A kit for identifying pig y sperm and application thereof

Abstract

The application provides a kit for identifying pig Y sperm and application thereof. Specifically, the kit takes pig membrane protein PPP4C or pig membrane protein CSNK2A2 as a molecular marker, tests protein expression amount by Western blotting or immunofluorescence, and identifies pig X and Y sperm according to the difference of the protein expression amount, and the specific identification standard is that the expression of pig membrane protein PPP4C or pig membrane protein CSNK2A2 in Y sperm is higher than that in X sperm. The application provides an effective scheme for realizing the identification of pig X and Y sperm by immunization, realizes the identification of pig X and Y sperm with low cost, high efficiency and simplicity, helps the sustainable development of animal husbandry, improves economic benefits, helps to alleviate the future food shortage problem, solves a series of animal welfare problems, and has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a kit for identifying pig Y sperm and application thereof. BACKGROUND

[0002] In livestock production, the sex ratio of newborn livestock has become an important factor affecting the economic benefits of livestock industry. For example, in dairy cattle breeding, people prefer to have a higher proportion of female offspring. In addition to alleviating food shortages, it can also effectively solve the problem of animal welfare such as castration. Sex control technology is a new biotechnology that selectively determines the sex of offspring by interfering with the normal reproductive process of animals. X / Y sperm separation and identification technology based on the differences in size, motility, charge, surface antigen and DNA content of X and Y sperm is one of the feasible solutions for sex control technology.

[0003] At present, the methods for X / Y sperm separation and identification include electrophoresis, albumin column separation, Percoll density gradient centrifugation and flow cytometry separation. Flow cytometry sperm separation technology separates sperm based on the difference in DNA content in the head of X and Y sperm, and is the most successful sperm separation and identification technology at present, which has been widely used in dairy cattle breeding practice. However, the use of flow cytometry to separate pig sperm can cause damage to the sperm cell membrane, resulting in a decrease in motility, fertilization ability and in vitro preservation ability (Garcia E M, Vazquez J M, Parrilla I, et al. Improving the fertilizing ability of sex sorted boar spermatozoa [J]. Theriogenology. 2007, 68(5):771-778. Maxwell W M, Johnson L A. Physiology of spermatozoa at high dilution rates: the influence of seminal plasma [J]. Theriogenology. 1999, 52(8):1353-1362.). In addition, the amount of insemination for sows is usually much higher than that for cows, and the flow cytometry equipment is expensive and costly. The yield of sperm separated and identified by flow cytometry cannot meet the actual breeding needs.

[0004] The immunization method realizes the identification of X and Y sperm by recognizing the differential proteins on the membranes of X and Y sperm, has the advantages of low cost, high efficiency and simplicity, helps the sustainable development of the livestock industry, improves the economic benefits, helps to alleviate the future food shortage problem, solves a series of animal welfare problems, and has important application value. Therefore, it is necessary to provide a method for identifying X and Y sperm by immunization. SUMMARY

[0005] The present application aims at the deficiencies of the prior art, and aims to provide a kit for identifying pig Y sperm and application thereof, so as to realize the identification of pig X and Y sperm by immunization and better control the gender of pigs.

[0006] The primary purpose of the present application is to provide the application of pig membrane protein PPP4C and / or pig membrane protein CSNK2A2 in identifying pig Y and X sperm.

[0007] Another purpose of the present application is to provide the application of a reagent for detecting pig membrane protein PPP4C and / or pig membrane protein CSNK2A2 in preparing a kit for identifying pig Y and X sperm.

[0008] Another purpose of the present application is to provide a kit for identifying pig X and Y sperm.

[0009] Another purpose of the present application is to provide a method for identifying pig X and Y sperm.

[0010] The present application realizes the above-mentioned application purposes through the following technical means:

[0011] The present application provides the application of pig membrane protein PPP4C and / or pig membrane protein CSNK2A2 in identifying pig Y and X sperm, wherein the protein ID corresponding to the pig membrane protein PPP4C in the Uniprot database is M3VK32, and the protein ID corresponding to the pig membrane protein CSNK2A2 in the Uniprot database is A0A287B496.

