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

Application of the SCAMP1 albumen in identification mammalian sperm gender Download PDF

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CN110187126A
CN110187126A CN201910455442.8A CN201910455442A CN110187126A CN 110187126 A CN110187126 A CN 110187126A CN 201910455442 A CN201910455442 A CN 201910455442A CN 110187126 A CN110187126 A CN 110187126A
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albumen
sperm
scamp1
antibody
mammal
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张胜利
姜力
沈丹
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China Agricultural University
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China Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0608—Germ cells
    • C12N5/061—Sperm cells, spermatogonia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

The invention discloses application of the SCAMP1 albumen in identification mammalian sperm gender.SCAMP1 albumen is in the surface specifically expressing of mammal y sperm cell, and the amino acid sequence of SCAMP1 albumen is as shown in sequence 2 in sequence table.The method of separation y sperm provided by the invention is not only simple and easy, but also entire separation process is not needed using dyestuff and radiation, smaller to the pressure of spermatoblast, also smaller to the adverse effect of sperm.Method provided by the invention can satisfy the wilderness demand in production to sexing semen.The present invention has important application value.

Description

Application of the SCAMP1 albumen in identification mammalian sperm gender
Technical field
The invention belongs to field of biotechnology, and in particular to SCAMP1 albumen answering in identification mammalian sperm gender With.
Background technique
Milk cow sexing semen technology is known as the leather of the third time breeding fast-propagation technology after artificial insemination and embryo transfer Life, mainly X, y sperm are effectively separated, using the sperm after separation to dam inseminate, thus produce meet it is pre- The offspring of phase gender.For milk cow group, semen deposition is carried out by separating the sperm of excellent milk cow, so that it is fast to reach dairy bread The purpose of speed improvement.The use of sexing semen significantly improves the ratio of female descendant simultaneously, for the selection intensity for increasing individual Possibility is brought, to accelerate fine breed cow genetic improvement process.
X, the separation of y sperm is the key that sexing semen technology.1980, the laboratory of California, USA was begun to Trial separates striping sperm.With the development of isolation technics, at present according to X, the size of y sperm, vigor, charge, table The difference of face antigen and DNA content etc., reported separation method mainly have density-gradient centrifugation method, electrophoresis, albumen Immunology partition method, flow cytometric sorting method etc., but all there is theoretical foundation deficiency, poor repeatability, result in most of method The problems such as unreliable, wherein flow cytometric sorting method be at present it is most successful to mammalian sperm carry out Gender Classification and In the commericially feasible method of gestation.However, there are still many drawbacks for flow cytometric sorting method, such as big to spermatoblast damage, Effective sperm count is less, conception rate is lower, higher to insemination requirements after artificial insemination, and due to equipment valuableness, separation Speed is slow, is far from satisfying the wilderness demand in production to sexing semen.In addition, bull X spermatozoa isolation purity is higher at present And it is basicly stable, but the separation purity of y sperm is extremely difficult to 90%.In contrast, using protein immunization method, (i.e. antigen-antibody is anti- Answer principle) to carry out X, the method for y sperm separation simple and easy, and is possible to meet in production to a large amount of need of sexing semen It asks；And it is not needed using dyestuff and radiation, therefore separates that the time it takes is less, to the pressure of spermatoblast also compared with It is small, to reduce the adverse effect to sperm.It, can if specific marker's albumen between X, y sperm can be identified It the antibody of x and y sperm can be separated for its exploitation, is expected to lower cost, efficient, easy realizes milk cow semen X, y sperm separates, and then reaches this target of sexual control.However, due to Researches on Sperm Membrane Proteins hydrophobicity itself and low abundance etc. The limitation of property and Identification of Fusion Protein technology leads to the very few nothing of sex-specific albumen for identifying at present and being able to be applied successfully It is several.
