CN104278011A - Application of RhoA recombinant protein in preparation of culture solution for improving embryo freezing resistance and recovery rate after thawing - Google Patents
Application of RhoA recombinant protein in preparation of culture solution for improving embryo freezing resistance and recovery rate after thawing Download PDFInfo
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- CN104278011A CN104278011A CN201410483815.XA CN201410483815A CN104278011A CN 104278011 A CN104278011 A CN 104278011A CN 201410483815 A CN201410483815 A CN 201410483815A CN 104278011 A CN104278011 A CN 104278011A
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Abstract
The invention discloses an application of RhoA recombinant protein in preparation of a culture solution for improving embryo freezing resistance and recovery rate after thawing. The research finds that by addition of in vitro recombined RhoA protein to a culture solution of normally hatched embryos of mice, compared with a control group, the thawing recovery rate of normally hatched embryos can be significantly increased. The RhoA recombinant protein is applied to the field of embryo freezing for the first time, through addition of the RhoA recombinant protein, the freezing resistance and the recovery rate after thawing of the embryos are significantly increased, and use of a toxic cryoprotectant is reduced to a certain extent, so that the adverse effects on the embryos are reduced, and the RhoA recombinant protein has a wide application prospect in the technical field of cryopreservation of the embryos.
Description
Technical field
The present invention relates to the novelty teabag of RhoA recombinant protein, particularly RhoA recombinant protein preparation improve embryo's frost resistance and after thawing anabiosis rate nutrient solution in application, the invention belongs to Embryo freezing preservation technical field.
Background technology
In domestic animal breeding field, the aspect development such as Embryo freezing preservation technology rapidly, in livestock industry, by thawing to the blastaea of freezen protective, being transplanted in acceptor dam at reasonable time afterwards and having been become a kind of very general working method.Up to the present, the polyoxyethylene glycol of import is adopted in the freeze applications of reality, to serve good effect as the use of refrigerating fulid in actual production process.But embryo through mistake freezing after 40% all to sustain damage in refrigerating process, embryo's surviving rate is after the transfer reduced, and these refrigerating fulids still can bring poisonous and harmful more or less to embryo.
Therefore, the method developing the frost resistance of a kind of embryo of raising and the anabiosis rate after thawing is current very exigence.
The present invention is by adding certain RhoA vitro recombination albumen in vitro culture liquid, after sequencing is freezing, its anabiosis rate that thaws is observed after embryo culture 6h, wish therefrom to find out a kind of toxicity low and the material of the freeze proof function of embryo can be improved, thus be applied in the freezing industry of livestock embryo.
Summary of the invention
The object of the present invention is to provide RhoA recombinant protein preparation improve embryo's frost resistance and after thawing anabiosis rate nutrient solution in application.
In order to achieve the above object, the technology used in the present invention means are:
The present invention carries out the vitro culture of embryo by the RhoA albumen adding in test vitro recombination, and whether the RhoA albumen exploring this vitro recombination makes mice embryonic frost resistance improve, thus whether can be applied in the embryo cryopreservation field of mass-producing.Research finds, adds the anabiosis rate that thaws (P<0.05) that vitro recombination RhoA albumen can significantly improve normal incubation embryo compared with control group in the nutrient solution of mouse normal incubation embryo.Be put into again cultivate 2h in Y-27632 after carry out after cultivating 6h in RhoA vitro recombination albumen freezing, thaw recovery after result reduce extremely significantly compared with control group.Illustrate that RhoA recombinant protein can not make Y-27632 reversible for the damage of embryo.More than study proof: the RhoA albumen of external interpolation restructuring can significantly improve the anabiosis rate that thaws of normal incubation embryo.
This research basis on, the present invention proposes RhoA recombinant protein preparation improve embryo's frost resistance and after thawing anabiosis rate nutrient solution in application.
In the present invention, preferably, described RhoA recombinant protein behaviour source RhoA recombinant protein, its gene bank accession number is Accession:NP_001655.1.
Further, the invention allows for a kind of embryo's of raising frost resistance and the cultural method of anabiosis rate after thawing, it is characterized in that comprising add RhoA recombinant protein in embryo medium.
