CN100368541C - Immune magnetic bead negtive-sieve method for sorting cow X sperm based on H-Y antigen - Google Patents
Immune magnetic bead negtive-sieve method for sorting cow X sperm based on H-Y antigen Download PDFInfo
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Abstract
The present invention relates to an immune magnetic bead negative sieve method for selecting cow X sperms on the basis of an H-Y antigen, which comprises: 4 antigenic determinants owned by the H-Y antigen are selected; the antigenic determinants are connected after being amplified by a reverse transcription polymerase chain reaction method to obtain gene recombinants which only encode the 4 antigenic determinants; the gene recombinants are subcloned into an expression vector pET28a; the gene recombinants are induced to express in colibacillus BL21 and purified the by Histrap columns to obtain fusion protein with molecular weight of 37kDa; the fusion protein is used for immunizing stable-breeding chicken, polyclonal chicken IgY antibodies of the H-Y antigen are extracted from yolk, and the yolk is labeled with nanometer grade magnetic beads; the magnetic bead labeled antibodies are added in fresh cow sperms, the reacted sperms slowly pass through magnetic posts in the magnetic field to obtain X sperms which are not adsorbed on the magnetic posts. The present invention does not need especial equipment and has low costs, and can largely select sperms in a short time, and obtained specific antibodies can not damage X sperms.
Description
Technical field
The present invention relates to a kind of method of sorting cow X sperm, be specifically related to a kind of negative sieve method of immunomagnetic beads of h y antigen sorting cow X sperm of expressing based on Y chromosome, be applicable to the domestic animal sex control in livestock industry and the milk cattle cultivating industry.
Background technology
The domestic animal sex control is meant by intervening artificially or operating and makes dam breed pupil's thing technology of required sex offspring according to people's hope.Under field conditions (factors), the milk cow female-male proportion of new birth is near 1: 1, serves as that the milk cattle cultivating industry of main means is then wished many breeder mother ox to give milk, to improve its economic benefit, so the sex control technology has great importance in milk cattle cultivating and herding production.Domestic and international many investigators have carried out extensive studies at the sex control technical elements.People utilize conceived early sex authenticate technology, in time termination of pregnancy; Or in all sorts of ways sperm is separated: as fine difference according to DNA between X and the Y chromosome, dyeing back selected by flow cytometry apoptosis or density gradient centrifugation; The somebody attempts the characteristics faster than the mobility speed of X sperm according to y sperm, select and run slowly the X-sperm (see document for details: Johnson LA.Sexing mammalian sperm for production of offspring:the state-of-the-art.AnimReprod Sci, Jul 2000; 60-61:93-107; Jiang Zhongling, kingdom's will. sex-controlled research approach of milk cow and present situation Heilungkiang animal reproduction 2004; 12 (2): 10-12).In these methods, can be except that to going flow cytometry again behind the staining of sperm to the effective sorting of sperm, other method is not all succeeded.Yet the utilization flow cytometry also exists does not allow the problem of avoiding, and as the teratogenesis of nucleic acid dye to gene, laser also has expensive equipment etc. to the damage of sperm during the selected by flow cytometry apoptosis sperm.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of immunomagnetic beads negative sieve method, by immunologic method, look for another way, thereby the X sperm of milk cow is sorted out from seminal fluid based on the h y antigen sorting cow X sperm.
For realizing this purpose, the present invention is a kind of male specific tissue compatibility antigen by the SMCY genes encoding on the Y chromosome according to h y antigen, be present in all and contain the cell surface that Y chromosome comprises y sperm, and the X sperm is not owing to contain Y chromosome, the feature of h y antigen so do not encode, proposition first utilizes antibody sorting from seminal fluid of anti-h y antigen to obtain the method for X-sperm.The present invention at first selects peculiar 4 antigenic determinants of h y antigen, utilize the amplification of reverse transcriptional PCR method and connect this 4 dna sequence dnas, the genetic recombinants of these 4 antigenic determinants and its subclone gone into expression vector pET28a obtains only to encode, abduction delivering in e. coli bl21, obtaining a part amount behind the Histrap column purification is the fusion rotein of 37kDa, again with fusion protein immunization stable breeding chicken, from yolk, extract the chicken IgY antibody of the anti-h y antigen of polyclone and use the nano level marked by magnetic bead, the antibody that in the fresh cattle sperm, adds marked by magnetic bead, slowly by being positioned over the magnetic post in the magnetic field, the sperm that is not adsorbed on the magnetic post is the X sperm to reacted sperm.