Nonstandard sizing technique (Label-free) is mass spectrum quantitative approach important in recent years, is responded by comparing mass signal Intensity or spectrogram number analyze the Plantago fengdouensis of separate sources sample protein.Nonstandard sizing technique is directly taken without marking sample Liquid chromatograph mass spectrography is analyzed by mass spectrometry peptide fragment after enzymatic hydrolysis, obtains the quantification of protein letter of a certain cell or tissue Breath.The basic principle is that calculating integral of each peptide segment signal on first mass spectrometric obtains quantitative result, then to all peptide fragments The second order ms information of signal carries out database retrieval and obtains qualitative results；By integral protein in different samples expression quantity number According to, achieve the purpose that identification in single group specific expressed albumen.Furthermore the high performance liquid chromatography that Label-free is utilized is A kind of efficient separation means and analytical technology have extraordinary reproducibility and sensitivity, single protein separation and Analysis aspect has had relatively broad application.
SCAMP1 albumen (Secretory carrier-associated membrane protein 1) is cell surface Circulating carrier, be concentrated mainly in the endocytosis and removal process of cell surface receptor, and neuron and other have regulation It transports in the cell of access, SCAMPs is also the component part of the regulation carrier such as synaptic vesicle, secretory granules and transporter vesica.
Summary of the invention
The purpose of the present invention is separate mammal X, y sperm.
At least one of the present invention protects the application of SCAMP1 albumen first, can be S1)-S4):
S1 mammalian sperm gender) is identified；
S2 the kit for identifying mammalian sperm gender) is prepared；
S3) separate or screen mammal y sperm；
S4 the kit for separating or screening mammal y sperm) is prepared.
At least one of the present invention also protects the application of the substance of detection SCAMP1 albumen, can be S1)-S4):
S1 mammalian sperm gender) is identified；
S2 the kit for identifying mammalian sperm gender) is prepared；
S3) separate or screen mammal y sperm；
S4 the kit for separating or screening mammal y sperm) is prepared.
In any of the above-described application, the SCAMP1 albumen identifies albumen as cell surface.The cell can be the food in one's mouth Newborn animal y sperm cell.
In any of the above-described application, the substance of the detection SCAMP1 albumen can be the antibody of SCAMP1 albumen.
The present invention also protects a kind of product, it may include the substance of detection SCAMP1 albumen；The function of the product can be S1) Or S3):
S1 mammalian sperm gender) is identified；
S3) separate or screen mammal y sperm.
The product specifically can be by the material composition of detection SCAMP1 albumen.
In the said goods, the substance of the detection SCAMP1 albumen can be the antibody of SCAMP1 albumen.
The present invention also protects a kind of identification mammalian sperm method for distinguishing, can be the cell of detection mammalian sperm Surface whether there is SCAMP1 albumen, then make the following judgment:
If the cell surface of mammalian sperm, there are SCAMP1 albumen, which is y sperm；
If SCAMP1 albumen is not present in the cell surface of mammalian sperm, which is X sperm.
In the above method, " cell surface of detection mammalian sperm whether there is SCAMP1 albumen " be can be used The antibody of SCAMP1 albumen carries out.
A kind of method that the present invention also protects separation or screening mammal y sperm, can be with for separation or screening The mammalian sperm that the antibody of SCAMP1 albumen combines.
Above, the principle of the Identification of the antibodies mammalian sperm gender of SCAMP1 albumen or SCAMP1 albumen is SCAMP1 Albumen therefore is adopted in the surface specifically expressing (i.e. SCAMP1 albumen is that cell surface identifies albumen) of mammal y sperm cell It can identify that mammalian sperm is y sperm or X sperm with protein immunization method (i.e. antigen-antibody reaction principle).
Above, the principle of the separation of the antibody of SCAMP1 albumen or SCAMP1 albumen or screening mammal y sperm is SCAMP1 albumen mammal y sperm cell surface specifically expressing (i.e. SCAMP1 albumen be cell surface identify albumen), Therefore mammal y sperm can be separated or screened using protein immunization method (i.e. antigen-antibody reaction principle).