[0012] The present application also provides the application of a reagent for detecting pig membrane protein PPP4C and / or pig membrane protein CSNK2A2 in preparing a kit for identifying pig Y and X sperm, wherein the protein ID corresponding to the pig membrane protein PPP4C in the Uniprot database is M3VK32, and the protein ID corresponding to the pig membrane protein CSNK2A2 in the Uniprot database is A0A287B496.

[0013] Preferably, the method for application is to take porcine membrane protein PPP4C and / or porcine membrane protein CSNK2A2 as a molecular marker, test the protein expression amount by Western blotting and / or immunofluorescence, and identify X and Y sperm according to the difference of the protein expression amount, and the specific identification standard is that the expression of porcine membrane protein PPP4C or porcine membrane protein CSNK2A2 in Y sperm is higher than that in X sperm.

[0014] Preferably, the reagent for detecting porcine membrane protein PPP4C and / or porcine membrane protein CSNK2A2 is a PPP4C antibody and / or a CSNK2A2 antibody.

[0015] The application also provides a kit for identifying porcine X and Y sperm, comprising a reagent for detecting porcine membrane protein PPP4C and / or porcine membrane protein CSNK2A2, wherein the protein ID corresponding to the porcine membrane protein PPP4C in the Uniprot database is M3VK32, and the protein ID corresponding to the porcine membrane protein CSNK2A2 in the Uniprot database is A0A287B496.

[0016] Preferably, the reagent comprises a PPP4C antibody and / or a CSNK2A2 antibody.

[0017] More preferably, the reagent further comprises a reagent required for Western blotting.

[0018] More preferably, the reagent further comprises a reagent required for immunofluorescence.

[0019] Preferably, the reagent comprises a reagent for detecting the relative expression amount of gene PPP4C and / or gene CSNK2A2, wherein the gene ID corresponding to the gene PPP4C in the NCBI database is 110260085, and the gene ID corresponding to the gene CSNK2A2 in the NCBI database is 100737256.

[0020] More preferably, the reagent is a primer for detecting the relative expression amount of gene PPP4C and / or gene CSNK2A2.

[0021] The application also provides a method for identifying porcine X and Y sperm, taking porcine membrane protein PPP4C and / or porcine membrane protein CSNK2A2 as a molecular marker, testing the protein expression amount by Western blotting or immunofluorescence, and identifying porcine X and Y sperm according to the difference of the protein expression amount, and the specific identification standard is that the expression of porcine membrane protein PPP4C or porcine membrane protein CSNK2A2 in Y sperm is higher than that in X sperm.

[0022] Preferably, the specific steps of taking porcine membrane protein PPP4C and / or porcine membrane protein CSNK2A2 as a molecular marker, and identifying porcine X and Y sperm by Western blotting and immunofluorescence are as follows:

[0023] S1. Extracting the membrane proteins from two semen samples after XY sperm separation;

[0024] S2. Detecting the expression amount of pig membrane protein PPP4C and / or pig membrane protein CSNK2A2 in the membrane proteins in step S1 by Western blotting and immunofluorescence;

[0025] S3. Result determination: the semen with high expression amount of pig membrane protein PPP4C and / or CSNK2A2 is Y semen, and the other semen sample is identified as X semen.

[0026] Preferably, the standard of high expression amount in step S3 is that the expression amount of pig membrane protein PPP4C in Y semen is more than 40% higher than that in X semen (more preferably more than 46% higher).

[0027] Preferably, the standard of high expression amount in step S3 is that the expression amount of pig membrane protein CSNK2A2 in Y semen is more than 50% higher than that in X semen (more preferably more than 57% higher).

[0028] Preferably, the reagents required for the Western blotting in S2 include a first antibody and a second antibody; the first antibody is an antibody specifically recognizing pig membrane protein PPP4C and / or an antibody specifically recognizing pig membrane protein CSNK2A2.

[0029] Preferably, the second antibody is a horseradish peroxidase-labeled second antibody.

[0030] Preferably, S2 determines the expression amount of pig membrane protein PPP4C and / or pig membrane protein CSNK2A2 in the membrane proteins in step S1 by Western blotting exposure and gray value statistics by a low-background chemiluminescence detection kit.

