in the identification and / or differentiation of porcine Y sperm. The porcine membrane protein EPG5 has the protein ID I3LSJ7 in the Uniprot database and the gene ID 100518639 in the NCBI database.

[0010] This invention provides a novel application of porcine membrane protein EPG5. Through DIA proteomics analysis of porcine X and Y sperm membrane proteins, differentially expressed porcine membrane protein EPG5 was screened in Y sperm membrane proteins. The expression of this protein in Y sperm is significantly higher than in X sperm, thus it can be used as a molecular marker to identify and distinguish porcine X and Y sperm. Further validation shows that differential expression analysis of porcine membrane protein EPG5 can accurately identify and distinguish porcine X and Y sperm, with results consistent with flow cytometry sorting results, demonstrating reliability and a more convenient identification method. This invention provides a novel application of porcine membrane protein EPG5 in identifying and distinguishing porcine Y sperm, expanding protein resources and providing more and better methods and options for identifying and distinguishing porcine Y sperm. Sperm sex sorting through differential expression of porcine Y sperm membrane protein EPG5 has the advantages of low cost, minimal damage, high efficiency, and simplicity, and shows promising application prospects.

[0011] Preferably, the identification and / or differentiation of porcine Y sperm is performed by: the expression level of porcine membrane protein EPG5 in porcine Y sperm is significantly higher than that in X sperm, thereby identifying and / or differentiating porcine Y sperm.

[0012] Preferably, the expression level of the membrane protein EPG5 in Y sperm is more than 50% higher than that in X sperm.

[0013] More preferably, the expression level of the membrane protein EPG5 in Y sperm is more than 53% higher than that in X sperm.

[0014] This invention provides the application of porcine membrane protein EPG5 in the preparation of products for identification and / or differentiation of porcine Y sperm.

[0015] This invention provides a reagent for detecting the expression level of porcine membrane protein EPG5 in the preparation, identification, and / or differentiation of porcine Y sperm products.

[0016] Furthermore, the expression level of porcine membrane protein EPG5 in porcine sperm was detected using a reagent to identify and differentiate porcine Y sperm.

[0017] This invention provides a method for identifying and / or distinguishing porcine Y sperm, which involves detecting the expression level of porcine membrane protein EPG5 in porcine sperm. The expression level of porcine membrane protein EPG5 in Y sperm is significantly higher than that in X sperm, thereby identifying and / or distinguishing porcine Y sperm.

[0018] Preferably, the expression level of the membrane protein EPG5 in Y sperm is more than 50% higher than that in X sperm.

[0019] Preferably, the method uses a reagent for detecting the expression level of porcine membrane protein EPG5.

[0020] Preferably, the expression level of EPG5 protein is detected by Western blotting or immunofluorescence.

[0021] More specifically, the steps for detecting EPG5 protein expression using the Western blot method are as follows: S1. Extract membrane proteins from the two semen samples after X and Y sperm separation, and determine the sperm protein concentration. S2. Denature sperm proteins and separate membrane proteins using 6% SDS-PAGE gel electrophoresis. S3. Transfer the film obtained in step S2 onto a methanol-activated PVDF membrane; wash the membrane with buffer, and then block it overnight at room temperature with TBST buffer (containing 0.1% Tween 20) containing 5% skim milk powder by mass. S4. Wash the membrane with TBST buffer, add EPG5 antibody and β-actin antibody, and incubate overnight at 4°C; then wash again with buffer, and incubate with secondary antibody at room temperature for 1-2 hours; the secondary antibody used is horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L). S5. Wash with buffer solution, then expose the blot using a low background chemiluminescence detection kit (cECL Western Blot Kit), and then use ImageJ statistical software to count the gray values ​​of the blot.

