CN110187127A - Application of the CLRN3 albumen as cell surface mark albumen in separation X sperm - Google Patents

Application of the CLRN3 albumen as cell surface mark albumen in separation X sperm Download PDF

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CN110187127A
CN110187127A CN201910455581.0A CN201910455581A CN110187127A CN 110187127 A CN110187127 A CN 110187127A CN 201910455581 A CN201910455581 A CN 201910455581A CN 110187127 A CN110187127 A CN 110187127A
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albumen
sperm
clrn3
antibody
mammal
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张胜利
沈丹
姜力
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China Agricultural University
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    • 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
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Abstract

Application the invention discloses CLRN3 albumen as cell surface mark albumen in separation X sperm.CLRN3 albumen is in the surface specifically expressing of mammal X spermatoblast, and the amino acid sequence of CLRN3 albumen is as shown in sequence 2 in sequence table.The method of separation X 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 CLRN3 albumen as cell surface mark albumen in separation X sperm
Technical field
The invention belongs to field of biotechnology, and in particular to CLRN3 albumen is as cell surface mark albumen in separation X essence Application in son.
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.
CLRN3 albumen is just regulating and controlling differentiation of the Barrett esophagus stem cell to columnar epithelial cell, is detection intestinal metaplasia One of biomarker.As the N- sugar chain albumen on primary liver cell surface, it is dry that CLRN3 facilitates purifying inductive pluripotent The quasi-liver cell homogeneous population in the source cell (iPSCs).
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 CLRN3 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 X sperm；
S4 the kit for separating or screening mammal X sperm) is prepared.
At least one of the present invention also protects the application of the substance of detection CLRN3 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 X sperm；
S4 the kit for separating or screening mammal X sperm) is prepared.
In any of the above-described application, the CLRN3 albumen identifies albumen as cell surface.The cell can be lactation Animal X spermatoblast.
In any of the above-described application, the substance of the detection CLRN3 albumen can be the antibody of CLRN3 albumen.
The present invention also protects a kind of product, it may include the substance of detection CLRN3 albumen；The function of the product can be S1) Or S3):
S1 mammalian sperm gender) is identified；
S3) separate or screen mammal X sperm.
The product specifically can be by the material composition of detection CLRN3 albumen.
In the said goods, the substance of the detection CLRN3 albumen can be the antibody of CLRN3 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 CLRN3 albumen, then make the following judgment:
If the cell surface of mammalian sperm, there are CLRN3 albumen, which is X sperm；
If CLRN3 albumen is not present in the cell surface of mammalian sperm, which is y sperm.
In the above method, " cell surface of detection mammalian sperm whether there is CLRN3 albumen " be can be used The antibody of CLRN3 albumen carries out.
A kind of method that the present invention also protects separation or screening mammal X sperm, can be with for separation or screening The mammalian sperm that the antibody of CLRN3 albumen combines.
Above, the principle of the Identification of the antibodies mammalian sperm gender of CLRN3 albumen or CLRN3 albumen is CLRN3 egg The white surface specifically expressing (i.e. CLRN3 albumen is that cell surface identifies albumen) in mammal X spermatoblast, therefore use egg White immunization (i.e. antigen-antibody reaction principle) can identify that mammalian sperm is X sperm or y sperm.
Above, the principle of the separation of the antibody of CLRN3 albumen or CLRN3 albumen or screening mammal X sperm is CLRN3 Albumen mammal X spermatoblast surface specifically expressing (i.e. CLRN3 albumen be cell surface identify albumen), therefore use Protein immunization method (i.e. antigen-antibody reaction principle) can separate or screen mammal X sperm.
The antibody of any of the above-described CLRN3 albumen can be the antibody prepared using CLRN3 albumen as immunogene.
Any of the above-described " antibody prepared using CLRN3 albumen as immunogene " can be A1) or A2):
A1) the Anti-TNF-α prepared using CLRN3 albumen as immunogen immune animal (such as mouse, rat, rabbit, sheep, people) Body；
A2) using CLRN3 albumen as immunogen immune animal (such as mouse, rat, rabbit, sheep, people), using hybridoma technology 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 CLRN3 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 CLRN3 albumen specific expressed in the total memebrane protein of X sperm identified Cellular localization prediction and Western Blotting verifying determine that CLRN3 albumen is cell table specific expressed in X sperm Face transmembrane protein can be used as the sex-specific mark albumen of separation milk cow X, y sperm.The present invention reflects in Niu Jingzi for the first time Surely CLRN3 albumen is arrived, which can be used as the cell surface mark albumen of protein immunization method separation X, y sperm, be expected to realize A kind of sperm sex identification method inexpensive, damage is small, efficient, easy.The method of separation X sperm provided by the invention is not only It is simple and easy, and entire separation process is not needed using dyestuff and radiation, it is smaller to the pressure of spermatoblast, have to sperm Evil influences also smaller.Method provided by the invention can satisfy the wilderness demand in production to sexing semen.The present invention has weight The application value wanted.