[0031] The present application has the following beneficial effects:

[0032] The present application studies pig membrane protein PPP4C and pig membrane protein CSNK2A2 specifically expressed on pig Y sperm, and uses pig membrane protein PPP4C or pig membrane protein CSNK2A2 as a molecular marker to identify pig X and Y sperm by Western blotting and immunofluorescence. The present application provides an effective scheme for identifying pig X and Y sperm by immunization, realizes low-cost, efficient and simple identification of pig X and Y sperm, helps the sustainable development of animal husbandry, improves economic benefits, helps to alleviate future food shortages, solves a series of animal welfare problems, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 A result analysis chart for the sperm DNA fluorescent quantitative PCR method for detecting the gender of pig sperm.

[0034] Figure 2 Graphical analysis of the results of the expression amount of porcine membrane protein PPP4C in X sperm and Y sperm.

[0035] Figure 3 Graphical analysis of the results of the expression amount of porcine membrane protein CSNK2A2 in X sperm and Y sperm.

[0036] Figure 4 Graphical analysis of the results of the expression amount of porcine membrane protein PPP4C in gender-controlled semen in Example 4 using Image J software for Western Blotting detection results.

[0037] Figure 5 Graphical analysis of the results of the expression amount of porcine membrane protein CSNK2A2 in gender-controlled semen in Example 5 using Image J software for Western Blotting detection results. DETAILED DESCRIPTION

[0038] The present application will be further described in conjunction with the following examples and drawings, but the embodiments of the present application are not limited thereto.

[0039] The test methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the examples are all purchased from conventional biochemical reagent stores unless otherwise specified; the quantitative tests in the examples are all set up with three repeated experiments, and the results are averaged.

[0040] Test instruments:

[0041] Agilent 1100 HPLC high-pH separation liquid chromatograph (Agilent);

[0042] EASY-nLC 1200 high-efficiency nanoliter liquid chromatograph (ThermoFisher);

[0043] Q Exactive HF mass spectrometer (ThermoFisher);

[0044] Fluorescent quantitative PCR instrument (ABI);

[0045] PowerPac universal electrophoresis instrument (Bio-rad);

[0046] PCR instrument (ThermoFisher);

[0047] Flow cytometer and gel imaging system (UVP), etc.

[0048] Test materials:

[0049] Mem-PER TMPlus membrane protein extraction kit (Thermo Fisher);

[0050] Protease inhibitor (Thermo Fisher);

[0051] BCA kit (Thermo Scientific);

[0052] SDS lysis buffer (Bioun);

[0053] iRT standard (Biognosys, Thermo Fisher);

[0054] PowerUp TM SYBR TM Green Master Mix (Thermo Fisher);

[0055] 6x Protein Loading Buffer (Quanshijin);

[0056] TBS buffer (Savillex), Tween20 (Sigma);

[0057] Skim milk powder (Shenguo);

[0058] PPP4C antibody (Novus);

[0059] CSNK2A2 antibody (Novus);

[0060] TUBULIN antibody (Servicebio);

[0061] Horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) (Bioun);

[0062] cECL Western Blot Kit low background chemiluminescence detection kit (CWBIO) and the like.

[0063] Example 1 Collection, separation and purity identification of boar semen samples

[0064] I. Collection and separation of boar semen samples

[0065] The collection and separation of boar semen samples were carried out in Inner Mongolia Saikesheng Livestock Breeding and Biotechnology Research Institute: The semen of three normal fertile Large White boars (provided by Wens Food Group Co., Ltd.) was collected by hand-holding method, and the collected boar semen was sorted by flow cytometry according to the conventional method to obtain gender-controlled X semen (X-1, X-2, X-3) and gender-controlled Y semen (Y-1, Y-2, Y-3).

[0066] II. Purity detection of pig semen sample

[0067] 1. Sperm DNA extraction, specifically comprising the following steps:

[0068] S1. Take part of the six separated sex-controlled semen samples, and control the sperm number to be 1000-2500 sperm, centrifuge at 2000 G value for 5 min, discard the supernatant, resuspend the cell precipitate with 5 μL lysis solution (containing 200 mM KOH, 50 mM DTT), incubate at 65°C for 20 min in a PCR instrument.

[0069] S2. Add 5 μL neutralization solution (containing 900 mM Tris-HCL, pH = 8.3) to terminate the lysis reaction, and dilute with ddH2O to 40 μL, to obtain the DNA extraction solution of the six sex-controlled semen samples.