Wherein, preferably, the final concentration of the RhoA recombinant protein added in nutrient solution is 1 μ g/ μ l.
Wherein, preferably, described cultural method comprises the following steps:
The normal incubation embryo obtained cleans 3 times with CZB solution, carries out vitro culture more afterwards, is specially at 37 DEG C, the CO of 5%
2under air and 100% humidity condition, vitro culture is carried out with four orifice plates, and respectively to adding RhoA recombinant protein in nutrient solution, its final concentration is made to be 1 μ g/ μ l, after vitro culture 6h, embryo is carried out sequencing freezing, after recovery of thawing, put into the recovery that equilibrated new CZB solution carries out 24h again.
Compared to prior art, beneficial effect of the present invention is:
1, by RhoA recombinant protein first Application in embryo cryopreservation field, significantly improve the frost resistance of embryo and the anabiosis rate after thawing by the interpolation of RhoA recombinant protein;
2, by cultivating in containing the nutrient solution of RhoA recombinant protein, enhancing the anti-seismic design of embryo in refrigerating process to a certain extent, reducing the damage that it is subject in refrigerating process, thus reducing the disadvantageous effect of refrigerating process to embryo.
3, RhoA recombinant protein is directly added to and namely in the nutrient solution of frozen embryo, the ability of its anti-low temperature injury will be strengthened.
Accompanying drawing explanation
Fig. 1 is the impact of anabiosis rate of normally thawing on mouse after adding inhibitor and recombinant protein;
Fig. 2 is normal incubation embryo before freezing, adds or 0h, interpolation or the figure after not adding people source vitro recombination RhoA protein frozen after 24h after not adding people source vitro recombination RhoA protein frozen;
Wherein, 1 ~ 3 figure be respectively normal incubation embryo freezing before, do not add people source vitro recombination RhoA protein frozen after 0h, do not add people source vitro recombination RhoA protein frozen after 24h, 4 ~ 6 figure be respectively normal incubation embryo freezing before, add people source vitro recombination RhoA protein frozen after 0h, add 24h after the vitro recombination RhoA protein frozen of people source.
Embodiment
Come to further describe the present invention with accompanying drawing below in conjunction with specific embodiment; advantage and disadvantage of the present invention will be more clear along with description " but these embodiments are only exemplary; and " what those skilled in the art should understand that is not form any restriction to scope of the present invention; can modify to the details of technical solution of the present invention and form lower without departing from the spirit and scope of the present invention or replace, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
1, material
1.1 key instruments and equipment
Pipettor: the brilliant pipettor in Beijing hundred (1mL, 200 μ L, 100 μ L, 10 μ L)
Medical centrifuge: Beijing Medical Centrifugal Machine Factory, LDZ4-0.8
CO
2incubator: Heal Force company, HF90
Stereoscopic microscope: south of the River microscope factory, X11-3
Bechtop: Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd., ZHJH-1112B
Electronic balance: Beijing Sai Duolisi instrument system company limited, BS124S
Acidometer: Thermo company, 868
Accurate hot-plate: Bei Da instrument company, MEH-2
Water-bath: Chang'an, Beijing scientific instrument factory, HHS1-Ni
1.2 main agents
1.2.1 people source vitro recombination RhoA albumen, purchased from Sino Biological company, article No.: 12441-H07B.
1.2.2 the outer nutrient solution of weight set proteoplast: the liquid storage being made into 10 μ g/ μ l at the vitro recombination albumen of 50 μ g.Be kept at 4 DEG C.
1.2.3 nutrient solution preparation (CZB liquid storage):
Note: after A, B liquid fully dissolves respectively, mixing, adds water to 250mL, 0.22 μm of frit, preserves January for 4 DEG C.
1.2.4 the preparation of Y-27632 solution
The white powder of Y-27632 is made into the liquid storage of 20 μm of ol/l.
1.3 test materials
This experiment selects 8 ~ 12 weeks rheological properties ripe, and body weight is the ICR system mouse of about 28 g, purchased from Beijing Animal Science company limited of dimension tonneau China.