The antibody of any of the above-described SCAMP1 albumen can be the antibody prepared using SCAMP1 albumen as immunogene.
Any of the above-described " antibody prepared using SCAMP1 albumen as immunogene " can be A1) or A2):
A1) the Anti-TNF-α prepared using SCAMP1 albumen as immunogen immune animal (such as mouse, rat, rabbit, sheep, people) Body；
A2) using SCAMP1 albumen as immunogen immune animal (such as mouse, rat, rabbit, sheep, people), using hybridoma skill Art or DNA recombinant technique, the monoclonal antibody of preparation.The monoclonal antibody can be Humanized monoclonal antibodies.
Any of the above-described mammal can be c1) or c2) or c3) c4) or c5): c1) artiodactylous animals；C2) Bovidae Animal；C3) bovine animals；C4) ox；C5) Holstein cow.
Any of the above-described SCAMP1 albumen can be a1) or a2) or a3):
A1) the protein that amino acid sequence shown in sequence 2 forms in sequence table；
A2) the fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；
A3) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or Obtain and and a1 is deleted and/or added) the protein protein with the same function.
The present invention carries out proteomics using the total memebrane protein of X, y sperm of the Label-free technology to Holstein bull Then analysis carries out transmembrane structure prediction, Asia to the SCAMP1 albumen specific expressed in the total memebrane protein of y sperm identified Cellular localization prediction and Western Blotting verifying determine that SCAMP1 albumen is cell specific expressed in y sperm Surface trans-membrane protein can be used as the sex-specific mark albumen of separation milk cow X, y sperm.The present invention is for the first time in Niu Jingzi SCAMP1 albumen is identified, which can be used as the cell surface mark albumen of protein immunization method separation X, y sperm, be expected to reality Small, efficient, the easy sperm sex identification method of a kind of existing low cost, damage.The method of separation y sperm provided by the invention is not It is only simple and easy, and entire separation process is not needed using dyestuff and radiation, it is smaller to the pressure of spermatoblast, to sperm Adverse effect is also smaller.Method provided by the invention can satisfy the wilderness demand in production to sexing semen.The present invention has Important application value.
Detailed description of the invention
Fig. 1 is the prediction result of SCAMP1 protein transmembrane structure.
Fig. 2 is the expression that Western Blotting detects SCAMP1 albumen in Niu Jingzi.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Test method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop It arrives.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
1200 efficient nanoliters of liquid chromatographs of EASY-nLC and FUSION LUMOUS mass spectrometer system are the U.S. The product of ThermoScientic company.Rabbit-anti SCAMP1 is the product of Britain Abcam company, catalog number ab3430. ECL reagent is the product of U.S. Thermo Fisher Scientific company.TUBLIN is that Beijing health is century biotechnology The product of Co., Ltd, catalog number CW0098A.MinuteTM Plasma Membrane Protein Isolation And Cell Fraction Kit is the product of U.S. Invent Biotechnologies company.Protease inhibitors is beauty The product of Thermo Scientific company, state.2.1 software of ThermoProteome Discoverer is U.S. Thermo The product of Scientific company.
Lysis buffer is the aqueous solution of urea containing 7M, 2M thiocarbamide and 0.1% (m/v) CHAPS.
The amino acid sequence of SCAMP1 albumen is as shown in sequence 2 in sequence table.Encode the gene of SCAMP1 albumen (i.e. SCAMP1 gene) nucleotide sequence as shown in sequence 1 in sequence table.
Embodiment 1, SCAMP1 albumen are specific expressed in y sperm
One, the acquisition of the total memebrane protein of sperm
1, the sample of sperm first and sample of sperm second for obtaining Holstein bull are separated using flow cytometric sorting method.Essence Subsample first and sample of sperm second are free of animal derived protein.
Contain 1.2 hundred million X sperms, separation purity >=95% of X sperm in sample of sperm first.