[0070] 2. PCR primer design

[0071] According to the basic principles of primer design, the Oligo7 software is used to design the forward primer SRY-F and the reverse primer SRY-R for SRY gene (Y chromosome specific gene) PCR amplification, and the forward primer AMELX-F and the reverse primer AMELX-R for AMELX gene (X chromosome specific gene) PCR amplification. The primer synthesis is completed by Huada Gene, and the primer sequences are shown in Table 1.

[0072] Table 1 Primer sequences for fluorescence quantitative PCR detection

[0073]

[0074]

[0075] 3. Fluorescence quantitative PCR reaction

[0076] The extracted DNA of the six sex-controlled semen samples is subjected to fluorescence quantitative PCR reaction.

[0077] The PCR reaction system is shown in Table 2:

[0078] Table 2 Fluorescence quantitative PCR reaction system

[0079] Component Amount (μL) Sperm DNA template 2 Primer F 0.2 Primer R 0.2 PowerUp TM SYBR TM Green Master Mix]]> 5.0 ddH2O 2.6 Total volume 10.0

[0080] The PCR reaction program is shown in Table 3:

[0081] Table 3 PCR reaction program

[0082]

[0083] Use 2-△△CT The relative expression levels of SRY and AMELX genes in different semen samples were calculated to determine the ratio of X sperm to Y sperm in each sample. Figure 1 As shown. The test results correspond one-to-one with the flow cytometry sorting results; sperm 1, 2, and 3 are three groups of sex-controlling X semen obtained by flow cytometry sorting, with the proportion of X sperm all above 80%; sperm 4, 5, and 6 are three groups of sex-controlling Y semen obtained by flow cytometry sorting, with the proportion of Y sperm all above 80%.

[0084] Example 2: Extraction of sperm membrane proteins from X and Y semen for sex control

[0085] Use Mem-PER TM The Plus Membrane Protein Extraction Kit (purchased from Thermo Fisher Scientific) was used to extract sperm membrane proteins from sex-controlled X semen and sex-controlled Y semen obtained in Example 1, respectively. The specific steps are as follows:

[0086] S1. Take a portion of each of the six sex-controlled semen samples obtained in Example 1, and control the sperm count to 100 million sperm. Centrifuge at 2000x G value for 5 min to separate the cell pellet, and then use 3 mL of Mem-PER. TM After washing the cell pellet with Plus cell washing buffer, centrifuge at 2000-fold G value for 5 min;

[0087] S2. Carefully remove and discard the supernatant, then redisperse the cell pellet in 1.5 mL of Mem-PER. TM The cells were washed with Plus cell washing solution, then transferred to a 2 mL centrifuge tube, centrifuged at 2000x G value for 5 min and the supernatant was discarded, while the cell pellet was retained.

[0088] S3. Add 0.75 mL of Mem-PER to the cell pellet retained in S2. TM Plus permeation buffer (before use with Mem-PER) TM Plus permeation buffer and protease inhibitor were mixed at a volume ratio of 100:1, and the mixture was briefly vortexed to obtain a homogeneous cell suspension. The suspension was then incubated at 4°C for 10 min under continuous mixing to obtain permeated cells.

[0089] S4. Centrifuge the permeabilized cells at 16000 times G value for 15 min, carefully remove the supernatant, and retain the cell pellet;

[0090] S5. Add 0.25 mL of Mem-PER to the cell pellet retained in S4. TM Plus solubilization buffer (before use, Mem-PER) TMPlus solubilization buffer and protease inhibitors in a volume ratio of 100:1, and use a pipette to suck up and down to re-disperse the cells, and incubate at 4°C for 60 min under continuous mixing;

[0091] S6. Centrifuge the cell suspension of S5 at 16000 G value for 15 min at 4°C; transfer the supernatant containing soluble membrane proteins and membrane-associated proteins to a new reaction tube to obtain 3 sets of gender-controlled X semen membrane protein sample solutions (X-1, X-2, X-3) and 3 sets of gender-controlled Y semen membrane protein sample solutions (Y-1, Y-2, Y-3), respectively.

