Application of Membrane Protein RALB in Identifying and Distinguishing Porcine X Sperm

Abstract

The present invention provides an application of the membrane protein RALB in identifying and differentiating porcine X sperm. The application method is as follows: by utilizing the characteristic of differential expression of the membrane protein RALB on the surface of porcine X sperm cells, X and Y sperm are identified and differentiated. The present invention uses DIA proteomics technology to perform proteomic analysis on porcine X and Y sperm membrane proteins, and screens out the protein RALB that is differentially expressed in X sperm membrane proteins. The expression of this protein on X sperm is significantly higher than that on Y sperm. Therefore, it can be used as a molecular marker to identify porcine X and Y sperm. The present invention performs sperm sex sorting through the differential expression of the porcine X sperm membrane protein RALB, which has the advantages of low cost, small damage, high efficiency and simplicity, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. More specifically, it relates to the application of the membrane protein RALB in differentiating and distinguishing porcine X sperm, specifically the application of differentiating or distinguishing porcine X and Y sperm by the differential expression of the porcine sperm membrane protein RALB on the surface of X sperm cells. Background Art

[0002] The technology of animal sex control is a biotechnology that artificially intervenes in the normal reproductive process of animals to enable adult female animals to produce offspring of the desired sex. The sex control technology is of great significance in livestock production. First, by controlling the sex ratio of offspring, the maximum economic benefits of production traits restricted by sex (such as lactation) and production traits affected by sex (such as growth rate, meat quality, etc.) can be fully exerted. Second, controlling the sex ratio of offspring can increase the selection intensity and accelerate the breeding process. By controlling the sex of embryos, the phenomenon of heterosexual twinning infertility in bovine embryo transfer can also be overcome, and the harm of sex-linked harmful genes can be excluded. Sex control can be achieved through sperm separation or embryo sex identification.

[0003] Currently, existing sex control technologies include changing the ratio of X sperm and Y sperm produced through gene editing technology during spermatogenesis, separating mature sperm based on the differences in size, motility, charge, surface antigens, and DNA content between X sperm and Y sperm, screening the sperm that finally reaches the oocyte by changing the female reproductive tract environment, and screening the sex of in vitro embryos through early embryo identification. However, due to problems such as high costs and limitations of existing technologies, most of the technologies still remain at the laboratory stage and cannot be used in livestock production practices. The flow cytometry sperm sorting technology is currently the most successful sperm sorting technology and has been widely applied in the production practice of dairy cows. However, in the pig industry, the flow cytometry sperm sorting is rarely used because the cell membrane of porcine sperm is damaged during the sorting process, resulting in a decrease in its motility, fertilization ability, and in vitro preservation ability. In addition, the amount of semen inseminated per time for sows is relatively large, usually much higher than that required for cows. Therefore, there is an urgent need for a new method to change the sex ratio of piglets.

[0004] Membrane proteins, usually referring to the proteins on the cell membrane, account for about 30% of the cell membrane components and are the main bearers of the functions of biological membranes. If the differential proteins on the membranes of X and Y sperm can be identified, then X and Y sperm can be sorted through antigen-antibody reactions (protein immunization methods) or membrane protein receptor signal stimulation. Compared with flow cytometry sorting, this method is simple and easy to implement, can meet the requirements of the single insemination volume in the pig industry, and has less harmful effects on sperm cells during the sorting process. Existing studies have identified the differential proteins of bovine X and Y sperm through proteomic techniques such as MALDI-TOF-MS (Chen X, J Proteomics. 2012, 77:59-67) and nUPLC-MS / MS (De Canio M, Molecular bioSystems. 2014, 10(6)). However, there is currently no report on successfully verified sex-differential sperm membrane proteins in pigs. Summary of the Invention

[0005] The technical problem to be solved by this invention is to explore a differential protein on porcine X sperm, so as to utilize the differential expression of this protein on the surface of X sperm cells to more simply and easily identify porcine X sperm and porcine Y sperm.

[0006] The purpose of this invention is to provide an application of porcine sperm membrane protein RALB in identifying or distinguishing porcine X sperm and porcine Y sperm.