Contain 1.2 hundred million y sperms, separation purity >=90% of y sperm in sample of sperm second.
2, after completing step 1, using MinuteTM Plasma Membrane Protein Isolation and Cell Fraction Kit extracts sperm (sample of sperm first or sample of sperm second) total memebrane protein.Specific step is as follows:
(1) it takes centrifuge tube (specification 15mL), sample of sperm is added, 4 DEG C, 850g centrifugation 20min abandon supernatant.
(2) after completing step (1), the centrifuge tube is taken, the aqueous sucrose solution (concentration 0.25M) that 5mL pre-cooling is added is clear Cell is washed, 4 DEG C, 850g centrifugation 20min abandon supernatant.
(3) after completing step (2), the centrifuge tube is taken, first adds 10.11 μ L protease inhibitors along wall, then rapidly It adds 1mL Buffer A and cell is resuspended, obtain cell suspension；Then cell suspension is transferred to another new centrifuge tube (specification For in 1.5mL), 4 DEG C of incubation 5-10min.
(4) after completing step (3), the centrifuge tube is taken, be vortexed big forced oscillation 10-30s, it is then transferred to centrifugation tubing string, 4 DEG C, 16000g be centrifuged 1min.
(5) after completing step (4), cell in centrifuge tube is resuspended, cell suspension is transferred back to centrifugation tubing string, 4 DEG C, 16000g It is centrifuged 1min.
(6) after completing step (5), pillar is discarded, cell, then 4 DEG C, 700g centrifugation is resuspended in the big forced oscillation 10s that is vortexed 1min collects supernatant.
(7) supernatant that step (6) are collected is transferred in another new centrifuge tube (specification 1.5mL), 4 DEG C, 16000g It is centrifuged 30min, is abandoned supernatant (i.e. endochylema), precipitating is collected；The precipitating is the total memebrane protein of sperm.
(8) 10 μ L protease inhibitors, -80 DEG C of preservations are added in the precipitating collected to step (7).
Buffer A and centrifugation tubing string are MinuteTM Plasma Membrane Protein Isolation and Component in Cell Fraction Kit.
Two, proteome analysis
1, the total memebrane protein of sperm (the total memebrane protein of sample of sperm first or the total memebrane protein of sample of sperm second) is taken, using Bradford Method measures concentration.Specific step is as follows:
(1) the standard protein solution (lysis buffer dissolves BSA and obtains) for taking 10 μ L various concentrations, is added 300 μ L eggs White quantitative stain, is protected from light 15-20min；
(2) after completing step (1), light absorption value of each standard protein solution at 595nm is detected with microplate reader；Then with The concentration of standard protein solution is abscissa, and corresponding light absorption value is ordinate, draws standard curve；
(3) the total memebrane protein of sperm is diluted to certain multiple with lysis buffer, obtains the total memebrane protein dilution of sperm (concentration of the total memebrane protein dilution of sperm is in standard curve range)；The 10 total memebrane protein dilutions of μ L sperm are taken, 300 μ L are added Protein quantification dyestuff, is protected from light 15-20min；
(4) after completing step (3), with light absorption value of the microplate reader detection total memebrane protein dilution of sperm at 595nm；Then According to the standard curve of step (2), the concentration of the total memebrane protein of sperm is calculated.
2, after completing step 1, the total memebrane protein of sperm (the total memebrane protein of sample of sperm first or the total memebrane protein of sample of sperm second) is taken SDS-PAGE electrophoresis is carried out, gel is obtained.Specific step is as follows:
(1) by the total memebrane protein of sperm and 2 × loading buffer, 5:1 is mixed by volume, and 100 DEG C of heating 8min make egg White matter denaturation；
(2) after completing step (1), loading, applied sample amount is 10 μ g；
(3) after completing step (2), electrophoretic apparatus is connected to power supply, voltage is adjusted to 150-160V, electric current should flow to Anode moves at the 0.5cm of separation gel bottom to bromophenol blue, closes power supply；
(4) after completing step (3), gel glass plate is unloaded from electrophoretic apparatus, then coomassie brilliant blue staining 1h is carried out Decoloration.