[0092] Example 3: Proteomics analysis

[0093] I. Protein concentration determination

[0094] Take the gender-controlled X semen and gender-controlled Y semen membrane protein sample solutions (X-1, X-2, X-3; Y-1, Y-2, Y-3) extracted in Example 2, respectively, and determine the protein concentration of the membrane protein sample solution by BCA method using BCA kit, and the specific steps are as follows:

[0095] S1. According to the description of BCA kit, prepare the required volume of color developing solution in a ratio of buffer A: Buffer B = 50:1 (v / v);

[0096] S2. Take part of the membrane protein sample solution and dilute it with Mem-PER TM Plus solubilization buffer (to prevent high concentration beyond the working range of the standard curve) to obtain the test protein solution (X-1, X-2, X-3; Y-1, Y-2, Y-3);

[0097] S3. Prepare a clean 96-well plate, and add the following volume gradient of BSA standard protein solution: 0, 1, 2, 4, 8, 12, 16, 20 μL, set 3 replicates for each volume gradient, then add the corresponding volume of ultrapure water to make up the volume to 20 μL per well;

[0098] S4. Add 2 μL of the test protein solution to the clean replicates of the 96-well plate, set three replicates for each sample, and use ultrapure water to make up the volume to 20 μL;

[0099] S5. Add 200 μL of pre-configured color developing solution (color developing solution must be prepared immediately before use) to each well, and react at 37°C for 30 min;

[0100] S6. Use a microplate reader to measure the absorbance value (wavelength 562 nm) of each replicate (including standard protein solution replicates and test protein solution replicates);

[0101] S7. Calculate the standard curve according to the known concentration and absorbance value of the standard protein solution, and substitute the absorbance value of the protein solution to be measured to calculate the concentration of each group of sperm membrane protein sample solution (see Table 4).

[0102] Table 4 Sperm membrane protein concentration

[0103] 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

[0104] II. Trypsin enzymolysis

[0105] The membrane protein sample solutions of the gender control X sperm and the gender control Y sperm obtained in Example 2 were subjected to trypsin enzymolysis, and the specific steps were as follows:

[0106] S1. According to the protein concentration obtained by the above protein concentration determination, 50 μg of protein was taken from each of the membrane protein samples extracted in Example 2, and different groups of samples were diluted and adjusted to the same concentration and volume with SDS lysis buffer;

[0107] S2. Add dithiothreitol (DTT) to the protein solution of the same volume and concentration obtained in S1 to make the final concentration of DTT 4.5 mM, mix well, and incubate at 55°C for 30 min;

[0108] S3. After the protein solution incubated in S2 is cooled to room temperature on ice, add iodoacetamide to make the final concentration of iodoacetamide 9 mM, mix well, and place in the dark at room temperature for 15 min;

[0109] S4. Add 6 times the volume of acetone to the solution after the operation in S3 to precipitate the protein, and place it at -20°C for more than 4 h;

[0110] S5. Centrifuge at 4°C and 8000 times G value for 10 min to collect the protein precipitate, and evaporate the acetone for 2-3 min;

[0111] S6. Add 100 μL of tetraethylammonium bromide (TEAB2) to resuspend the protein precipitate, then add 1 mg / mL of trypsin-TPCK in an amount of 1 / 50 of the sample mass, and digest overnight at 37°C;

[0112] S7. Add phosphoric acid to terminate the enzymolysis reaction at a pH value of about 3 to obtain the peptide segment sample solutions after enzymolysis (X-1, X-2, X-3; Y-1, Y-2, Y-3).

[0113] III. Desalination of peptide segments

[0114] The peptide segment sample solutions after enzymolysis were desalted using SOLA TM SPE 96-well plates (Thermo Fisher) according to the following specific steps:

[0115] S1. Activation: 200 μL methanol activated column, repeated 3 times;

[0116] S2. Equilibrium: 200 μL pure water activated column, repeated 3 times;

[0117] S3. After adjusting the pH of the enzymatic peptide sample solution to 7, it was loaded, and then adjusted vacuum column, the drop speed was kept at 1 mL / min, a total of 2 times, so that the peptide was adsorbed in the column;

[0118] S4. Wash column with 200 μL 5% (V / V) methanol, repeated 3 times;

[0119] S5. Elution: 150 μL 60% (V / V) methanol eluted peptide, repeated 3 times, 450 μL eluent was obtained, vacuum dried, and the desalted peptide sample (X-1, X-2, X-3; Y-1, Y-2, Y-3) was obtained.

[0120] Four, LC-MS / MS high-resolution mass spectrometry detection (Shanghai Luming Biological Company)

[0121] Before the experiment, the desalted peptide sample was mixed with iRT standard according to the volume ratio of 10:1, and the iRT standard was used as an internal standard.