[0007] Another purpose of this invention is to provide a method for identifying porcine X sperm and porcine Y sperm.

[0008] The above purposes of this invention are achieved through the following technical solutions:

[0009] This invention provides an application of porcine sperm membrane protein RALB in identifying or distinguishing porcine X sperm and porcine Y sperm. The method of identification or distinction is to utilize the characteristic of differential expression of RALB protein on the surface of X sperm cells for identification or distinction, and the expression of this protein in X sperm is higher than that in Y sperm.

[0010] Preferably, the expression level of membrane protein RALB in X sperm is more than 50% higher than that in Y sperm.

[0011] More preferably, the expression level of membrane protein RALB in X sperm is more than 53% higher than that in Y sperm.

[0012] Specifically, the protein ID corresponding to the amino acid sequence of the porcine sperm membrane protein RALB in the Uniprot database is: I3LV17.

[0013] Specifically, the gene ID corresponding to the nucleotide sequence of the gene encoding the RALB protein in the NCBI database is: 100624229.

[0014] The present invention also provides a method for identifying porcine X sperm and porcine Y sperm. The identification method is to identify or distinguish by using the characteristic that RALB protein is differentially expressed on the surface of X sperm cells, and the expression of this protein in X sperm is significantly higher than that in Y sperm.

[0015] Preferably, the identification method is to use RALB protein as a molecular marker, detect the expression level of RALB protein, and judge by the difference in the expression level of RALB protein.

[0016] More specifically, the expression level of membrane protein RALB in X sperm is more than 50% higher than that in Y sperm.

[0017] More specifically, the expression level of membrane protein RALB in X sperm is more than 53% higher than that in Y sperm.

[0018] More preferably, the identification method for detecting the expression level of RALB protein is Western blotting or immunofluorescence.

[0019] More specifically, the steps for detecting the expression level of RALB protein by Western blotting are as follows:

[0020] S1. Extract membrane proteins from two semen samples after XY sperm separation respectively, and measure the protein concentration of sperm.

[0021] S2. Denature sperm proteins, and separate membrane proteins by SDS-PAGE gel electrophoresis with a mass ratio of 10%.

[0022] S3. Transfer the film obtained in step S2 to a nitrocellulose membrane; wash the membrane with buffer, and then block it with TBST buffer containing 5% mass ratio of skim milk powder at room temperature for 1 h - 2 h.

[0023] S4. Wash the membrane with buffer, add RALB antibody and GAPDH antibody, and incubate overnight at 4°C; then wash with buffer again, and then incubate with secondary antibody at room temperature for 1 h - 2 h.

[0024] S5. Wash with buffer, then perform blot exposure using a low-background chemiluminescence detection kit, and then count the gray value of the blot.

[0025] More preferably, the protein denaturation method in S2 is: take 30 μg of sperm protein as the sample loading amount, add 6×Protein Loading Buffer with a volume ratio of 17% and Mem-PER TM Plus solubilization buffer to make up to 20 μL, and incubate at 99°C for 10 min.

[0026] More preferably, the buffer is a TBST solution, and 0.1% (by volume) of Tween 20 is added to the solution.

[0027] More preferably, the secondary antibody used in S4 is horseradish peroxidase-labeled goat anti-rabbit IgG (H+L).

[0028] More preferably, the luminescence kit used in S5 is the cECL Western Blot Kit.

[0029] More preferably, the statistical software used in S5 is Image J.

[0030] As some alternative embodiments, the application of RALB protein in pig breeding:

[0031] 1. A method for identifying porcine X and Y sperm, comprising the following steps: using RALB protein as a molecular marker, and identifying porcine X and Y sperm by Western blotting or immunofluorescence, wherein RALB protein is differentially expressed on the surface of X sperm cells.

[0032] 2. A method for separating porcine X and Y sperm, comprising the following steps: using RALB protein as an antigen or receptor, and differentially changing the functions of porcine X or Y sperm by antibody-antigen reaction or ligand reaction, thereby separating X or Y sperm, wherein RALB protein is differentially expressed on the surface of X sperm cells.