[0022] As some alternative implementation methods, the application of porcine membrane protein EPG5 in pig breeding is provided: 1. A method for identifying porcine X and Y sperm, comprising the following steps: using EPG5 protein as a molecular marker, Western blotting is used to identify porcine X and Y sperm, wherein EPG5 protein is differentially expressed on the surface of Y sperm cells; 2. A method for separating pig X and Y sperm, comprising the following steps: using EPG5 protein as an antigen or receptor, differentially altering the function of pig X or Y sperm through antibody-antigen reaction or ligand reaction, thereby separating X or Y sperm, wherein the EPG5 protein is differentially expressed on the surface of Y sperm cells.

[0023] In addition, the present invention also provides the application of a reagent for detecting the expression level of porcine membrane protein EPG5 in the breeding of male pig herds. The reagent for detecting the expression level of porcine membrane protein EPG5 is used to separate Y sperm based on the difference in expression level, thereby breeding male pig herds; the difference in expression level is that the expression level of porcine membrane protein EPG5 in Y sperm is more than 50% higher than the expression level in X sperm.

[0024] The present invention has the following beneficial effects: This invention provides a novel application of porcine membrane protein EPG5. Using DIA proteomics technology, proteomic analysis of porcine X and Y sperm membrane proteins was performed to screen for differentially expressed porcine membrane protein EPG5 in Y sperm membrane proteins. The expression of this protein in Y sperm is significantly higher than in X sperm, and it can be used as a molecular marker to identify and distinguish porcine X and Y sperm. Further validation showed that differential expression analysis of porcine membrane protein EPG5 can accurately identify and distinguish porcine X and Y sperm, with results consistent with flow cytometry sorting results, demonstrating reliability and convenience. This invention provides a new application of porcine membrane protein EPG5 in identifying and distinguishing porcine Y sperm, expanding protein resources and providing more and better methods and options for identifying and distinguishing porcine Y sperm. Sperm sex sorting through differential expression of porcine Y sperm membrane protein EPG5 has the advantages of low cost, minimal damage, high efficiency, and simplicity, and shows promising application prospects. Attached Figure Description

[0025] Figure 1 This is a graph showing the results of Western blotting analysis of the expression of the membrane protein EPG5 in porcine sperm.

[0026] Figure 2 This is a graph showing the results of the expression level analysis of the membrane protein EPG5 in porcine sperm. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] Example 1: Collection and Identification of Pig Sperm 1. Experimental materials Semen was collected from three normally fertile Duroc boars (provided by the Shuitai Breeding Pig Farm of Guangdong Huanong Wenshi Livestock Co., Ltd.). Semen sample collection was completed at the Shuitai Breeding Pig Farm of Guangdong Huanong Wenshi Livestock Co., Ltd., and separation was performed at the Guizhou Provincial Bull Breeding Station.

[0030] 2. Sperm sorting The collected boar semen was sorted by flow cytometry using conventional methods to obtain three X-semen samples and three Y-semen samples for sex control.

[0031] 3. Semen identification and purity testing (1) Sperm DNA extraction: Six collected semen samples were used for DNA extraction. Semen containing 1000-2500 sperm was centrifuged at 2000×g for 5 min, the supernatant was discarded, and the sample was resuspended in 5 μL of lysis buffer (150 mM KOH, 30 mM DTT, 1 mM EDTA) and incubated in a PCR instrument at 65℃ for 20 min. The reaction was terminated by adding 5 μL of neutralization solution (900 mM Tris-HCl (pH=8.3)) and diluted to 40 μL with ddH2O to obtain the DNA extract from the semen samples.

[0032] (2) Real-time PCR reaction Using the extracted sperm DNA as a template, quantitative real-time PCR was performed using primers for the differentially expressed SRY gene on the Y chromosome and the differentially expressed AMELX gene on the X chromosome, respectively. The specific sequences of the primers are shown in Table 1. The reaction system and procedure for the quantitative real-time PCR are shown in Tables 2 and 3 below.

