CN110187126A - Application of the SCAMP1 albumen in identification mammalian sperm gender - Google Patents

Abstract

The invention discloses the application of SCAMP1 protein in identifying the sex of mammalian sperm. The SCAMP1 protein is specifically expressed on the surface of mammalian Y sperm cells, and the amino acid sequence of the SCAMP1 protein is shown in Sequence 2 in the sequence listing. The method for separating Y sperm provided by the present invention is not only simple and easy, but also does not need to use dyes and radiation in the whole separation process, has less pressure on sperm cells, and has less harmful influence on sperm. The method provided by the invention can meet the large demand for sexually controlled semen in production. The invention has important application value.

Description

Application of SCAMP1 protein in sex identification of mammalian sperm

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of SCAMP1 protein in identifying the sex of mammalian sperm.

Background technique

Sex-controlled semen technology for dairy cows is known as the third technological revolution in the rapid multiplication of fine breeds after artificial insemination and embryo transfer. offspring of the intended sex. For dairy cows, insemination is carried out by separating the semen of excellent dairy cows, so as to achieve the purpose of rapid improvement of dairy cow breeds. At the same time, the use of sex-controlled semen significantly increased the proportion of female offspring, which made it possible to increase the intensity of individual selection, thereby accelerating the process of genetic improvement of fine-bred dairy cows.

The separation of X and Y sperm is the key to the technology of sexually controlled semen. In 1980, laboratories in California, USA began to try to separate demembraned sperm. With the development of separation technology, according to the differences in the size, motility, charge, surface antigen and DNA content of X and Y sperm, the separation methods reported mainly include density gradient centrifugation, electrophoresis, and protein immunology separation. , flow cytometry separation method, etc., but most of the methods have problems such as insufficient theoretical basis, poor repeatability, and unreliable results. Among them, flow cytometry separation method is currently the most successful method for sex classification of mammalian sperm and pregnancy. commercially viable method. However, there are still many disadvantages in the separation method of flow cytometry, such as large damage to sperm cells, low effective sperm count, low conception rate after artificial insemination, high requirements for insemination technology, and slow separation speed due to expensive equipment , It is far from being able to meet the large demand for sexually controlled semen in production. In addition, at present, the separation purity of bull X sperm is relatively high and basically stable, but the separation purity of Y sperm is difficult to reach 90%. In contrast, the method of separating X and Y sperm using the protein immunization method (i.e., the principle of antigen-antibody reaction) is simple and feasible, and it is possible to meet the large demand for sexually controlled semen in production; and it does not require the use of dyes and radiation , so the separation takes less time and puts less stress on the sperm cells, thereby reducing harmful effects on the sperm. If the specific marker protein between X and Y sperm can be identified, then an antibody that can separate X and Y sperm can be developed against it, and it is expected to achieve efficient and simple separation of X and Y sperm from dairy cow semen at a lower cost , so as to achieve the goal of gender control. However, due to the hydrophobicity and low abundance of sperm membrane proteins and the limitations of protein identification techniques, few sex-specific proteins have been identified and successfully applied.

The non-standard quantitative method (Label-free) is an important mass spectrometry quantitative method in recent years. By comparing the mass spectrometry signal response intensity or the number of spectra, the abundance changes of samples from different sources are analyzed. The non-standard quantitative method does not need to label samples, and directly adopts liquid chromatography-mass spectrometry to perform mass spectrometry analysis on the peptides after enzymatic hydrolysis to obtain protein quantitative information of a certain cell or tissue. The basic principle is to calculate the integral of each peptide signal on the primary mass spectrum to obtain quantitative results, and then perform database retrieval on the secondary mass spectrometry information of all peptide signals to obtain qualitative results; by integrating protein expression data in different samples, To achieve the purpose of identifying proteins specifically expressed in a single group. In addition, the high-performance liquid chromatography used by Label-free is an efficient separation method and analysis technology with very good reproducibility and sensitivity, and has been widely used in the separation and analysis of single proteins.

SCAMP1 protein (Secretory carrier-associated membrane protein 1) is a circulating carrier on the cell surface, mainly concentrated in the process of endocytosis and recycling of cell surface receptors, while in neurons and other cells with regulatory transport pathways, SCAMPs are also synaptic Components of regulatory vectors such as haptic vesicles, secretory granules, and transporter vesicles.