[0033] The present invention has the following beneficial effects:

[0034] The present invention uses DIA proteomics technology to discover the porcine sperm membrane protein RALB that is differentially expressed in X sperm membrane proteins, which can be used as a differential identification protein for separating porcine X and Y sperm. This protein can be used as a molecular marker, or as an antigen for protein immunization or as a receptor for ligand action to change sperm function, thereby separating X and Y sperm. It is expected to achieve a low-cost, efficient, and simple sperm sex sorting method, which is helpful for the sustainable development of animal husbandry, helps to alleviate the future food shortage problem, and also helps to solve a series of animal welfare problems, and has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a result analysis diagram of detecting the sex of porcine sperm by sperm DNA fluorescence quantitative PCR method.

[0036] Figure 2 It is a result analysis diagram of the expression levels of membrane protein RALB in X sperm and Y sperm.

[0037] Figure 3 It is a result analysis diagram of detecting the expression of membrane protein RALB in porcine sperm by Western Blotting.

[0038] Figure 4 It is a result analysis chart of the expression level of membrane protein RALB in porcine sperm. Specific implementation manners

[0039] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0040] Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0041] Example 1 Collection, sorting and purity identification of boar sperm

[0042] 1. Experimental materials: Semen from three normally fertile Large White boars (provided by Wen's Food Group Co., Ltd.).

[0043] 2. Sampling method and location: The semen of boars was collected by the hand-held method, and the sample collection and separation work was completed at Inner Mongolia Saikexing Livestock Breeding and Biotechnology Research Institute.

[0044] 3. Sperm sorting:

[0045] (1) Experimental instrument: Flow cytometer;

[0046] (2) Experimental steps: The collected boar semen was sorted by a flow cytometer according to the conventional method to obtain 3 portions of sex-controlled X semen and 3 portions of sex-controlled Y semen.

[0047] 4. Identification and purity test of sex-controlled X semen and sex-controlled Y semen:

[0048] (1) Experimental method: DNA fluorescence quantitative PCR method

[0049] (2) Experimental steps:

[0050] ① Sperm DNA extraction:

[0051] Take the 6 semen samples collected in step 3 and perform DNA extraction respectively according to the following methods:

[0052] A. Centrifuge the semen containing 1000 - 2500 sperm at 2000×g for 5 min, discard the supernatant, resuspend with 5 μL of lysis solution (200 mM KOH, 50 mM DTT), and incubate at 65 °C in a PCR instrument for 20 min;

[0053] B. Add 5 μL of neutralization solution (900 mM Tris-HCL, pH = 8.3) to terminate the reaction, and dilute to 40 μL with ddH2O to obtain the DNA extract of the semen sample.

[0054] ② PCR primer design:

[0055] Using Oligo7 software, according to the basic principles of primer design, design PCR amplification primers for SRY (Y chromosome differential gene) and AMELX (X chromosome differential gene) genes (Table 1), and submit them to BGI for synthesis.

[0056] Table 1 Primers for fluorescence quantitative PCR detection

[0057]

[0058] ③ Fluorescence quantitative PCR reaction

[0059] The PCR reaction system is as follows:

[0060] Table 2 Fluorescence quantitative PCR reaction system

[0061]

[0062] The PCR reaction program is as follows:

[0063] Table 3 PCR reaction program

[0064]

[0065]

[0066] Use the 2 -△△CT method to calculate the relative expression levels of SRY and AMELX genes in different sperm samples. The ratios of X sperm and Y sperm in each sample are as Figure 1 shown. The detection results correspond one-to-one with the sorting results of flow cytometry, and at the same time, the concentration of each sample is above 80%.

[0067] Example 2 Extraction of sperm membrane proteins

[0068] 1. Experimental reagents: Mem-PER TM Plus Membrane Protein Extraction Kit (purchased from Thermo Fisher Scientific).