3, after completing step 2, with the abundant digestion peptide fragment of trypsase.Specific step is as follows:
(1) it takes the gel in step 2 to carry out coomassie brilliant blue staining again, adhesive tape is cut into 1-2mm with scalpel2Size Film be put into tubule；
(2) after completing step (1), the tubule is taken, 500 μ L acetonitrile destainers are added and impregnate, vibrate 10min, abandon waste liquid； The step is repeated 1-2 times until blue takes off to the greatest extent；
(3) after completing step (2), the tubule is taken, 100 μ L DTT reducing solutions are added, 56 DEG C of oscillation 30min abandon waste liquid, 500 μ L acetonitriles dehydration 5-10min is added；
(4) after completing step (3), the tubule is taken, 100 μ L iodoacetamide subsequentlies are added, are placed in dark place 30min；
(5) after completing step (4), the tubule is taken, 500 μ L acetonitrile destainers is added to impregnate, vibrates 10min, abandons waste liquid, is used Water and acetonitrile clean 3 times, freeze dried 20min；
(6) after completing step (5), the tubule is taken, 50 μ L trypsin solutions (concentration is 0.01 μ g/ μ L) is added, 4 DEG C put Set 30min；Be absorbed completely to enzyme solution, be added 50-100 μ L enzymatic hydrolysis buffer (i.e. concentration be 25mM NH4HCO3Aqueous solution), It is totally submerged glue, 37 DEG C of heat preservation 15h or more；
(7) after completing step (6), the tubule is taken, 100 μ L extracting solution I (aqueous solution for containing 5% (v/v) TFA) is added, 40 DEG C of heated water bath 1h (in 30min, ultrasonic 3min), obtain extracting solution first, extracting solution first are drawn onto another clean pipe, It freezes dried；
(8) after completing step (7), the former tubule in step (6) equipped with blob of viscose is added 200 μ L extracting solution II and (contains 50% (v/v) aqueous solution of acetonitrile and 2.5% (v/v) TFA), 30 DEG C of heat preservation 1h (in 30min, ultrasonic 3min) obtain extracting solution Second；
(9) after completing step (7) and (8), extracting solution second is poured into the extracting solution first tubule equipped with lyophilization, is merged After freeze dried；
(10) after completing step (9), the aqueous solution that 5-10 μ L contains 0.1% (v/v) TFA is added, mixes, obtains mixed liquor.
4, after completing step 3, take the mixed liquor, using 1200 efficient nanoliters of liquid chromatographs of EASY-nLC and Orbitrap Fusion Lumos mass spectrometer system carries out LC-MS/MS mass spectral analysis, obtains mass spectrum original document.
5, after completing step 4, the mass spectrum original document is used into 2.1 software of ThermoProteome Discoverer Processing, the database used are ox UniProt protein sequence library (network address are as follows: https: //www.uniprot.org/ Uniprot/? query=taxonomy:9913, download date: 2018-12-10, protein quantity: 32506).Quantification of protein According to first mass spectrometric AREA method, Differential expression analysis is carried out to the albumen identified.
Partial analysis the results are shown in Table 1.The result shows that SCAMP1 albumen is expressed in sample of sperm second, in sample of sperm first In do not express, i.e., SCAMP1 albumen is only specific expressed in sample of sperm second, it may also be said to which SCAMP1 albumen is only in y sperm Specifically expressing.