Contents of the invention

The purpose of the present invention is to separate mammalian X, Y sperm.

In the present invention, the first application of protecting the SCAMP1 protein can be at least one of S1)-S4):

S1) identifying the sex of mammalian sperm;

S2) preparing a kit for identifying the sex of mammalian sperm;

S3) separating or screening mammalian Y sperm;

S4) Prepare a kit for isolating or screening mammalian Y sperm.

The present invention also protects the application of substances for detecting SCAMP1 protein, which can be at least one of S1)-S4):

S1) identifying the sex of mammalian sperm;

S2) preparing a kit for identifying the sex of mammalian sperm;

S3) separating or screening mammalian Y sperm;

S4) Prepare a kit for isolating or screening mammalian Y sperm.

In any of the above applications, the SCAMP1 protein is used as a cell surface marker protein. The cells may be mammalian Y sperm cells.

In any of the above applications, the substance for detecting SCAMP1 protein may be an antibody to SCAMP1 protein.

The present invention also protects a product, which may include substances for detecting SCAMP1 protein; the function of the product may be S1) or S3):

S1) identifying the sex of mammalian sperm;

S3) Separating or screening mammalian Y sperm.

Said product may in particular consist of a substance for detecting the SCAMP1 protein.

In the above product, the substance for detecting SCAMP1 protein may be an antibody to SCAMP1 protein.

The present invention also protects a method for identifying the sex of mammalian sperm, which can detect whether there is SCAMP1 protein on the cell surface of mammalian sperm, and then make the following judgment:

If there is SCAMP1 protein on the cell surface of mammalian sperm, the mammalian sperm is Y sperm;

If the SCAMP1 protein is not present on the cell surface of mammalian sperm, the mammalian sperm is X sperm.

In the above method, the "detecting whether there is SCAMP1 protein on the cell surface of mammalian sperm" can be carried out by using an antibody to SCAMP1 protein.

The present invention also protects a method for isolating or screening mammalian Y sperm, which can be used for isolating or screening mammalian sperm that can bind to the antibody of SCAMP1 protein.

In the above, the principle of SCAMP1 protein or SCAMP1 protein antibody identification of mammalian sperm sex is that SCAMP1 protein is specifically expressed on the surface of mammalian Y sperm cells (that is, SCAMP1 protein is a cell surface marker protein), so the protein immunization method (that is, antigen antibody Reaction principle) can identify mammalian sperm is Y sperm or X sperm.

In the above, the principle of SCAMP1 protein or SCAMP1 protein antibody separation or screening of mammalian Y sperm is that SCAMP1 protein is specifically expressed on the surface of mammalian Y sperm cells (that is, SCAMP1 protein is a cell surface marker protein), so the protein immunization method (ie, Antigen-antibody reaction principle) can separate or screen mammalian Y sperm.

The antibody to any one of the aforementioned SCAMP1 proteins can be an antibody prepared using the SCAMP1 protein as an immunogen.

Any of the above "antibodies prepared using SCAMP1 protein as an immunogen" can be A1) or A2):

A1) Polyclonal antibodies prepared by immunizing animals (such as mice, rats, rabbits, sheep, and humans) with SCAMP1 protein as an immunogen;

A2) Monoclonal antibody prepared by using SCAMP1 protein as an immunogen to immunize animals (such as mice, rats, rabbits, sheep, and humans) using hybridoma technology or DNA recombinant technology. The monoclonal antibody may be a humanized monoclonal antibody.

Any of the mammals mentioned above may be c1) or c2) or c3) or c4) or c5): c1) artiodactyl; c2) bovid; c3) bovine; c4) bovine; c5) Holstein Cattle.

Any of the aforementioned SCAMP1 proteins may be a1) or a2) or a3):

a1) A protein composed of the amino acid sequence shown in Sequence 2 in the sequence listing;

a2) a fusion protein obtained by connecting a tag to the N-terminal or/and C-terminal of the protein shown in Sequence 2 in the sequence listing;

a3) A protein obtained by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the sequence listing and having the same function as the protein described in a1).