[0122] 1. High pH liquid separation

[0123] (1) Sample: Equal amounts of desalted peptide samples were mixed (a portion of the desalted peptide sample was left for subsequent experiments), and Agilent 1100 HPLC system was used to separate the components in the mobile phase at pH = 10.

[0124] (2) Separation conditions:

[0125] Chromatographic column: Agilent Zorbax Extend-C18 narrow diameter column, 2.1 x 150 mm, 5 μm.

[0126] Detection wavelength: ultraviolet 210 nm and 280 nm.

[0127] 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 water), flow rate: 250 μL / min.

[0128] Gradient elution conditions:

[0129] 0-10 min, 2% mobile phase B;

[0130] 10-10.01 min, 2-5% B;

[0131] 10.01-37 min, 5-20% mobile phase B;

[0132] 37-48 min, 20-40% mobile phase B;

[0133] 48-48.01 min, 40-90% mobile phase B;

[0134] 48.01-58 min, 90% mobile phase B;

[0135] 58-58.01 min, 90-2% mobile phase B;

[0136] 58.01-63 min, 2% mobile phase B.

[0137] Component collection: Starting at the 10th minute, eluent was collected sequentially into centrifuge tubes 1-10 every minute, and the eluent was 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.

[0138] 2. Conditions for liquid chromatography-mass spectrometry (LC-MS)

[0139] Ten components obtained by high-pH liquid chromatography separation were scanned by data-dependent acquisition (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.

[0140] Table 5 Data-dependent acquisition (DDA) mass spectrometry conditions

[0141] Items Full MS MS2 Resolution 120000 30000 AGC target 3e6 1e5 Maximum injection time 100 ms 50 ms Scan range 350-1650 m / z 200-2000 m / z Isolation window - 1.4 m / z Normalized Collision Energy - 27

[0142] Separation conditions for the EASY-nLC 1200 high-performance nano-liquid chromatograph:

[0143] Mobile phase: flow rate 300 nL / min, mobile phase A is 0.1% FA (trifluoroacetic acid) aqueous solution, mobile phase B is 0.1% FA / 80% ACN / 20% water (the percentages in this section are volume percentages).

[0144] The liquid phase gradient elution conditions are shown in Table 6.

[0145] Table 6 Data-dependent acquisition (DDA) liquid phase gradient elution conditions

[0146] Time (min) Gradient 0 4％B 5 4％B 8 9％B 80 28％B 94 44％B 97 90％B 104 90％B 108 5％B

[0147] In addition, the remaining 6 samples of the desalted peptide fragments were individually subjected to data-independent acquisition (DIA) mass spectrometry scanning by using an EASY-nLC 1200 high-efficiency nanoliter liquid chromatograph and a Q Exactive HF mass spectrometer, with a scanning range of 350-1250 m / z and an isolation window of 26 m / z. The mass spectrometry scanning parameters are shown in Table 7.

[0148] Table 7. Data-independent acquisition (DIA) mass spectrometry conditions

[0149] Items Full MS MS2 Resolution 120000 30000 AGC target 3e6 1e6 Maximum injection time 100 ms Auto Scan range 350-1250 m / z - Isolation window - 26 m / z Normalized Collision Energy - 28

[0150] The DIA liquid phase gradient elution conditions are shown in Table 8.

[0151] Table 8. Data-independent acquisition (DIA) chromatography conditions

[0152] Time (min) Gradient 0 2％B 82 44％B 84 90％B 90 90％B

[0153] Mobile phase: flow rate 300 nL / min, mobile phase A is 0.1% FA aqueous solution, and mobile phase B is 0.1% FA / 80% ACN / 20% water.

[0154] V. Data processing and analysis

[0155] The LC-MS / MS mass spectrum raw file was imported into the Spectronaut Pulsar software and matched with the theoretical spectrum generated by the fasta library to convert the machine signal into peptide fragment and protein sequence information. Then, sequence information, peptide retention time, fragment ion information, etc. were combined for DDA library construction, and the main parameters are shown in Table 9, which facilitates subsequent DIA analysis.