[0033] Table 1 Primers for Real-Time PCR Detection

[0034] Table 2. Real-time PCR reaction system

[0035] Table 3. Quantitative Real-Time PCR Reaction Procedure

[0036] Then use 2 -ΔΔCT The relative expression levels of SRY and AMELX genes in different sperm samples were calculated. The results showed that the expression level of AMELX gene in sex-controlled X sperm and the expression level of SRY gene in sex-controlled Y sperm were both above 80%, indicating that the sex-controlled sperm reached experimental purity.

[0037] Example 2: Extraction and determination of sperm membrane proteins 1. Extraction of sperm membrane proteins Membrane proteins were extracted from the six semen samples identified in Example 1 using a cell membrane protein extraction kit (purchased from Shanghai Yamei Biomedical Technology Co., Ltd.). The specific steps were as follows: (1) Centrifuge the semen containing 100 million sperm at 2500×g for 5 min, then resuspend the cell pellet with 3 mL Cell WashSolution and centrifuge again at 2500×g for 5 min, and discard the supernatant. (2) Add 1 ml of Cell Wash Solution to transfer the cells to a 1.5 mL centrifuge tube, centrifuge at 2500 × g for 5 min and discard the supernatant; (3) Add 0.75 mL of permeation buffer containing 1% protease inhibitor to the cell pellet, pipette and briefly vortex to obtain a homogeneous cell suspension, and incubate in a rotary mixer at 4°C for 10 min. (4) Centrifuge the permeabilized cells at 4°C and 16000×g for 15 min and discard the supernatant; (5) Add 0.5 mL of solubilization buffer containing 1% protease inhibitor to the precipitate, pipette and briefly vortex to obtain a homogeneous cell suspension, and incubate in a rotary mixer at 4°C for 45 min. (6) Centrifuge the incubated solution at 4°C and 16000×g for 15 min and take the supernatant. Transfer the supernatant containing soluble membrane proteins and membrane-associated proteins to a new reaction tube to obtain X sperm sample membrane protein solution and Y sperm sample membrane protein solution, respectively.

[0038] 2. Determination of protein concentration in membrane protein solution The concentration of the sperm membrane protein solution (X sperm sample membrane protein solution or Y sperm sample membrane protein solution) prepared above was determined using a BCA (bicinchoninnc acid) kit (purchased from Thermo Fisher Scientific). The specific steps are as follows: (1) Prepare the required volume of colorimetric solution according to the BCA kit instructions, based on the ratio of buffer A: buffer B = 50:1 (v / v); (2) Take out a portion of the protein solution to be tested and dilute it with Mem-PER™Plus solubilization buffer (to prevent the concentration from being too high and exceeding the working range of the standard curve). (3) Prepare a clean 96-well plate and prepare BSA standard protein solutions with concentrations of 0, 5, 10, 15 and 20 μg / μL. (4) Add 2 μL of the protein solution to be tested to a 96-well plate, set up three replicates for each sample, and add volume to 20 μL; (5) Add 200 μL of pre-prepared colorimetric solution to each well (the colorimetric solution must be prepared fresh for use) and react at 37°C for 30 min; (6) Use an ELISA reader to measure the absorbance (wavelength 562 nm); (7) Calculate the standard curve based on the known concentration and absorbance value of the standard protein solution, and substitute the absorbance value of the sample to be tested to obtain the protein concentration value.

[0039] The results of sperm membrane protein concentration measurements for each group are shown in Table 4.