The present invention utilizes Label-free technology to carry out proteomic analysis on the X and Y sperm total membrane proteins of Holstein bulls, and then performs transmembrane structure prediction on the identified SCAMP1 protein specifically expressed in the Y sperm total membrane proteins, Subcellular localization prediction and Western Blotting verification confirmed that SCAMP1 protein is a cell surface transmembrane protein specifically expressed in Y sperm, which can be used as a sex-specific marker protein for separating cow X and Y sperm. The present invention identifies the SCAMP1 protein in bovine sperm for the first time, and the protein can be used as a cell surface marker protein for separating X and Y sperm by protein immunology, and is expected to realize a low-cost, less-damaging, efficient and simple sperm sex identification method. The method for separating Y sperm provided by the present invention is not only simple and easy, but also does not need to use dyes and radiation in the whole separation process, has less pressure on sperm cells, and has less harmful influence on sperm. The method provided by the invention can meet the large demand for sexually controlled semen in production. The invention has important application value.

Description of drawings

Fig. 1 is the prediction result of SCAMP1 protein transmembrane structure.

Figure 2 shows the expression of SCAMP1 protein detected in bovine sperm by Western Blotting.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

The test methods in the following examples are conventional methods unless otherwise specified.

The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

EASY-nLC 1200 high-efficiency nanoliter liquid chromatography and FUSION LUMOUS mass spectrometry system are both products of ThermoScientic Corporation in the United States. Rabbit anti-SCAMP1 is a product of Abcam, UK, and its catalog number is ab3430. ECL reagents are products of Thermo Fisher Scientific, USA. TUBLIN is a product of Beijing Kangwei Century Biotechnology Co., Ltd., and the product catalog number is CW0098A. Minute ^TM Plasma Membrane Protein Isolation and Cell Fraction Kit is a product of Invent Biotechnologies in the United States. Protease inhibitors are products of Thermo Scientific, USA. ThermoProteome Discoverer 2.1 software is the product of American ThermoScientific Company.

The analysis buffer is an aqueous solution containing 7M urea, 2M thiourea and 0.1% (m/v) CHAPS.

The amino acid sequence of SCAMP1 protein is shown as sequence 2 in the sequence listing. The nucleotide sequence of the gene encoding SCAMP1 protein (ie SCAMP1 gene) is shown as sequence 1 in the sequence listing.

Example 1, the specific expression of SCAMP1 protein in Y sperm

1. Acquisition of sperm total membrane protein

1. The sperm sample A and the sperm sample B of the Holstein bull were separated by flow cytometry. Neither sperm sample A nor sperm sample B contained protein of animal origin.

Sperm sample A contains 120 million X sperm, and the separation purity of X sperm is ≥95%.

Sperm sample B contains 120 million Y sperm, and the separation purity of Y sperm is ≥90%.

2. After completing step 1, use the Minute ^TM Plasma Membrane Protein Isolation and CellFraction Kit to extract the total membrane protein of sperm (sperm sample A or sperm sample B). Specific steps are as follows:

(1) Take a centrifuge tube (specification: 15 mL), add a sperm sample, centrifuge at 850 g for 20 min at 4°C, and discard the supernatant.

(2) After completing step (1), take the centrifuge tube, add 5 mL of pre-cooled sucrose aqueous solution (concentration: 0.25 M) to wash the cells, centrifuge at 850 g for 20 min at 4° C., and discard the supernatant.

(3) After completing step (2), take the centrifuge tube, first add 10.11 μL of protease inhibitor along the wall, then quickly add 1 mL of Buffer A to resuspend the cells to obtain a cell suspension; then transfer the cell suspension to another In a new centrifuge tube (1.5mL), incubate at 4°C for 5-10min.

(4) After completing step (3), take the centrifuge tube, vortex vigorously for 10-30 s, then transfer to a centrifuge column, and centrifuge at 16,000 g for 1 min at 4°C.

(5) After completing step (4), resuspend the cells in the centrifuge tube, transfer the cell suspension back to the centrifuge column, and centrifuge at 16,000 g for 1 min at 4°C.

(6) After completing step (5), discard the column, resuspend the cells by vortexing vigorously for 10 seconds, then centrifuge at 700g at 4°C for 1 minute, and collect the supernatant.

(7) Transfer the supernatant collected in step (6) to another new centrifuge tube (1.5mL), centrifuge at 16000g for 30min at 4°C, discard the supernatant (cytoplasm), and collect the precipitate; the precipitate is is the total membrane protein of sperm.