[0069] 3. The 6 semen samples in Example 1 were subjected to membrane protein extraction respectively according to the following steps:

[0070] (1) Centrifuge the semen containing 100 million sperm at 2000×g for 5 min, and then wash the cell pellet with 3 mL of Mem-PER TM Plus Cell Wash Solution and centrifuge at 2000×g for 5 min;

[0071] (2) Carefully remove and discard the supernatant, and then resuspend the cells in 1.5 mL of Mem-PERTM Add cell washing solution, then transfer it to a 2 mL centrifuge tube, centrifuge at 2000×g for 5 min and discard the supernatant;

[0072] (3) Add 0.75 mL of Mem-PER TM Plus permeabilization buffer (previously mixed with protease inhibitor at a volume ratio of 100:1), vortex briefly to obtain a homogeneous cell suspension, and incubate at 4 °C for 10 min under continuous mixing;

[0073] (4) Centrifuge the permeabilized cells at 16000×g for 15 min, carefully remove the supernatant containing cytoplasmic proteins;

[0074] (5) Add 0.25 mL of Mem-PER TM Plus solubilization buffer (previously mixed with protease inhibitor at a volume ratio of 100:1) and resuspend by pipetting up and down, incubate at 4 °C for 60 min under continuous mixing;

[0075] (6) At 4 °C, centrifuge the reaction tube at 16000×g for 15 min; 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.

[0076] Example 3 Determination of Protein Concentration in Membrane Protein Solution

[0077] 1. Experimental materials: Sperm membrane protein solution (X sperm sample membrane protein solution or Y sperm sample membrane protein solution) prepared in Example 2; BCA (bicinchonininc acid) kit (purchased from Thermo Fisher Scientific).

[0078] 2. The specific steps are as follows:

[0079] (1) According to the BCA kit instructions, prepare the required volume of chromogenic solution according to the ratio of buffer A:Buffer B = 50:1 (v / v);

[0080] (2) Take out part of the protein solution to be measured and dilute it with Mem-PER TM Plus solubilization buffer (to prevent the concentration from being too high and exceeding the working range of the standard curve);

[0081] (3) Prepare a clean 96-well plate, add the following gradient of BSA standard protein solution: 0, 1, 2, 4, 8, 12, 16, 20 μL, and then add the corresponding volume of ultrapure water to each well to make up the volume to 20 μL;

[0082] (4) Add 2 μL of the protein solution to be tested to a 96-well plate, set three replicates for each sample, and also supplement the volume to 20 μL;

[0083] (5) Add 200 μL of the pre-prepared chromogenic solution (the chromogenic solution must be prepared immediately before use) to each well, and react at 37 °C for 30 min;

[0084] (6) Use an enzyme-labeled instrument to measure the absorbance value (wavelength 562 nm);

[0085] (7) Calculate the standard curve based on the known concentration and absorbance value of the standard protein solution, substitute the absorbance value of the sample to be tested, and the protein concentration value can be calculated (see Table 4).

[0086] Table 4 Sperm membrane protein concentration

[0087] Sperm number Protein concentration (ug / uL) X-1 0.581 X-2 0.533 X-3 0.571 Y-1 0.543 Y-2 0.609 Y-3 0.552

[0088] Example 4 Trypsin digestion of membrane proteins

[0089] 1. Experimental materials: The sperm membrane protein solution prepared in Example 2 (X sperm sample membrane protein solution or Y sperm sample membrane protein solution).

[0090] 2. Experimental steps:

[0091] (1) According to the protein concentration measured in Example 3, take 50 μg of protein from each sample, and use SDS lysis buffer to dilute and adjust the samples in different groups to the same concentration and volume;

[0092] (2) Add dithiothreitol (DTT) to the above protein solution to make the final concentration of DTT 4.5 mM, mix well, and incubate at 55 °C for 30 min;

[0093] (3) Cool on ice until reaching room temperature, then add the corresponding volume of iodoacetamide to make the final concentration of iodoacetamide 9 mM, mix well, and place in the dark at room temperature for 15 min;

[0094] (4) Add 6 volumes of acetone to precipitate the protein in the above solution, and place it at -20 °C for more than 4 h or overnight;

[0095] (5) Centrifuge at 4 °C and 8000×g for 10 min to collect the precipitate, and evaporate the acetone for 2 - 3 min;

[0096] (6) Add 100 μL of tetraethylammonium bromide (TEAB2) to redissolve the precipitate, then add 1 mg / mL trypsin Trypsin-TPCK at 1 / 50 of the sample mass, and digest overnight at 37 °C;

[0097] (7) Adjust the pH value to about 3 with phosphoric acid to terminate the enzymatic hydrolysis reaction, complete the enzymatic hydrolysis of the membrane protein, and obtain the peptide sample solution.