[0040] Table 4 Sperm membrane protein concentration

[0041] Example 3: Purification of porcine sperm membrane proteins 1. Trypsin digests membrane proteins Based on the protein concentration determined in Example 2, 50 μg of protein was taken from each sample, and the samples from different groups were diluted with SDS lysis buffer to adjust to the same concentration and volume. Dithiothreitol (DTT) was added to the above protein solution to make the final DTT concentration 4.5 mM, mixed well, and incubated at 55 °C for 30 min. After cooling on ice until room temperature, the corresponding volume of iodoacetamide was added to make the final iodoacetamide concentration 9 mM, mixed thoroughly, and placed at room temperature in the dark for 15 min. Six times the volume of acetone was added to the above solution to precipitate the protein, and it was placed at -20 °C for more than 4 h or overnight. The precipitate was collected by centrifugation at 8000 × g for 10 min at 4 °C, and the acetone was evaporated for 2-3 min. 100 μL of tetraethylammonium bromide (TEAB) was added to reconstitute the precipitate, followed by the addition of 1 mg / mL trypsin-TPCK at 1 / 50 sample mass, and digestion was carried out overnight at 37 °C. The enzymatic digestion reaction was terminated by adding phosphate to adjust the pH to about 3, thus completing the enzymatic digestion of membrane proteins and obtaining peptide sample solutions.

[0042] 2. Desalting of membrane proteases by digesting peptides The peptide sample solution after membrane protein hydrolysis was desalted using a SOLA™ SPE column (purchased from Thermo Fisher Scientific) in a 96-well plate. The specific steps are as follows: (1) Activation: Activate the column with 200 μL of methanol, repeat 3 times; (2) Equilibration: Activate the column with 200 μL of pure water, repeat 3 times; (3) After adjusting the pH of the peptide sample solution after enzymatic hydrolysis in Example 4 to 7, the sample was loaded onto the column, and then vacuum was adjusted to pass through the column, with the droplet speed maintained at 1 mL / min, and this was repeated once; (4) Wash with 200 μL of 5% (V / V) methanol, repeat 3 times; (5) Elution: Elute the peptide with 150 μL of 60% (V / V) methanol, repeat 3 times to obtain 450 μL of eluent, evaporate under vacuum to obtain the desalted peptide sample.

[0043] Example 4: LC-MS / MS High-Resolution Mass Spectrometry Detection and Result Analysis 1. Testing method conditions The peptide samples prepared in Example 3 were detected by high-resolution LC-MS / MS at Hangzhou Jingjie Biotechnology Co., Ltd. Before mass spectrometry injection, each sample was mixed at a volume ratio of iRT standard to analyte sample = 1:10 as an internal standard. The six groups of peptide samples desalted in Example 3 were each divided into two portions; one portion was mixed and used for high-pH LC-MS separation, while the other portion was used individually for LC-MS separation.

[0044] (1) High pH liquid phase separation Samples: Equal amounts of peptides from the 6 groups of peptide samples after desalting in Example 3 were mixed, and the mixed samples were separated into components using an Agilent 1100 HPLC system in a mobile phase of pH=10. Separation conditions: Column: Agilent Zorbax Extend–C18 narrow-bore column, 2.1×150mm, 5μm; Detection wavelengths: ultraviolet 210nm and 280nm; Mobile phase A: Acetonitrile (ACN)-H2O (2:98, v / v), Mobile phase B: ACN-H2O (90:10, v / v) (Both mobile phases were adjusted to pH 10 with ammonia), Flow rate: 250 μL / min; Gradient elution conditions: 0-10 min, 2% B; 10-10.01 min, 2-5% B; 10.01-37 min, 5-20% B; 37-48 min, 20-40% B; 48-48.01 min, 40-90% B; 48.01-58 min, 90% B; 58-58.01 min, 90-2% B; 58.01-63 min, 2% B. Component collection: Starting at the 10th minute, eluent was collected sequentially into centrifuge tubes 1-10 every minute, and the fractions were collected in the order of 1→10. A total of 10 components were collected, freeze-dried under vacuum, and the samples were cryopreserved for mass spectrometry later.