(8) Add 10 μL of protease inhibitor to the precipitate collected in step (7), and store at -80°C.

Both Buffer A and the spin column are components in the Minute ^TM Plasma Membrane Protein Isolation and Cell Fraction Kit.

2. Proteomic analysis

1. Take the total sperm membrane protein (sperm sample A total membrane protein or sperm sample B total membrane protein), and use the Bradford method to measure the concentration. Specific steps are as follows:

(1) Take 10 μL of standard protein solutions of different concentrations (obtained by dissolving BSA in lysis buffer), add 300 μL of protein quantification dye, and react in the dark for 15-20 minutes;

(2) After completing step (1), detect the absorbance value of each standard protein solution at 595nm with a microplate reader; then take the concentration of the standard protein solution as the abscissa, and the corresponding absorbance value as the ordinate, and draw a standard curve;

(3) Dilute the total sperm membrane protein to a certain multiple with lysis buffer to obtain a sperm total membrane protein dilution (the concentration of the sperm total membrane protein dilution is within the range of the standard curve); take 10 μL of the sperm total membrane protein dilution and add 300 μL Protein quantification dye, avoid light reaction for 15-20min;

(4) After completing step (3), use a microplate reader to detect the absorbance at 595 nm of the sperm total membrane protein dilution; then calculate the concentration of the sperm total membrane protein according to the standard curve in step (2).

2. After step 1 is completed, take the total sperm membrane protein (total membrane protein A of the sperm sample or total membrane protein B of the sperm sample) for SDS-PAGE electrophoresis to obtain a gel. Specific steps are as follows:

(1) Mix the total sperm membrane protein with 2×loading buffer at a volume ratio of 5:1, and heat at 100°C for 8 minutes to denature the protein;

(2) After completing step (1), load the sample with a sample volume of 10 μg;

(3) After completing step (2), connect the electrophoresis device to the power supply, adjust the voltage to 150-160V, and the current should flow to the anode. After the bromophenol blue migrates to the bottom 0.5cm of the separation gel, turn off the power supply;

(4) After completing step (3), the gel glass plate was removed from the electrophoresis apparatus, stained with Coomassie brilliant blue for 1 hour, and then decolorized.

3. After completing step 2, fully digest the peptide with trypsin. Specific steps are as follows:

(1) Take the gel in step ² and perform Coomassie Brilliant Blue staining again, cut the adhesive tape into 1-2mm films with a scalpel and put them into small tubes;

(2) After completing step (1), take the small tube, add 500 μL of acetonitrile decolorization solution to soak, shake for 10 minutes, and discard the waste solution; repeat this step 1-2 times until the blue color fades away;

(3) After completing step (2), take the small tube, add 100 μL of DTT reducing solution, shake at 56°C for 30 minutes, discard the waste solution, and add 500 μL of acetonitrile for dehydration for 5-10 minutes;

(4) After completing step (3), take the small tube, add 100 μL of iodoacetamide for alkylation, and place it in the dark for 30 minutes;

(5) After completing step (4), take the small tube, add 500 μL of acetonitrile decolorization solution to soak, shake for 10 minutes, discard the waste solution, wash with water and acetonitrile for 3 times, and freeze-dry for 20 minutes;

(6) After completing step (5), take the small tube, add 50 μL of trypsin solution (concentration: 0.01 μg/μL), and place it at 4°C for 30 minutes; when the enzyme solution is completely absorbed, add 50-100 μL of enzymatic buffer ( That is, NH ₄ HCO ₃ aqueous solution with a concentration of 25mM), so that the glue is completely submerged, and kept at 37°C for more than 15h;

(7) After completing step (6), take the small tube, add 100 μL of extract solution I (aqueous solution containing 5% (v/v) TFA), heat the water bath at 40°C for 1 hour (at 30 minutes, sonicate for 3 minutes), and obtain the extracted Liquid A, suck the extracted liquid A into another clean tube, freeze-dry;

(8) After completing step (7), add 200 μL of extract solution II (aqueous solution containing 50% (v/v) acetonitrile and 2.5% (v/v) TFA) to the original small tube containing the gel block in step (6). , kept at 30°C for 1h (at 30min, ultrasonic for 3min), to obtain extract B;

(9) After completing steps (7) and (8), extract solution B is poured into the small tube of extract solution A equipped with lyophilization and drying, and freeze-dry after merging;

(10) After completing step (9), add 5-10 μL of an aqueous solution containing 0.1% (v/v) TFA and mix well to obtain a mixed solution.