Detailed description of the invention
Fig. 1 is the prediction result of CLRN3 protein transmembrane structure.
Fig. 2 is the expression that Western Blotting detects CLRN3 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 CLRN3 is the product of Britain Abcam company, catalog number ab94993. 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 CLRN3 albumen is as shown in sequence 2 in sequence table.Encode gene (the i.e. CLRN3 of CLRN3 albumen Gene) nucleotide sequence as shown in sequence 1 in sequence table.
Embodiment 1, CLRN3 albumen are specific expressed in X 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 FUSION LUMOUS 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 CLRN3 albumen is expressed in sample of sperm first, in sample of sperm second It does not express, i.e., CLRN3 albumen is only specific expressed in sample of sperm first, it may also be said to which CLRN3 albumen is only special in X sperm Expression.
Table 1
UniProt searching number A6H7D9
Protein name CLRN3 albumen
Gene Name CLRN3
Species Bostaurus(Bovine)
Length protein 229
Exp.q-value 0.001
Peptide fragment coverage 3.49345
Match peptide number of segment 1
Peptide spectrum matching 3
Unique peptide number of segment 1
Theoretical molecular weight (kDa) 25.4
Theoretical isoelectric point 9.51
The average abundance in X sample of sperm first 2.31E+07
The average abundance in y sperm sample second NA
Embodiment 2, the transmembrane structure for predicting CLRN3 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 CLRN3 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 CLRN3 albumen.
Prediction result is shown in Fig. 1.The result shows that the cross-film number of CLRN3 albumen is 4 times.
2, the subcellular localization of CLRN3 albumen is predicted
Utilize the subcellular of WoLF PSORT (network address https: //wolfpsort.hgc.jp/) prediction CLRN3 albumen Positioning, the higher expression CLRN3 albumen of score are more possibly positioned at the cellular component.
Prediction result is as follows: extr (extracellular): 17；Plas (plasma membrane): 10；E.R. (endoplasmic reticulum): 3；Lyso (lyase Body) 1；Golg (golgiosome): 1.The result shows that CLRN3 albumen is likely located on extracellular.
The above results show that CLRN3 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 CLRN3 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 CLRN3,4 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 CLRN3 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 CLRN3 albumen and Mass Spectrometer Method result are complete Unanimously, only specific expressed in sample of sperm first, it may also be said to CLRN3 albumen specifically expressing in X sperm.
The above results show that CLRN3 albumen can be used as the cell surface mark albumen of separation X, y sperm.
<110>China Agricultural University
<120>application of the CLRN3 albumen as cell surface mark albumen in separation X sperm
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 690
<212> DNA
<213> Bovine
<400> 1
atgccgacgg caaagaaaac actgacgttc ttactcagct tcgtcaccag cctgggggct 60
ttcatcgtcc tttgcttcgt tctggcgacc caacagtgga tccgcagtac gatcgccatc 120
agcgactctt cttctaatgg tagtgtgatc atcacctatg gactccttcg tgggaagagt 180
attcaagaat tgaatcacgg acttgcagag tcggacaaaa attttgaagt tttagggaca 240
ttgaccaatt cttctccgaa gactctgcac tcggtggtca tcgtgttcct ggcccttggt 300
ttgtttagtt cgctgctgag tgctgggttc accttctgca acagcgtcag caacccctac 360
cagacgttcc tggggccgac gggcgtgtac acctggagcg ggctcagcgc atccttcaca 420
ttcataacca tggtgctctt tgtggggaac acgcagtcca atcgtctctc ggaagagctg 480
gcccagaggc tgtacccact ctatccagct gccactcagc aagggacgac ccacgcttac 540
agatactcgt tctggctcac cctgctggtc atcctcctaa acgtggtcac cacggtcatc 600
atcatcttct accagaaggc cagataccag cggaagcagg agcagagaaa gcccatggag 660
tctgcgccga gggatggaat tttattctga 690
<210> 2
<211> 229
<212> PRT
<213> Bovine
<400> 2
Met Pro Thr Ala Lys Lys Thr Leu Thr Phe Leu Leu Ser Phe Val Thr
1 5 10 15
Ser Leu Gly Ala Phe Ile Val Leu Cys Phe Val Leu Ala Thr Gln Gln
20 25 30
Trp Ile Arg Ser Thr Ile Ala Ile Ser Asp Ser Ser Ser Asn Gly Ser
35 40 45
Val Ile Ile Thr Tyr Gly Leu Leu Arg Gly Lys Ser Ile Gln Glu Leu
50 55 60
Asn His Gly Leu Ala Glu Ser Asp Lys Asn Phe Glu Val Leu Gly Thr
65 70 75 80
Leu Thr Asn Ser Ser Pro Lys Thr Leu His Ser Val Val Ile Val Phe
85 90 95
Leu Ala Leu Gly Leu Phe Ser Ser Leu Leu Ser Ala Gly Phe Thr Phe
100 105 110
Cys Asn Ser Val Ser Asn Pro Tyr Gln Thr Phe Leu Gly Pro Thr Gly
115 120 125
Val Tyr Thr Trp Ser Gly Leu Ser Ala Ser Phe Thr Phe Ile Thr Met
130 135 140
Val Leu Phe Val Gly Asn Thr Gln Ser Asn Arg Leu Ser Glu Glu Leu
145 150 155 160
Ala Gln Arg Leu Tyr Pro Leu Tyr Pro Ala Ala Thr Gln Gln Gly Thr
165 170 175
Thr His Ala Tyr Arg Tyr Ser Phe Trp Leu Thr Leu Leu Val Ile Leu
180 185 190
Leu Asn Val Val Thr Thr Val Ile Ile Ile Phe Tyr Gln Lys Ala Arg
195 200 205
Tyr Gln Arg Lys Gln Glu Gln Arg Lys Pro Met Glu Ser Ala Pro Arg
210 215 220
Asp Gly Ile Leu Phe
225