[0045] (2) Liquid chromatography-mass spectrometry separation Liquid chromatography-mass spectrometry (LC-MS) conditions: The 10 components collected in step (1) were scanned by DDA mass spectrometry using an EASY-nLC 1200 high performance nano-liquid chromatograph and a Q Exactive HF mass spectrometer. The scanning parameters are shown in Table 5. Liquid phase elution gradient: flow rate 300 nL / min, buffer A is 0.1% FA (trifluoroacetic acid) aqueous solution, buffer B is 0.1% FA / 80% ACN / 20% water (the percentages in this section are volume percentages); The remaining six peptide fragments after desalting in Example 5 were individually tested using an EASY-nLC 1200 high-performance nano-liquid chromatograph and a Q Exactive HF mass spectrometer. The DIA mass spectrometry scan was performed with the scan range set to 350-1250 m / z and the isolation window set to 26 m / z. The mass spectrometry scan parameters are shown in Table 6. The DIA liquid phase elution gradient is shown in Table 7: flow rate 300 nL / min, buffer A is 0.1% FA aqueous solution, and buffer B is 0.1% FA / 80% ACN / 20% water.

[0046] Table 5. Data-dependent acquisition (DDA) mass spectrometry conditions

[0047] Table 6. Data Independent Acquisition (DIA) Mass Spectrometry Conditions

[0048] Table 7 Chromatographic conditions for data independent acquisition (DIA)

[0049] The purpose of this operation is to match the mass spectrometry output spectrum with the theoretical spectrum generated by the FASTA library, converting the machine signal into peptide and protein sequence information. Then, by combining the sequence information, peptide retention time, fragment ion information, etc., a spectral library is established to facilitate subsequent DIA analysis.

[0050] 2. Results Analysis Import the raw LC-MS / MS mass spectrometry files into Spectronaut Pulsar software for DDA library construction. The main parameters are shown in Table 8.

[0051] Table 8 Data Dependency Acquisition (DDA) Database Construction Parameters

[0052] The processing of raw DIA data was completed using Spectronaut Pulsar software, and quantitative data were exported; key parameters for DIA data parsing are shown in Table 9.

[0053] Table 9 Key Parameters for Data Parsing in Data Independent Acquisition (DIA)

[0054] Subsequently, using the quantitative data obtained from the database, proteins with an expression rate ≥50% were retained from any sample group. Proteins with missing values ​​≤50% were filled with the mean of the same sample group, and after Median Normalization and log2 transformation, reliable proteins were obtained. Then, differential expression analysis was performed on the identified proteins, with the differential expression screening criteria being Foldchange = 1.2-fold and p-value < 0.05.

[0055] Finally, porcine membrane protein EPG5 (protein ID: I3LSJ7 in the Uniprot database, gene ID: 100518639 in the NCBI database) was identified as a differential membrane protein marker for Y sperm.

[0056] Example 5: Identification and Differentiation of Pig Y Sperm Following the method in Example 1, semen was collected again from three normally fertile Large White boars. The semen was sorted using flow cytometry to obtain six samples. These six samples were anonymized and their order was shuffled. Membrane proteins were extracted from the six anonymized samples following the method in Example 2, and the concentration of membrane proteins in each sample was determined. Then, 30 μg of protein from each sample was added and diluted to the same concentration and volume with 6× Protein Loading Buffer and Mem-PER™ Plus solubilization buffer. The proteins were incubated at 99°C for 10 min to denature them. The protein denaturation system is shown in Table 10 below.

[0057] Table 10 Protein denaturation reaction system

[0058] Subsequently, Western blotting was used to identify the expression of EPG5 in porcine sperm membrane proteins. The specific steps were as follows: (1) Prepare TBST buffer: Dissolve TBS buffer powder (Saiwell Biotechnology Co., Ltd.) completely in 2L of double-distilled water, then add 2mL of Tween20 and stir for 25min; (2) Membrane proteins were separated by 6% (w / w) SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After the transfer, the membrane was washed three times with TBST buffer for 5 min each time, and then blocked overnight at room temperature with TBST buffer containing 5% (w / w) skim milk powder. (3) Wash the membrane three times with TBST buffer for 5 min each time, add EPG5 antibody and β-actin antibody respectively, and incubate at 4°C overnight; (4) Wash three times with TBST buffer for 5 min each time, and then incubate with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) at room temperature for 2 h; (5) Wash three times with TBST buffer for 5 min each time, and then expose the blot using the cECL Western Blot Kit with low background chemiluminescence detection kit; (6) Use ImageJ software (http: / / imagej.net / ImageJ) to count grayscale values.