4. After completing step 3, take the mixed solution, and use EASY-nLC 1200 high-performance nanoliter liquid chromatograph and Orbitrap Fusion Lumos mass spectrometry system for LC-MS/MS mass spectrometry analysis to obtain the original file of mass spectrometry.

5. After step 4 is completed, the mass spectrometry original file is processed with ThermoProteome Discoverer 2.1 software, and the database used is the bovine UniProt protein sequence library (URL is: https://www.uniprot.org/uniprot/?query=taxonomy: 9913, download date: 2018-12-10, number of proteins: 32506). Protein quantification According to the AREA method of primary mass spectrometry, the differential expression analysis of the identified proteins was carried out.

Some analysis results are shown in Table 1. The results showed that SCAMP1 protein was expressed in sperm sample B but not in sperm sample A, that is, SCAMP1 protein was specifically expressed only in sperm sample B, and it could also be said that SCAMP1 protein was specifically expressed only in Y sperm.

Table 1

UniProt accession number Q3T0D2 protein name SCAMP1 protein (Secretory carrier membrane protein 1) gene name SCAMP1 species Bostaurus (Bovine) protein length 354 Exp.q-value 0 Peptide coverage 2.8249 Number of matching peptides 1 Peptide Spectrum Matching 3 number of unique peptides 1 Theoretical Molecular Weight (kDa) 40 theoretical isoelectric point 7.94 Average abundance in sperm sample A NA Average abundance in sperm sample B 3.31E+06

Example 2, predicting the transmembrane structure and subcellular localization of SCAMP1 protein

The antigens suitable for separating X and Y sperm by protein immunization must be cell surface proteins, so that they will not destroy the cell structure and affect the sperm motility after combining with the antibody. Having a transmembrane structure is the basis for ensuring the stability of the antigen. The more transmembrane times, the less likely the cell surface antigen is to escape.

1. Predict the transmembrane structure of SCAMP1 protein

Using TMHMM Server v.2.0 (URL: http://www.cbs.dtu.dk/services/TMHMM/) to predict the transmembrane structure of SCAMP1 protein.

The predicted results are shown in Figure 1. The results showed that the transmembrane times of SCAMP1 protein was 4 times.

2. Predict the subcellular localization of SCAMP1 protein

WoLF PSORT (https://wolfpsort.hgc.jp/) was used to predict the subcellular localization of SCAMP1 protein, and the higher the score, the more likely SCAMP1 protein was localized in this cell component.

The predicted results are as follows: plas (plasma membrane): 13; cyto (cytoplasm): 8; E.R. (endoplasmic reticulum): 7.5; E.R._golg (junction of endoplasmic reticulum and Golgi apparatus): 5; golg (Golgi apparatus): 1.5; nucl (nucleus): 1; pero (peroxisome): 1. The results indicated that the SCAMP1 protein might be localized on the plasma membrane.

The above results indicated that the SCAMP1 protein is a cell surface protein and can be used as an antigen for separating X and Y sperm by protein immunization.

Embodiment 3, Western Blotting detects the expression of SCAMP1 protein in bovine X, Y sperm

TBST buffer: pH 7.5, 20 mol/L Tris-HCl buffer containing 0.05% (v/v) Tween20 and 140 mol/L NaCl.

1. Perform 10% SDS-PAGE gel electrophoresis on the total sperm membrane protein (sperm sample A total membrane protein or sperm sample B total membrane protein), and then transfer it to a nitrocellulose membrane. (v/v) Skim milk TBST buffer blocking 1h.

2. After completing step 1, add rabbit anti-SCAMP1 and incubate overnight at 4°C.

3. After completing step 2, wash 4 times with TBST buffer, and then incubate with horseradish peroxidase (HRP)-coupled secondary antibody for 1 h at room temperature.