Method of the present invention specifically comprises the steps:
1, according to the feature of h y antigen, the present invention has at first selected peculiar 4 antigenic determinants of h y antigen, utilize increase the respectively dna sequence dna of this 4 antigenic determinants of reverse transcriptional PCR method, and these 4 dna sequence dnas are linked together, thereby obtained new, the encode genetic recombinants of this 4 HY-antigenic determinants only.This recombinant chou confirms length overall 1079bp, 4 the selected h y antigen determinants of encoding through dna sequence analysis.
2, with this recombinant chou after subclone is gone into expression vector pET28a, in intestinal bacteria (BL21), obtained the fusion rotein that a part amount is about 37kDa behind abduction delivering, the Histrap column purification, its molecular weight conforms to desired value.
3, with the fusion protein immunization stable breeding chicken of above-mentioned acquisition, totally 3 times, each 14 days at interval, the last immunity began to collect egg after 10 days.From yolk, extract the chicken IgY antibody of the anti-h y antigen of polyclone with chloroform-polyoxyethylene glycol method.The methods such as titre, Western-blot method, immunohistochemical methods method, flow cytometry and polymerase chain reaction of measuring this antibody by the agarose double-diffusion process confirm that this antibody can specificly be incorporated into the joint portion end to end of dairy cow sperms.
4, with the nano level marked by magnetic bead of chicken IgY antibody of the anti-h y antigen of above-mentioned acquisition, get fresh cattle sperm sample then, per 10
8The antibody that adds the 0.5mg marked by magnetic bead in the individual spermatid fully after the reaction, slowly by being positioned over the magnetic post in the magnetic field, is collected the sperm that is not adsorbed on the magnetic post with the sperm sample, promptly obtains the X sperm of milk cow.
The present invention has following characteristics with respect to other sperm partition methods:
1, because the sequence of the SMCX on SMCY and the X chromosome on the Y chromosome is originated identical, the present invention has selected 4 special antigenic determinants of h y antigen targetedly, the method of utilizing gene recombination links together the dna sequence dna of these 4 antigenic determinants, thereby obtained new, the encode genetic recombinants of this 4 antigenic determinants only.Like this, avoided and other antigenic cross reaction.
2, because h y antigen is a poor antigen, so the present invention has selected the chicken far away with mammiferous sibship, in the hope of obtaining reactive stronger antibody.Therefore the present invention has obtained the very high antibody of specificity with above-mentioned reorganization h y antigen immunity stable breeding chicken.
3, do not have the damage of nucleic acid staining agent to DNA, antibody acts on the Y-sperm and can not cause damage to the X-sperm, do not need simultaneously special equipment, processing ease, with low cost, can carry out the sperm sorting at short notice in enormous quantities.
Embodiment
Below by specific embodiment technical scheme of the present invention is further described.
1, the clone of h y antigen genetic recombinants and evaluation thereof
According to the feature of h y antigen, at first selected peculiar 4 antigenic determinants of h y antigen, designed 3 pairs of primers.First pair of primer upstream and downstream added the restriction enzyme site of BamHI and SacI respectively, second pair of primer upstream and downstream added the restriction enzyme site of SacI and SalI respectively, the 3rd pair of primer upstream and downstream added the restriction enzyme site of SalI and EaeI respectively, primer sequence (F1 GGA TCC TGT TAT GAC TGT CCA GAT AG; R1GAG CTC AAA ATG GGC CTT TTC CTC CCA; F2GAG CTC CAC AGT TTTGAA CCT TGT ATG; R2GTC GAC ATC ACA GAG CAG AAA CTT ATC; F3GTC GAC CAG GAT GAA TTG AGA CAT AAG; R3TGG CCA CCT TTC TTCATT TAG TAC CAA C).Wherein, contain the 1st～2 antigenic determinant of h y antigen in the 1st segment, contain the 3rd antigenic determinant in the 2nd segment, contain the 4th antigenic determinant in the 3rd segment.
The male mice in 6 ages in week is put to death, aseptic its cerebral tissue of getting, after the TRIzol test kit extracted total RNA, reverse transcription polymerize chain reaction method (RT-PCR) above-mentioned 3 sheets that increase were respectively had no progeny, the PCR product that purifying is reclaimed is connected with the pMD18-T carrier respectively, is transformed into JM109.The clone of blue hickie reacting positive identifies through order-checking, its sequence and the complete homology of former sequence.To be cloned into the pMD18-T carrier again after these 3 fragments connections, capable again sequencing confirms length overall 1079bp, 4 the selected h y antigen determinants of encoding.