[0156] Table 9. Data-dependent acquisition (DDA) library construction parameters

[0157] Items Para. Missed cleavage 2 Fixed modification Carbamidomethyl (C) Variable modification Oxidation (M) Enzyme Trypsin / P Protein FDR Cut Off 0.01 Peptide FDR Cut Off 0.01 PSM FDR Cut Off 0.01 Database pig-uniprot-proteome%3A UP000008227.fasta

[0158] The processing of the DIA raw data was completed using the Spectronaut Pulsar software, and the key steps are as follows:

[0159] S1. Open the Analysis module of the Spectronaut Pulsar software and select "+" to create a new analysis;

[0160] S2. Complete the parameter setting step by step according to the software prompt, and the key parameters are shown in Table 10. Start analysis;

[0161] S3. After analysis, enter the "Report" module to export the quantitative data.

[0162] Table 10 Key parameters for data-dependent acquisition (DIA) data analysis

[0163] Items Para. Precursor Qvalue cutoff 0.01 Protein Qvalue cutoff 0.01 Normalization Strategy Local Normalization Quantity MS-Level MS2

[0164] Subsequently, the quantitative data obtained by database retrieval was used to retain proteins with expression values accounting for ≥50% in any one group of samples. Proteins with missing values ≤50% were filled with the mean value of the same group of samples, and after Median Normalization and log2 logarithmic conversion, reliable proteins were obtained. Subsequently, differential expression analysis was performed on the identified proteins, and the differential screening conditions were Fold change = 1.2 times and p-value < 0.05. Finally, pig membrane protein PPP4C and pig membrane protein CSNK2A2 were identified as Y sperm specific membrane proteins. The expression amount of pig membrane protein PPP4C and pig membrane protein CSNK2A2 in Y sperm was significantly higher than that in X sperm; the results are shown in Figure 2 、 3 , wherein, Figure 2 is a result analysis diagram of the expression amount of pig membrane protein PPP4C in X sperm and Y sperm, Figure 3 is a result analysis diagram of the expression amount of pig membrane protein CSNK2A2 in X sperm and Y sperm.

[0165] The protein ID corresponding to the pig membrane protein PPP4C in the Uniprot database is M3VK32; the gene ID corresponding to the gene encoding the pig membrane protein PPP4C in the NCBI database is 110260085.

[0166] The protein ID corresponding to the pig membrane protein CSNK2A2 in the Uniprot database is A0A287B496; the gene ID corresponding to the gene encoding the pig membrane protein CSNK2A2 in the NCBI database is 100737256.

[0167] Example 4 Identification of sperm gender by pig membrane protein PPP4C quantitative method

[0168] Using pig membrane protein PPP4C as a molecular marker, Western Blotting and immunofluorescence methods were used to identify pig X and Y sperm, and the specific steps are as follows:

[0169] S1. According to the protein concentration obtained by the protein concentration determination in Example 3, 30 μg of protein was taken from each sample extracted in Example 2, and the gender information of the protein sample was hidden during the detection process. 3.4 μL of 6×ProteinLoading Buffer solution was added, and Mem-PER TM Plus solubilization buffer was added to dilute and adjust each sample solution to 20 μL, and the protein was denatured at 99℃ for 10 min. The protein denaturation system is shown in Table 11:

[0170] Table 11 Protein denaturation reaction system

[0171] Component Amount Sperm protein 30 μg 6x Protein Loading Buffer 3.4 μL Mem-PER TM Plus solubilization buffer To 20 μL Total volume 20 μL

[0172] S2. Prepare TBST buffer.

[0173] Dissolve the TBS buffer powder completely in 2L of double-distilled water, then add 2mL of Tween20 and stir for 25 minutes.

[0174] S3. Separation of membrane proteins

[0175] Membrane proteins were separated by 10% (w / w) SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membranes. After the transfer, the membranes were washed three times with TBST buffer for 5 min each time, and then blocked with TBST buffer containing 5% (w / w) skim milk powder at room temperature for 2 h.

[0176] S4. Wash the membrane three times with TBST buffer for 5 min each time, add PPP4C antibody and TUBULIN antibody respectively, and incubate overnight at 4°C;

[0177] S5. 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.

[0178] S6. 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.

[0179] S7. Use ImageJ software (http: / / imagej.net / ImageJ) to count the grayscale values, as shown below. Figure 4 The experimental results are shown.