Table 1
UniProt searching number Q3T0D2
Protein name SCAMP1 albumen (Secretory carrier membrane protein 1)
Gene Name SCAMP1
Species Bostaurus(Bovine)
Length protein 354
Exp.q-value 0
Peptide fragment coverage 2.8249
Match peptide number of segment 1
Peptide spectrum matching 3
Unique peptide number of segment 1
Theoretical molecular weight (kDa) 40
Theoretical isoelectric point 7.94
Average abundance in sample of sperm first NA
Average abundance in sample of sperm second 3.31E+06
Embodiment 2, the transmembrane structure for predicting SCAMP1 albumen and subcellular localization
Suitable for Western Immuno method separation X, y sperm antigen must be cell cortex protein, in this way with antibody knot Eucaryotic cell structure will not be just destroyed after conjunction, influence sperm motility.The basis of Antigen Stability, cross-film time are to ensure that with transmembrane structure Number is more, and cell surface antigen is less susceptible to be detached from.
1, the transmembrane structure of SCAMP1 albumen is predicted
It is v.2.0 (network address is http://www.cbs.dtu.dk/services/TMHMM/) pre- using TMHMM Server Survey the transmembrane structure of SCAMP1 albumen.
Prediction result is shown in Fig. 1.The result shows that the cross-film number of SCAMP1 albumen is 4 times.
2, the subcellular localization of SCAMP1 albumen is predicted
Utilize the subcellular of WoLF PSORT (network address https: //wolfpsort.hgc.jp/) prediction SCAMP1 albumen Positioning, the higher expression SCAMP1 albumen of score are more possibly positioned at the cellular component.
Prediction result is as follows: plas (plasma membrane): 13；Cyto (cytoplasm): 8；E.R. (endoplasmic reticulum): 7.5；E.R._golg (endoplasmic reticulum and golgiosome have a common boundary): 5；Golg (golgiosome): 1.5；Nucl (nucleus): 1；Pero (peroxisome): 1.The result shows that SCAMP1 albumen is likely located on plasma membrane.
The above results show that SCAMP1 albumen is cell cortex protein, can be used as Western Immuno method separation X, Y essence The antigen of son.
The expression of embodiment 3, Western Blotting detection SCAMP1 albumen in ox X, y sperm
TBST buffer: contain pH7.5,20mol/L Tris- of 0.05% (v/v) Tween20 and 140mol/L NaCl HCl buffer.
1, the total memebrane protein of sperm (the total memebrane protein of sample of sperm first or the total memebrane protein of sample of sperm second) is subjected to 10%SDS- PAGE gel electrophoresis, is then transferred on nitrocellulose filter, at room temperature, with the TBST for containing 5% (v/v) skim milk Buffer blind 1h.
2, after completing step 1, rabbit-anti SCAMP1,4 DEG C of overnight incubations are added.
3, it after completing step 2, is washed 4 times with TBST buffer, then uses horseradish peroxidase at room temperature (HRP) secondary antibody being coupled is incubated for 1h.
4, it after completing step 3, is washed 4 times with TBST buffer, is then detected with ECL reagent.
According to above-mentioned steps, the rabbit-anti SCAMP1 in step 2 is replaced with into TUBLIN, other steps are constant, as interior Ginseng.
Testing result is shown in that (X1, X2 and X3 are three Duplicate Samples of the total memebrane protein of sample of sperm first to Fig. 2, and Y1, Y2 and Y3 are essence Three Duplicate Samples of the total memebrane protein of subsample second).The result shows that the expression of SCAMP1 albumen and Mass Spectrometer Method result are complete Unanimously, only specific expressed in sample of sperm second, it may also be said to SCAMP1 albumen specifically expressing in y sperm.
The above results show that SCAMP1 albumen can be used as the cell surface mark albumen of separation X, y sperm.
<110>China Agricultural University
<120>application of the SCAMP1 albumen in identification mammalian sperm gender
<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
ccacctcttc 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 aggggacaat 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. At least one of the application of 1.SCAMP1 albumen is S1)-S4):
    S1 mammalian sperm gender) is identified；
    S2 the kit for identifying mammalian sperm gender) is prepared；
    S3) separate or screen mammal y sperm；
    S4 the kit for separating or screening mammal y sperm) is prepared.