2, h y antigen Recombinant Protein Expression
With the above-mentioned dna fragmentation that contains 4 h y antigen determinants, subclone is gone into prokaryotic expression plasmid pET-28a, imports in the competence e. coli bl21, after the kantlex screening, selects positive recombinant plasmid.With the final concentration is that 1mMol/L sec.-propyl-thiogalactoside (IPTG) is induced down the bacterial cultures after expressed fusion protein, N,O-Diacetylmuramidase add decontamination method cracking abduction delivering, and obtaining a part amount behind the Histrap column purification is the fusion rotein of 37kDa.This albumen conforms to the proteic molecular weight of expection.
3, the preparation of anti-h y antigen polyclonal antibody chicken IgY
With complete freund adjuvant and after containing the fusion rotein mixing of h y antigen determinant, the recessive Cold boiled chicken of immunity stable breeding, the full freund adjuvant of toing many or too much for use in per afterwards 14 days mixes the fusion rotein booster immunization 2 times that contains the h y antigen determinant, and begins to collect egg in the last immunity after 10 days.Collect altogether surplus the egg 200.
Separate yolk, chloroform-polyoxyethylene glycol (molecular weight 6000) method is extracted the antibody IgY of the anti-h y antigen of chicken.Get the IgY of anti-h y antigen, after the dilution of 1: 1,1: 2,1: 4 and 1: 8, as antigen, the agarose immune double diffusion method is measured it and is tired with the bull, ox cerebral tissue, when antibody dilution to 1: in the time of 8, visible reaction precipitation line clearly still.And female milk cattle is not seen reaction.
The Western-blot detected result can detect h y antigen at the bull, ox cerebral tissue, and does not detect in the female cow brain tissue.Illustrate that this polyclone IgY antibody can combine by h y antigen specific and in the male tissue.
Get the cover glass of a poly-lysine bag quilt, will contain 10 among the 200 μ l
5The solution of individual dairy cow sperms covers, and hatches 2 hours PBS washing 3 times under the room temperature in the wet box.Again 37 ℃ down with the anti-h y antigen IgY antibody response that dilutes at 1: 30 1 hour, use the anti-chicken IgG of rabbit of FITC (fluorescein isothiocyanate) mark of dilution in 1: 50 to hatch 30 minutes at last at 37 ℃, observe under fluorescent microscope then, the result is at the head of sperm and the intersection visible fluorescence of afterbody.The antibody that this anti-h y antigen IgY is described can combine by specific h y antigen with spermatid film surface.
The dairy cow sperms sample was hatched following 1 hour for 37 ℃ with the anti-h y antigen IgY antibody of dilution in 1: 5, and PBS washes 2 times.And then the anti-chicken IgG37 of rabbit ℃ of adding the FITC mark hatched 30 minutes, and PBS washes 2 times.The spermatid that is resuspended among the PBS detects with flow cytometer, sorting, and the spermatid after the recovery sorting.The flow cytometer result shows that the positive cell proportion is 38.7%, gets rid of the noise jamming of machine itself, the ratio between positive cell and the negative cells approaching with 1: 1.Positive cell after the recovery and negative cells extract genomic dna respectively, primer with ZFY and ZFX carries out the PCR reaction then, PCR result confirms, can detect ZFY in H-Y antibody labeling male spermatid, and detects less than ZFY in negative spermatid.Illustrate that this antibody can be used for sorting sperms.
4, the sorting of sperm
With the nano level marked by magnetic bead of chicken IgY antibody of the anti-h y antigen of above-mentioned acquisition, get fresh cattle sperm sample then, per 10
8The antibody that adds the 0.5mg marked by magnetic bead in the individual spermatid, fully after the reaction, slowly by being positioned over the magnetic post in the magnetic field, the sperm that is marked with magnetic bead is attracted in the middle of the magnetic post, is not collected with centrifuge tube by the spermatid of marked by magnetic bead with the sperm sample.The sperm of collecting is repeated the magnetic post 1 time again, collected the sperm that adsorb/is not adsorbed on the magnetic post respectively.The sperm that is adsorbed on the magnetic post is the positive sperm of H-Y, is the Y-sperm, otherwise is negative sperm, i.e. X-sperm of H-Y.
Add the two anti-of FITC mark in the negative sperm of above-mentioned H-Y, 37 ℃ hatch 30 minutes after, flow cytometer detects, the positive rate of H-Y antibody is 3.10～8.4% in the middle of the spermatid suspension that the result finally collects, average out to 7.8.Explanation is in the middle of the negative sieve method of immunomagnetic beads is separated the spermatid that obtains, and the ratio of X sperm has reached more than 90%.
The sperm that above-mentioned sorting obtains extracts genomic dna, and the primer with ZFY and ZFX carries out the PCR reaction detection then, and H-Y is positive, and sperm can detect ZFY, and the H-Y negative cells then can only detect the ZFY of minute quantity, illustrates that its overwhelming majority is the X sperm.