[0059] Test results as follows Figure 1 and Figure 2 As shown, the results indicate that the EPG5 protein quantification method identification results are consistent with the flow cytometry sorting results; the EPG5 protein expression level in sperm cells 4, 5, and 6 is 53% higher than that in sperm cells 1, 2, and 3. Sperm cells 4, 5, and 6 are identified as Y sperm, while sperm cells 1, 2, and 3 are identified as X sperm, which is consistent with the flow cytometry sorting results.

[0060] The results showed that porcine sperm membrane protein EPG5 can serve as a cell surface marker protein for identifying and distinguishing between X and Y sperm. It can be used to prepare more products that can efficiently identify and distinguish porcine Y sperm, which can be better used for the breeding and control of male pig herds. It also expands the protein resources that can efficiently identify and distinguish porcine Y sperm. Sperm sex sorting through differential expression of porcine Y sperm membrane protein EPG5 has the advantages of low cost, minimal damage, high efficiency and simplicity, and has good application prospects.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of porcine membrane protein EPG5 in the identification and / or differentiation of porcine Y sperm, characterized in that, The porcine membrane protein EPG5 has the protein ID I3LSJ7 in the Uniprot database and the gene ID 100518639 in the NCBI database.

2. The application according to claim 1, characterized in that, The identification and / or differentiation of porcine Y sperm is based on the fact that the expression level of porcine membrane protein EPG5 in porcine Y sperm is significantly higher than that in X sperm, thereby identifying and / or differentiating porcine Y sperm.

3. The application according to claim 1, characterized in that, The expression level of the membrane protein EPG5 in Y sperm is more than 50% higher than that in X sperm.

4. The application of porcine membrane protein EPG5 in the preparation, identification, and / or differentiation of porcine Y sperm products, characterized in that, The porcine membrane protein EPG5 has the protein ID I3LSJ7 in the Uniprot database and the gene ID 100518639 in the NCBI database.

5. The application of a reagent for detecting the expression level of porcine membrane protein EPG5 in the preparation, identification, and / or differentiation of porcine Y sperm products, characterized in that... The porcine membrane protein EPG5 has the protein ID I3LSJ7 in the Uniprot database and the gene ID 100518639 in the NCBI database.

6. A method for identifying and / or distinguishing porcine Y sperm, characterized in that, The expression level of porcine membrane protein EPG5 in porcine sperm was detected. The expression level of porcine membrane protein EPG5 in Y sperm was significantly higher than that in X sperm, which was used to identify and / or distinguish porcine Y sperm.

7. The method according to claim 6, characterized in that, The expression level of the membrane protein EPG5 in Y sperm is more than 50% higher than that in X sperm.

8. The method according to claim 6, characterized in that, The method uses a reagent for detecting the expression level of porcine membrane protein EPG5.

9. The method according to claim 6, characterized in that, The expression level of EPG5 protein was detected by Western blotting or immunofluorescence.

10. The application of a reagent for detecting the expression level of porcine membrane protein EPG5 in the breeding of male pig herds, characterized in that... Y sperm were isolated based on the difference in expression level using a reagent that detects the expression level of porcine membrane protein EPG5, thereby breeding male pig herds. The difference in expression level was defined as the expression level of porcine membrane protein EPG5 in Y sperm being more than 50% higher than that in X sperm. The protein ID of porcine membrane protein EPG5 in the Uniprot database is I3LSJ7, and the gene ID in the NCBI database is 100518639.

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