[0098] Example 5 Desalination of Trypsin-Digested Peptides

[0099] 1. Experimental Materials: Peptide sample solution after trypsin digestion in Example 4; SOLA TM SPE column (purchased from Thermo Fisher Scientific); 96-well plate.

[0100] 2. Experimental Procedures:

[0101] (1) Activation: Activate the column with 200 μL of methanol, repeat 3 times;

[0102] (2) Equilibration: Equilibrate the column with 200 μL of pure water, repeat 3 times;

[0103] (3) Adjust the pH of the peptide sample solution after enzymatic digestion in Example 4 to 7 and then load the sample. Subsequently, adjust the vacuum to pass the column, and keep the droplet speed at 1 mL / min, and repeat once;

[0104] (4) Wash with 200 μL of 5% (V / V) methanol, repeat 3 times;

[0105] (5) Elution: Elute the peptides with 150 μL of 60% (V / V) methanol, repeat 3 times, obtain 450 μL of eluate, and evaporate to dryness under vacuum to obtain the desalted peptide sample.

[0106] Example 6 LC-MS / MS High-Resolution Mass Spectrometry Detection

[0107] 1. Detection Institution: Shanghai Luminary Biotechnology Co., Ltd.

[0108] 2. Sample Pretreatment: Before mass spectrometry injection, mix each sample with the iRT standard at a volume ratio of iRT standard: test sample = 1:10 as an internal standard.

[0109] 3. Detection Procedures:

[0110] The 6 groups of peptide samples after desalination in Example 5 are each divided into two parts. One part is mixed and used for high-pH liquid separation, and the other part is used for liquid chromatography-mass spectrometry (LC-MS) separation alone.

[0111] (1) High-pH Liquid Separation

[0112] Sample: Take equal amounts of peptides from the 6 groups of peptide samples after desalination in Example 5 and mix them. Use the Agilent 1100 HPLC system to separate the components in the mobile phase with pH = 10.

[0113] Separation Conditions: Chromatographic Column: Agilent Zorbax Extend–C18 narrow-bore column, 2.1×150 mm, 5 μm.

[0114] Detection wavelengths: UV 210 nm and 280 nm.

[0115] Mobile phase A: Acetonitrile (ACN) - H2O (2:98, v / v), mobile phase B: ACN - H2O (90:10, v / v) (both mobile phases are adjusted to pH 10 with ammonia water), flow rate: 250 μL / min.

[0116] 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.

[0117] Component collection: Starting from the 10th minute, the eluate is collected into centrifuge tubes numbered 1 - 10 at intervals of one minute in sequence, and the fractions are collected in a cycle according to the order of 1 → 10. A total of 10 components are collected, vacuum freeze-dried and dried, and the samples are stored frozen for mass spectrometry.

[0118] (2) Liquid chromatography - mass spectrometry separation

[0119] Liquid chromatography - mass spectrometry conditions:

[0120] Use an EASY-nLC 1200 high-performance nano liquid chromatography instrument and a Q Exactive HF mass spectrometer to perform DDA mass spectrometry scans on the 10 components collected in step (1). The scanning parameters are shown in Table 5:

[0121] Table 5 Data-dependent acquisition (DDA) mass spectrometry conditions

[0122] Items Full MS MS2 Resolution 120000 30000 AGC target 3e6 1e5 Maximum injection time 100ms 50ms Scan range 350 - 1650m / z 200 - 2000m / z Isolation window - 1.4m / z Normalized Collision Energy - 27

[0123] The liquid chromatography elution gradient is shown in Table 6: The flow rate is 300 nL / min, buffer A is 0.1% FA (trifluoroacetic acid) aqueous solution, and buffer B is 0.1% FA / 80% ACN / 20% water (the percentages in this section are volume percentages).