2, method
The dormancy of 2.1 mouse obtains and collects
Select the Female Rats of vaginal orifice pale pink, abdominal injection injection serum gonadotrophin (PMSG) 10IU/ only, only human chorionic gonadotrophin (hCG) 10IU/ is injected after 48h, mating of spending the night is mated immediately afterwards with adult public mouse 1:1, next day is 8 inspection mating situations early, only anti-pregnant mare serum (A-PMSG) 10IU/ is injected after seeing vaginal suppository
The normal incubation embryo obtained cleans 3 times with CZB, carries out vitro culture more afterwards, is specially at 37 DEG C, the CO of 5%
2under air and 100% humidity condition, vitro culture is carried out with import four orifice plate, and cultivate 6h respectively to adding people source RhoA recombinant protein (final concentration is 1 μ g/ μ l) or Y-27632 (final concentration is 2 μm of ol/L) in CZB nutrient solution or being put into again cultivate 2h in interpolation Y-27632 (final concentration is 2 μm of ol/l) after to add in people source RhoA vitro recombination albumen (final concentration is 1 μ g/ μ l), establish simultaneously separately do not add any inhibitor nutrient solution as a control group.After vitro culture 6h, respectively each group of embryo is carried out sequencing freezing, after recovery of thawing, put into the recovery that equilibrated new CZB nutrient solution carries out 24h again.Its anabiosis rate that thaws is added up after 24h.
2.2 sequencing are freezing
The embryo obtained is cleaned twice with PBS, then sloughs PBS gradually with gradient concentration and be put into cleaning twice in refrigerating fulid again, then tubulature.By obtaining embryo cleans twice with PBS, then cross liquid (A liquid: 1/3 refrigerating fulid+2/3PBS, B liquid: 1/2 refrigerating fulid+1/2PBS, C liquid: 2/3 refrigerating fulid+1/3PBS) successively, then be put into cleaning twice in refrigerating fulid, then tubulature.Suck in order with the plastic straw of 0.25ml.Liquid nitrogen put into by refrigerating fulid (3cm) → air (0.5cm) → refrigerating fulid (3cm) → air (0.5cm) → refrigerating fulid (3cm) frigorimeter of refrigerating fulid (3cm) → air (0.5cm) → refrigerating fulid (3cm) → air (0.5cm) → containing embryo, the straw installed is put into frigorimeter, with 1 DEG C/min obtain speed drop to-5 DEG C time, balance 10min, then the refrigerating fulid place 3 ~ 5s clamping the straw upper end that embryo is housed with the tweezers of precooling occurs that ice crystal is planted ice and completed, reequilibrate 10min, then with the speed of 0.3 DEG C/min until drop to-35 DEG C.When thawing, freezing tubule is taken out from liquid nitrogen, after at room temperature rocking 5s ~ 10s gently, put into 10s in 35 DEG C of water and take out.Cut the spigot at straw two ends, the embryo in tubule is placed in the watch-glass containing 1.0M/L sucrose solution, sloughs refrigerating fulid by three-step approach.
3, data analysis
All experimental data SAS 9.0 statistical softwares carry out single factor test variance significance.Data analysis figure is GraphPad software development.
4, result
The impact on mouse normal incubation embryo thawing anabiosis rate after different inhibitor is added in 4.1 nutrient solutions
Result is as shown in following table 1 and Fig. 1:
The anabiosis rate that thaws (%) of blastaea cultivated by table 1 after adding different inhibitor
Note: numerical representation is mean ± SD.Be shown as extremely significantly (P<0.01) between capitalizations different in each treatment group; Be expressed as significantly (P<0.05) between different lowercases; Be not designated as not significantly (P>0.05).
Repeating groups is 3 groups.
4.2 RhoA recombinant proteins are for the impact of form after normal incubation embryo cryopreservation and recovery of thawing
Fig. 2 is normal incubation embryo before freezing, adds or 0h, interpolation or the figure after not adding people source vitro recombination RhoA protein frozen after 24h after not adding people source vitro recombination RhoA protein frozen; Wherein, 1 ~ 3 figure be respectively normal incubation embryo freezing before, do not add people source vitro recombination RhoA protein frozen after 0h, do not add people source vitro recombination RhoA protein frozen after 24h, 4 ~ 6 figure be respectively normal incubation embryo freezing before, add people source vitro recombination RhoA protein frozen after 0h, add 24h after the vitro recombination RhoA protein frozen of people source.