[0180] The expression level of porcine membrane protein PPP4C in sperm cells 4, 5, and 6 was 46% higher than that in sperm cells 1, 2, and 3, and these cells were identified as Y sperm. Sperm cells 1, 2, and 3 were identified as X sperm, consistent with the results obtained by quantitative real-time PCR. These results indicate that the quantitative method for identifying porcine membrane protein PPP4C is accurate and reliable, consistent with the quantitative real-time PCR method.

[0181] The above results indicate that porcine membrane protein PPP4C can serve as a cell surface marker protein for identifying X and Y sperm.

[0182] Example 5: Quantitative method for identifying sperm sex using porcine membrane protein CSNK2A2

[0183] The pig membrane protein CSNK2A2 was used as a molecular marker to identify pig X and Y sperm by Western Blotting and immunofluorescence. The specific experimental steps were the same as in Example 4, except that CSNK2A2 antibody was added in S4. The experimental results are shown in Table 4. Figure 5

[0184] The expression amount of pig membrane protein CSNK2A2 in sperm 4, 5, and 6 was 57% higher than that in sperm 1, 2, and 3, and the sperm were identified as Y sperm, and sperm 1, 2, and 3 were identified as X sperm, which was the same as the result detected by DNA fluorescent quantitative PCR. The results showed that the identification result of pig membrane protein CSNK2A2 quantitative method was the same as that of DNA fluorescent quantitative PCR, and the use of pig membrane protein CSNK2A2 to identify X sperm and Y sperm was accurate and reliable.

[0185] The above results showed that pig membrane protein CSNK2A2 could be used as a cell surface marker protein for identifying X and Y sperm.

[0186] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.​

Claims

1. Application of porcine membrane protein PPP4C in the identification of porcine Y and X sperm, wherein the protein ID of porcine membrane protein PPP4C in the Uniprot database is M3VK32.

2. The application according to claim 1, characterized in that, The method described involves using porcine membrane protein PPP4C as a molecular marker, testing the protein expression level using Western blotting or immunofluorescence, and identifying X and Y sperm based on the differences in protein expression levels. Specifically, the identification criterion is that porcine membrane protein PPP4C is expressed higher in Y sperm than in X sperm.

3. Application of reagents for detecting porcine membrane protein PPP4C in the preparation of kits for identifying porcine Y and X sperm, wherein the protein ID of porcine membrane protein PPP4C in the Uniprot database is M3VK32.

4. The application according to claim 3, characterized in that, The method described involves using porcine membrane protein PPP4C as a molecular marker, testing the protein expression level using Western blotting or immunofluorescence, and identifying X and Y sperm based on the differences in protein expression levels. Specifically, the identification criterion is that porcine membrane protein PPP4C is expressed higher in Y sperm than in X sperm.

5. The application according to claim 3, characterized in that, The reagent used to detect porcine membrane protein PPP4C is a PPP4C antibody.

6. The application according to claim 3, characterized in that, The reagents also include those required for Western blotting.

7. The application according to claim 3, characterized in that, The reagents also include those required for immunofluorescence assays.

8. Application of porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 in the identification of porcine Y and X sperm, wherein the protein ID of porcine membrane protein PPP4C in the Uniprot database is M3VK32, and the protein ID of porcine membrane protein CSNK2A2 in the Uniprot database is A0A287B496.

9. The application according to claim 8, characterized in that, The method described involves using porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 as molecular markers, testing protein expression levels using Western blotting or immunofluorescence, and identifying X and Y sperm based on differences in protein expression levels. Specifically, the identification criteria are that the expression of porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 is higher in Y sperm than in X sperm.

10. Application of reagents for detecting porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 in the preparation of kits for identifying porcine Y and X sperm, wherein the protein ID of porcine membrane protein PPP4C in the Uniprot database is M3VK32, and the protein ID of porcine membrane protein CSNK2A2 in the Uniprot database is A0A287B496.

11. The application according to claim 10, characterized in that, The method described involves using porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 as molecular markers, testing protein expression levels using Western blotting or immunofluorescence, and identifying X and Y sperm based on differences in protein expression levels. Specifically, the identification criteria are that the expression of porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 is higher in Y sperm than in X sperm.

12. The application according to claim 10, characterized in that, The reagents used to detect porcine membrane protein PPP4C and porcine membrane protein CSNK2A2 are PPP4C antibody and CSNK2A2 antibody.

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