[0124] Table 6 Data-dependent acquisition (DDA) chromatography conditions

[0125]

[0126]

[0127] Take six aliquots of the desalted peptides from Example 5 and separately load them onto the instrument (EASY-nLC 1200 high-performance nano liquid chromatography instrument and Q Exactive HF mass spectrometer) for DIA mass spectrometry scanning. The scanning range is set to 350 - 1250 m / z, the isolation window is 26 m / z, and the mass spectrometry scanning parameters are shown in Table 7:

[0128] Table 7 Data-independent acquisition (DIA) mass spectrometry conditions

[0129] Items Full MS MS2 Resolution 120000 30000 AGC target 3e6 1e6 Maximum injection time 100ms Auto Scan range 350 - 1250m / z - Isolation window - 26m / z Normalized Collision Energy - 28

[0130] The DIA liquid elution gradient is shown in Table 8:

[0131] Table 8 Data-independent acquisition (DIA) chromatography conditions

[0132] Time (min) Gradient 0 2％B 82 44％B 84 90％B 90 90％B

[0133] The flow rate is 300 nL / min, buffer A is 0.1% FA aqueous solution, and buffer B is 0.1% FA / 80% ACN / 20% water.

[0134] Example 7 Data processing and analysis

[0135] The purpose of this operation is to match the mass spectrometry output spectra with the theoretical spectra generated by the fasta library, convert the machine signals into peptide and protein sequence information, and then establish a spectral library by combining sequence information, peptide retention time, fragment ion information, etc. for subsequent DIA analysis. Import the LC-MS / MS mass spectrometry raw files into the Spectronaut Pulsar software for DDA library construction. The main parameters are shown in the following table.

[0136] Table 9 Data-dependent acquisition (DDA) library construction parameters

[0137]

[0138]

[0139] The processing of DIA raw data is completed using the Spectronaut Pulsar software. The key steps are as follows:

[0140] (1) Open the Analysis module of the Spectronaut Pulsar software and select "+" to create a new analysis;

[0141] (2) Complete the parameter settings step by step according to the software prompts and start the analysis;

[0142] (3) Analysis is completed, and enter the "Report" module to export quantitative data.

[0143] Table 10 Key parameters for data-independent acquisition (DIA) data analysis

[0144] Items Para. Precursor Qvalue cutoff 0.01 Protein Qvalue cutoff 0.01 Normalization Strategy Local Normalization Quantity MS - Level MS2

[0145] Subsequently, using the quantitative data obtained from database retrieval, retain any group of samples with a protein expression value ratio ≥ 50%. Proteins with missing values ≤ 50% are filled with the mean value of the same group of samples. After Median Normalization and log2 logarithmic transformation, reliable proteins are obtained. Subsequently, differential expression analysis is performed on the identified proteins, and the differential screening conditions are Foldchange = 1.2-fold and p-value < 0.05. Finally, RALB is identified as the differential membrane protein marker of X sperm. The molecular marker RALB is significantly higher in X sperm than in Y sperm (see Figure 2 as shown).

[0146] Example 8 Identification of RALB in porcine sperm membrane proteins by Western Blotting

[0147] 1. Pretreatment before experiment:

[0148] (1) Sample collection: Referring to the method of Example 1, collect semen from three normally fertile large white boars again, sort it using a flow cytometer to obtain 6 samples, anonymize the 6 samples and shuffle the order.