  2. At least one of it is S1 2. detecting the application of the substance of SCAMP1 albumen)-S4):
    S1 mammalian sperm gender) is identified；
    S2 the kit for identifying mammalian sperm gender) is prepared；
    S3) separate or screen mammal y sperm；
    S4 the kit for separating or screening mammal y sperm) is prepared.
  3. 3. a kind of product, the substance including detecting SCAMP1 albumen；The function of the product is S1) or S3):
    S1 mammalian sperm gender) is identified；
    S3) separate or screen mammal y sperm.
  4. 4. application as claimed in claim 1 or 2 or product as claimed in claim 3, it is characterised in that: the detection The substance of SCAMP1 albumen is the antibody of SCAMP1 albumen.
  5. 5. a kind of identification mammalian sperm method for distinguishing whether there is to detect the cell surface of mammalian sperm SCAMP1 albumen, then makes the following judgment:
    If the cell surface of mammalian sperm, there are SCAMP1 albumen, which is y sperm；
    If SCAMP1 albumen is not present in the cell surface of mammalian sperm, which is X sperm.
  6. 6. method as claimed in claim 5, it is characterised in that: described " whether the cell surface of detection mammalian sperm is deposited In SCAMP1 albumen " it is carried out using the antibody of SCAMP1 albumen.
  7. 7. a kind of method of separation or screening mammal y sperm, can be with the antibody knot of SCAMP1 albumen to separate or screening The mammalian sperm of conjunction.
  8. 8. the application as described in claim 1,2 or 4, or, product described in claim 3 or 4, or, claim 6 or 7 institutes The method stated, it is characterised in that: the antibody of the SCAMP1 albumen is the antibody prepared using SCAMP1 albumen as immunogene.
  9. 9. application, product or method as claimed in claim 8, it is characterised in that: described " using SCAMP1 albumen as immunogene The antibody of preparation " is A1) or A2):
    A1) the polyclonal antibody prepared using SCAMP1 albumen as immunogen immune animal；
    A2) using SCAMP1 albumen as immunogen immune animal, using hybridoma technology or DNA recombinant technique, the Dan Ke of preparation Grand antibody.
  10. 10. the application as described in claim 1,2,4,8 or 9, or, product described in claim 3,4,8 or 9, or, right is wanted Seek 5 to 9 any methods, it is characterised in that: the mammal is c1) or c2) or c3) c4) or c5): c1) artiodactyl Mesh animal；C2) bovid；C3) bovine animals；C4) ox；C5) Holstein cow.
CN201910455442.8A 2019-05-29 2019-05-29 Application of the SCAMP1 albumen in identification mammalian sperm gender Pending CN110187126A (en)

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CN201910455442.8A CN110187126A (en) 2019-05-29 2019-05-29 Application of the SCAMP1 albumen in identification mammalian sperm gender

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CN110187126A true CN110187126A (en) 2019-08-30

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104862273A (en) * 2003-03-28 2015-08-26 英格朗公司 Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US20170146508A1 (en) * 2012-12-16 2017-05-25 Satish Deshpande System to distinguish between x sperm and y sperm

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104862273A (en) * 2003-03-28 2015-08-26 英格朗公司 Apparatus, methods and processes for sorting particles and for providing sex-sorted animal sperm
US20170146508A1 (en) * 2012-12-16 2017-05-25 Satish Deshpande System to distinguish between x sperm and y sperm

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JINGCHUN LI等: "Application of a microfluidic sperm sorter to in vitro production of dairy cattle sex-sorted embryos", 《THERIOGENOLOGY》 *
李建斌等: "影响荷斯坦种公牛精液生产性状的环境因素分析", 《中国畜牧杂志》 *
许晓玲等: "奶牛性控精液技术研究进展", 《黑龙江畜牧兽医》 *

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Application publication date: 20190830