The various embodiments described above prove absolutely, can remove y sperm with method of the present invention from seminal fluid.
Claims (1)
1. the negative sieve method of the immunomagnetic beads based on the h y antigen sorting cow X sperm is characterized in that comprising the steps:
1) selects peculiar 4 antigenic determinants of h y antigen, design three pairs of primers, first pair of primer upstream and downstream added the restriction enzyme site of BamHI and SacI respectively, second pair of primer upstream and downstream added the restriction enzyme site of SacI and SalI respectively, the 3rd pair of primer upstream and downstream added the restriction enzyme site of SalI and EaeI respectively, and primer sequence is: F1 GGA TCC TGT TAT GAC TGT CCA GAT AG; R1 GAG CTC AAAATG GGC CTT TTC CTC CCA; F2 GAG CTC CAC AGT TTT GAA CCTTGT ATG; R2 GTC GAC ATC ACA GAG CAG AAA CTT ATC; F3 GTCGAC CAG GAT GAA TTG AGA CAT AAG; R3 TGG CCA CCT TTC TTCATT TAG TAC CAA C; Wherein, contain the 1st～2 antigenic determinant of h y antigen in the 1st fragment, contain the 3rd antigenic determinant in the 2nd fragment, contain the 4th antigenic determinant in the 3rd fragment; Utilize increase the respectively dna sequence dna of this 4 antigenic determinants of reverse transcriptional PCR method, and these 4 dna sequence dnas are linked together, thereby obtain the genetic recombinants of 4 selected h y antigen determinants of a length overall 1079bp, coding;
2) with this genetic recombinants after subclone is gone into expression vector pET28a, obtaining a part amount behind abduction delivering, the Histrap column purification in e. coli bl21 is the fusion rotein of 37kDa;
3) with the fusion protein immunization stable breeding chicken of above-mentioned acquisition, totally 3 times, each 14 days at interval, the last immunity began to collect egg after 10 days; From yolk, extract the chicken IgY antibody of the anti-h y antigen of polyclone with chloroform-polyoxyethylene glycol method;
4) with the nano level marked by magnetic bead of chicken IgY antibody of the anti-h y antigen of above-mentioned acquisition, get fresh cattle sperm sample then, per 10
8The antibody that adds the 0.5mg marked by magnetic bead in the individual spermatid fully after the reaction, slowly by being positioned over the magnetic post in the magnetic field, is collected the sperm that is not adsorbed on the magnetic post with the sperm sample, promptly obtains the X sperm of milk cow.
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| CN105331516B (en) * | 2015-12-01 | 2018-03-06 | 苏州浚惠生物科技有限公司 | rare cell enrichment device and method |
| CN105646707B (en) * | 2016-02-26 | 2019-01-22 | 西北农林科技大学 | For ox, the preparation method and application of the UTY antibody nanoparticles of sheep X/Y sperm quick separating |
| CN106544411A (en) * | 2016-08-27 | 2017-03-29 | 华中农业大学 | A kind of method for identifying mammal y sperm typing using mark analysis of protein |
| CN111349158B (en) * | 2020-03-06 | 2022-11-08 | 西北农林科技大学 | Preparation method of sex-controlled semen of milk goat |
| CN112481198A (en) * | 2020-12-18 | 2021-03-12 | 宁波市博坤生物科技有限公司 | Method for realizing separation of sperm cells of mixed spot inspection material based on polypeptide modified membrane |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990013315A1 (en) * | 1989-05-12 | 1990-11-15 | Cytogam, Inc. | Sex-associated membrane proteins and methods for increasing the probability that offspring will be of a desired sex |
| CN1414975A (en) * | 1999-12-07 | 2003-04-30 | 圭尔夫大学 | Sex-chromosome-specific proteins, species specific and sperm specific protein and method for their identification and isolation |
| CN1562354A (en) * | 2004-04-07 | 2005-01-12 | 魏宏泉 | Method for controlling mammal to give birth of female mammal based on H-Y antigen |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990013315A1 (en) * | 1989-05-12 | 1990-11-15 | Cytogam, Inc. | Sex-associated membrane proteins and methods for increasing the probability that offspring will be of a desired sex |
| CN1414975A (en) * | 1999-12-07 | 2003-04-30 | 圭尔夫大学 | Sex-chromosome-specific proteins, species specific and sperm specific protein and method for their identification and isolation |
| CN1562354A (en) * | 2004-04-07 | 2005-01-12 | 魏宏泉 | Method for controlling mammal to give birth of female mammal based on H-Y antigen |
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