Above result shows: in mouse normal incubation embryo, add the anabiosis rate that thaws (P<0.05) that vitro recombination RhoA albumen can significantly improve normal blastaea compared with control group.Be put into again cultivate 2h in Y-27632 after carry out after cultivating 6h in RhoA vitro recombination albumen freezing, thaw recovery after result reduce extremely significantly compared with control group.Illustrate that RhoA recombinant protein can not make Y-27632 reversible for the damage of embryo.More than test proof: the RhoA albumen of external interpolation restructuring can significantly improve the anabiosis rate that thaws of normal incubation embryo.
Claims (6)
1.RhoA recombinant protein preparation improve embryo's frost resistance and after thawing anabiosis rate nutrient solution in application.
2. apply as claimed in claim 1, it is characterized in that described RhoA recombinant protein behaviour source RhoA recombinant protein, its gene bank accession number is Accession:NP_001655.1.
3. apply as claimed in claim 1, it is characterized in that described embryo is normal incubation embryo.
4. improve embryo's frost resistance and the cultural method of anabiosis rate after thawing, it is characterized in that comprising add RhoA recombinant protein in embryo medium.
5. cultural method as claimed in claim 4, is characterized in that the final concentration of added RhoA recombinant protein in nutrient solution is 1 μ g/ μ l.
6. cultural method as claimed in claim 5, is characterized in that comprising the following steps:
The normal incubation embryo obtained cleans 3 times with CZB solution, carries out vitro culture more afterwards, is specially at 37 DEG C, the CO of 5%
2under air and 100% humidity condition, vitro culture is carried out with four orifice plates, and respectively to adding RhoA recombinant protein in nutrient solution, its final concentration is made to be 1 μ g/ μ l, after vitro culture 6h, embryo is carried out sequencing freezing, after recovery of thawing, put into the recovery that equilibrated new CZB solution carries out 24h again.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106479962A (en) * | 2016-12-13 | 2017-03-08 | 北京农学院 | A kind of embryo in vitro culture fluid comprising Cathepsin L inhibitor and its application in freezen protective |
| CN106520675A (en) * | 2016-11-08 | 2017-03-22 | 北京农学院 | In-vitro embryo culture solution containing Clusterin protein and application of in-vitro embryo culture solution in embryo cryopreservation |
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| CN103460038A (en) * | 2011-02-23 | 2013-12-18 | 里兰斯坦福初级大学理事会 | Methods of detecting aneuploidy in human embryos |
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| DUVAL D ET AL: "transforming protein RhoA precursor [Homo sapiens]", 《NCBI REFERENCE SEQUENCE: NP_001655.1》 * |
| HOSSEIN BAHARVAND ET AL: "An efficient and easy-to-use crypreservation protocol for human ES and iPS cells", 《 PROTOCOL》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520675A (en) * | 2016-11-08 | 2017-03-22 | 北京农学院 | In-vitro embryo culture solution containing Clusterin protein and application of in-vitro embryo culture solution in embryo cryopreservation |
| CN106520675B (en) * | 2016-11-08 | 2020-10-13 | 北京农学院 | In-vitro embryo culture solution containing Clusterin protein and application thereof in embryo cryopreservation |
| CN106479962A (en) * | 2016-12-13 | 2017-03-08 | 北京农学院 | A kind of embryo in vitro culture fluid comprising Cathepsin L inhibitor and its application in freezen protective |
| CN106479962B (en) * | 2016-12-13 | 2019-10-15 | 北京农学院 | A kind of embryo in vitro culture solution comprising Cathepsin L inhibitor and its application in freezen protective |
| US10624336B2 (en) | 2016-12-13 | 2020-04-21 | Beijing University Of Agriculture | In-vitro embryonic culture medium comprising an inhibitor of cathepsin L and the use of the same in embryo cryopreservation |
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