Claims (10)

  1. At least one of the application of 1.CLRN3 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 X sperm；
    S4 the kit for separating or screening mammal X sperm) is prepared.
  2. At least one of it is S1 2. detecting the application of the substance of CLRN3 albumen)-S4):
    S1 mammalian sperm gender) is identified；
    S2 the kit for identifying mammalian sperm gender) is prepared；
    S3) separate or screen mammal X sperm；
    S4 the kit for separating or screening mammal X sperm) is prepared.
  3. 3. a kind of product, the substance including detecting CLRN3 albumen；The function of the product is S1) or S3):
    S1 mammalian sperm gender) is identified；
    S3) separate or screen mammal X 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 CLRN3 albumen is the antibody of CLRN3 albumen.
  5. 5. a kind of identification mammalian sperm method for distinguishing whether there is to detect the cell surface of mammalian sperm CLRN3 albumen, then makes the following judgment:
    If the cell surface of mammalian sperm, there are CLRN3 albumen, which is X sperm；
    If CLRN3 albumen is not present in the cell surface of mammalian sperm, which is y 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 CLRN3 albumen " it is carried out using the antibody of CLRN3 albumen.
  7. 7. a kind of method of separation or screening mammal X sperm, can be in conjunction with the antibody of CLRN3 albumen to separate or screening Mammalian sperm.
  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 CLRN3 albumen is the antibody prepared using CLRN3 albumen as immunogene.
  9. 9. application, product or method as claimed in claim 8, it is characterised in that: described " using CLRN3 albumen as immunogene The antibody of preparation " is A1) or A2):
    A1) the polyclonal antibody prepared using CLRN3 albumen as immunogen immune animal；
    A2) using CLRN3 albumen as immunogen immune animal, using hybridoma technology or DNA recombinant technique, the monoclonal of preparation 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.
CN201910455581.0A 2019-05-29 2019-05-29 Application of the CLRN3 albumen as cell surface mark albumen in separation X sperm Pending CN110187127A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111323361A (en) * 2020-03-17 2020-06-23 南通大学 Method for quickly separating sperm head, sperm tail and normal viable sperm

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CN102711628A (en) * 2009-11-13 2012-10-03 医药研究委员会 Cell sampling device
WO2013148984A1 (en) * 2012-03-30 2013-10-03 National University Of Singapore Dual function markers for diagnostics and therapeutics for upper gastrointestinal tract precancer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111323361A (en) * 2020-03-17 2020-06-23 南通大学 Method for quickly separating sperm head, sperm tail and normal viable sperm
CN111323361B (en) * 2020-03-17 2022-05-10 南通大学 Method for quickly separating sperm head, sperm tail and normal viable sperm

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Application publication date: 20190830