[0149] (2) Protein denaturation: Referring to the method of Example 2, extract the membrane proteins of 6 anonymous samples, and then refer to Example 3 to measure the membrane protein concentration of each sample; then take 30 μg of protein from each sample, add 6×Protein Loading Buffer and Mem-PER TM Plus solubilization buffer to dilute to the same concentration and volume, and incubate at 99 °C for 10 min to denature the protein. The protein denaturation system is shown in Table 11 below:

[0150] Table 11 Protein denaturation reaction system

[0151] Component Dosage Sperm protein 30μg 6×Protein Loading Buffer 3.4μL <![CDATA[Mem-PER TM Plus solubilization buffer]]> Make up to 20μL Total volume 20μL

[0152] 2. Steps of Western Blotting experiment:

[0153] (1) Prepare TBST buffer: Thoroughly dissolve the dry powder of TBS buffer (Saiwei'er Biotechnology Co., Ltd.) in 2 L of double-distilled water, and then add 2 mL of Tween20 and stir for 25 min;

[0154] (2) Separate membrane proteins by 10% (mass percentage) SDS-PAGE gel electrophoresis, and then transfer them to a nitrocellulose membrane. After the transfer is completed, wash the membrane 3 times with TBST buffer for 5 minutes each time. Subsequently, block it with TBST buffer containing 5% (mass percentage) skim milk powder at room temperature for 2 hours.

[0155] (3) Wash the membrane 3 times with TBST buffer for 5 minutes each time, add RALB antibody and GAPDH antibody respectively, and incubate overnight at 4°C.

[0156] (4) Wash the membrane 3 times with TBST buffer for 5 minutes each time, and then incubate it with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) at room temperature for 2 hours.

[0157] (5) Wash the membrane 3 times with TBST buffer for 5 minutes each time, and then perform blot exposure using the cECL Western Blot Kit low-background chemiluminescence detection kit (CW Biotech Co., Ltd.).

[0158] (6) Use Image J software (http: / / imagej.net / ImageJ) to statistically analyze the gray value.

[0159] The detection results are shown in Figure 3 and Figure 4 . The results show that the identification results of the RALB protein quantification method are consistent with the sorting results of the flow cytometer.

[0160] The expression level of RALB protein in sperm 1, 2, and 3 is 53% higher than that in sperm 4, 5, and 6. Sperm 1, 2, and 3 are identified as X sperm, and sperm 4, 5, and 6 are identified as Y sperm, which is consistent with the sorting results of the flow cytometer.

[0161] The above results indicate that the porcine sperm membrane protein RALB can be used as a cell surface marker protein for identifying and differentiating X and Y sperm.

[0162] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Application of pig sperm membrane protein RALB in identifying or distinguishing pig X sperm from pig Y sperm, characterized in that: The method for identification or differentiation is to use the fact that the expression of pig sperm membrane protein RALB in pig X sperm is higher than that in pig Y sperm for identification or differentiation; the amino acid sequence of the pig sperm membrane protein RALB corresponds to the protein ID: I3LV17 in the Uniprot database; the nucleotide sequence of the gene encoding the pig sperm membrane protein RALB corresponds to the gene ID: 100624229 in the NCBI database.

2. The application according to claim 1, characterized in that The expression level of pig sperm membrane protein RALB in pig X sperm was more than 53% higher than that in pig Y sperm.

3. A method for distinguishing pig X sperm from pig Y sperm, characterized in that: Identification is performed by utilizing the fact that the expression of the porcine sperm membrane protein RALB in pig X sperm is higher than that in pig Y sperm; the amino acid sequence of the porcine sperm membrane protein RALB corresponds to the protein ID: I3LV17 in the Uniprot database; the nucleotide sequence of the gene encoding the porcine sperm membrane protein RALB corresponds to the gene ID: 100624229 in the NCBI database.

4. The method according to claim 3, characterized in that the porcine sperm membrane protein RALB is used as a molecular marker to detect the expression level of the porcine sperm membrane protein RALB, and the judgment is made based on the difference in the expression level of the porcine sperm membrane protein RALB.

5. The method according to claim 4, characterized in that the method for detecting the expression level of pig sperm membrane protein RALB is Western blotting.

6. The method according to claim 4, characterized in that the method for detecting the expression level of pig sperm membrane protein RALB is immunofluorescence method.

7. The method according to claim 4, wherein the expression level of the porcine sperm membrane protein RALB in pig X sperm is more than 53% higher than that in pig Y sperm.

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