4. After completing step 3, wash 4 times with TBST buffer, and then detect with ECL reagent.

According to the above steps, replace the rabbit anti-SCAMP1 in step 2 with TUBLIN, and keep other steps unchanged, as an internal reference.

The detection results are shown in Fig. 2 (X1, X2 and X3 are three parallel samples of the total membrane protein of the sperm sample, Y1, Y2 and Y3 are three parallel samples of the total membrane protein of the sperm sample). The results showed that the expression of SCAMP1 protein was completely consistent with the results of mass spectrometry, and it was only specifically expressed in sperm sample B. It can also be said that SCAMP1 protein was specifically expressed only in Y sperm.

The above results indicate that SCAMP1 protein can be used as a cell surface marker protein for separating X and Y sperm.

<110> China Agricultural University

<120> Application of SCAMP1 protein in sex identification of mammalian sperm

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1065

<212> DNA

<213> Bovine

<400> 1

atgtcggact tcgacagtaa cccgtttgcc gacccggacc tcaacaaccc tttcaaggac 60

ccatcagtta cacaggtgac aagaaatgtc ccaccaggac ttgatgaata caatccattc 120

tcagattcta gaacacctcc accaggtaat gtgaaaatgc ctaacgtacc cagtacacag 180

ccagcaataa tgaagccaac agaagaacat ccagcttata cacagattgc aaaggaacat 240

gccttggccc aagctgaact tcttaagcgc caggaagaac tagaaagaaa agcagcggag 300

ttagaccgtc gagaacgaga aatgcagaac ctcagtcaac atggcagaaa aaacaattgg 360

ccaccctcttc ctgacaactt tcctgtgggc ccttgttttt atcaggattt ttctgtagat 420

atccctgtag aattccaaaa aacagtaaag attatgtact acttatggat gttccatgct 480

gtaacactat ttctaaatat cttcggatgc ttggcttggt tttgtgttga tcctccaaga 540

ggggttgatt ttggattgag tatcctgtgg ttcttacttt ttactccttg ttcatttgtc 600

tgttggtaca gaccacttta cggagctttc aggagtgaca gctccttccg gttcttcgtg 660

ttcttttttg tctatatctg tcagtttgct gtgcacgtac tccaggctgc aggatttcat 720

aactggggta actgtggttg gatttcatcc cttactggtc tcaacaaaag tattcctgtt 780

ggaatcatga tgattatcat agcagcactt ttcacagcat cagcagtcat ctcactagtt 840

atgtttaaaa aggtacatgg actgtatcgc acaacaggtg ctagttttga gaaggcccag 900

caggagtttg caacaggtgt gatgtccaac aaaactgtcc agaccgcggc cgcaaacgca 960

gcttcaactg cagcgagcag tgcagctcag aatgcgttca agggggacaat ggactctaga 1020

aaacaattca gtaatcaaga aaaaaagtac agcgtgtatt tttga 1065

<210> 2

<211> 354

<212> PRT

<213> Bovine

<400> 2

Met Ser Asp Phe Asp Ser Asn Pro Phe Ala Asp Pro Asp Leu Asn Asn

1 5 10 15

Pro Phe Lys Asp Pro Ser Val Thr Gln Val Thr Arg Asn Val Pro Pro

20 25 30

Gly Leu Asp Glu Tyr Asn Pro Phe Ser Asp Ser Arg Thr Pro Pro Pro

35 40 45

Gly Asn Val Lys Met Pro Asn Val Pro Ser Thr Gln Pro Ala Ile Met

50 55 60

Lys Pro Thr Glu Glu His Pro Ala Tyr Thr Gln Ile Ala Lys Glu His

65 70 75 80

Ala Leu Ala Gln Ala Glu Leu Leu Lys Arg Gln Glu Glu Leu Glu Arg

85 90 95

Lys Ala Ala Glu Leu Asp Arg Arg Glu Arg Glu Met Gln Asn Leu Ser

100 105 110

Gln His Gly Arg Lys Asn Asn Trp Pro Pro Leu Pro Asp Asn Phe Pro

115 120 125

Val Gly Pro Cys Phe Tyr Gln Asp Phe Ser Val Asp Ile Pro Val Glu

130 135 140

Phe Gln Lys Thr Val Lys Ile Met Tyr Tyr Leu Trp Met Phe His Ala

145 150 155 160

Val Thr Leu Phe Leu Asn Ile Phe Gly Cys Leu Ala Trp Phe Cys Val

165 170 175

Asp Pro Pro Arg Gly Val Asp Phe Gly Leu Ser Ile Leu Trp Phe Leu

180 185 190

Leu Phe Thr Pro Cys Ser Phe Val Cys Trp Tyr Arg Pro Leu Tyr Gly

195 200 205

Ala Phe Arg Ser Asp Ser Ser Phe Arg Phe Phe Val Phe Phe Phe Val

210 215 220

Tyr Ile Cys Gln Phe Ala Val His Val Leu Gln Ala Ala Gly Phe His

225 230 235 240

Asn Trp Gly Asn Cys Gly Trp Ile Ser Ser Leu Thr Gly Leu Asn Lys

245 250 255

Ser Ile Pro Val Gly Ile Met Met Ile Ile Ile Ala Ala Leu Phe Thr

260 265 270

Ala Ser Ala Val Ile Ser Leu Val Met Phe Lys Lys Val His Gly Leu

275 280 285

Tyr Arg Thr Thr Gly Ala Ser Phe Glu Lys Ala Gln Gln Glu Phe Ala

290 295 300

Thr Gly Val Met Ser Asn Lys Thr Val Gln Thr Ala Ala Ala Asn Ala

305 310 315 320

Ala Ser Thr Ala Ala Ser Ser Ala Ala Gln Asn Ala Phe Lys Gly Thr

325 330 335

Met Asp Ser Arg Lys Gln Phe Ser Asn Gln Glu Lys Lys Tyr Ser Val

340 345 350

Tyr Phe

Claims (10)

1. The application of SCAMP1 protein is at least one of S1)-S4): S1) identifying the sex of mammalian sperm; S2) preparing a kit for identifying the sex of mammalian sperm; S3) separating or screening mammalian Y sperm; S4) Prepare a kit for isolating or screening mammalian Y sperm. 2. The application of the substance for detecting SCAMP1 protein, which is at least one of S1)-S4): S1) identifying the sex of mammalian sperm; S2) preparing a kit for identifying the sex of mammalian sperm; S3) separating or screening mammalian Y sperm; S4) Prepare a kit for isolating or screening mammalian Y sperm. 3. A product comprising a substance for detecting SCAMP1 protein; the function of said product is S1) or S3): S1) identifying the sex of mammalian sperm; S3) Separating or screening mammalian Y sperm. 4. The application according to claim 1 or 2, or the product according to claim 3, characterized in that: the substance for detecting SCAMP1 protein is an antibody to SCAMP1 protein. 5. A method for identifying the sex of mammalian sperm is to detect whether there is SCAMP1 protein on the cell surface of mammalian sperm, and then judge as follows: If there is SCAMP1 protein on the cell surface of mammalian sperm, the mammalian sperm is Y sperm; If the SCAMP1 protein is not present on the cell surface of mammalian sperm, the mammalian sperm is X sperm. 6. The method according to claim 5, characterized in that: said "detecting whether there is SCAMP1 protein on the cell surface of mammalian sperm" is carried out by using an antibody to SCAMP1 protein. 7. A method for isolating or screening mammalian Y sperm, which is to isolate or screen mammalian sperm that can bind to the antibody of SCAMP1 protein. 8. The application according to claim 1, 2 or 4, or the product according to claim 3 or 4, or the method according to claim 6 or 7, wherein the antibody to the SCAMP1 protein is Antibody prepared by using SCAMP1 protein as immunogen. 9. The application, product or method according to claim 8, characterized in that: the "antibody prepared using SCAMP1 protein as an immunogen" is A1) or A2): A1) polyclonal antibodies prepared by immunizing animals with SCAMP1 protein as an immunogen; A2) A monoclonal antibody prepared by immunizing animals with SCAMP1 protein as an immunogen, using hybridoma technology or DNA recombinant technology. 10. The application of claim 1, 2, 4, 8 or 9, or the product of claim 3, 4, 8 or 9, or the method of any one of claims 5 to 9, wherein It is characterized in that: the mammal is c1) or c2) or c3) or c4) or c5): c1) artiodactyl; c2) bovid; c3) bovine; c4) bovine; c5) Holstein cattle .