CN105925541A - Blue tongue virus cryoprotective agent and cryopreservation method - Google Patents
Blue tongue virus cryoprotective agent and cryopreservation method Download PDFInfo
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- CN105925541A CN105925541A CN201610314546.3A CN201610314546A CN105925541A CN 105925541 A CN105925541 A CN 105925541A CN 201610314546 A CN201610314546 A CN 201610314546A CN 105925541 A CN105925541 A CN 105925541A
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- protective agent
- blue tongue
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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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12111—Orbivirus, e.g. bluetongue virus
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Abstract
The invention relates to a blue tongue virus cryoprotective agent and a cryopreservation method. The blue tongue virus cryoprotective agent disclosed by the invention consists of the following raw materials in parts by weight: 80-120ml of a minimal medium, 1-5ml of a PH stabilizer, 2.5-11ml of a cryoprotective agent, 0.5-2ml of serum albumin, 0.5-2ml of chlorothalonil and 5-10ml of horse serum. The prescription of the cryoprotective agent disclosed by the invention, through appropriate combination and proportioning, can effectively protect viruses, so that the activity of the cryoprotective agent is not declined in a virus cryopreservation process.
Description
Technical field
The present invention relates to the frozen field of blue tongue virus, particularly relate to a kind of blue tongue virus freezing protective agent.
Background technology
Blue tongue virus (Blue Tongue Virus, TV) it is to cause bluetongue (Blue Tongue, BT) cause of disease, this disease is the ruminant viral infectious propagated by storehouse midge, heavy economic losses can be caused, be classified as A class animal epidemic for OIE (OIE).Preserve blue tongue virus strain is by bluetongue vaccine research, the exploitation of bluetongue diagnostic method, the primary condition of bluetongue Control Technology research.
Bluetongue is the most stable with condition of ultralow temperature at ambient temperature, can preserve a period of time, is the mode of current main preservation virus.But once carrying out frozen, blue tongue virus activity can rapid decrease.This characteristic of blue tongue virus brings the biggest difficulty to the stored frozen of virus.VP2 and VP7 that blue tongue virus is mainly virus coat to the reason of frozen sensitivity is easily destroyed by ice crystal.Appropriate freezing protective agent can protect virus not to be damaged.But have not seen the freezing protective agent for viral design in the market, the freezing protective agent for cell is had to be mainly permeability cryoprotective agent (DMSO, ethylene glycol, propylene glycol, acetamide, methanol etc.) and impermeability cryoprotective agent (polyvinylpyrrolidone (PVP), sucrose, Polyethylene Glycol, glucosan, albumin and hetastarch etc.), these protective agent compositions can effectively hinder the brilliant injury to cell of ice making, but owing to not carrying out formulation optimization and screening, virus can may be caused irreversible injury, it is to be not used in blue tongue virus preservation.
Summary of the invention
In view of current technology above shortcomings; the present invention provides a kind of blue tongue virus freezing protective agent and cryopreservation methods; the freezing protective agent of the present invention is after appropriate combination and proportioning, and the formula of formation can be effectively protected virus, and the vigor during virus is frozen that is allowed to will not decline.
The present invention adopts the following technical scheme that
A kind of blue tongue virus freezing protective agent, the raw material including following weight portion forms: minimal medium 80-120ml, PH stabilizer 1-5ml, freezing protective agent 2.5-11ml, serum albumin 0.5-2ml, and hundred bacterium remove 0.5-2ml, horse serum 5-10ml.
As the preferred technical solution of the present invention, the raw material including following weight portion forms: minimal medium 100ml, PH stabilizer 3ml, freezing protective agent 7ml, serum albumin 1ml, and hundred bacterium remove 1ml, horse serum 7ml.
As the preferred technical solution of the present invention, described freezing protective agent is at least one in DMSO, sucrose.
As the preferred technical solution of the present invention, described freezing protective agent is made up of DMSO and sucrose, and wherein said DMSO is 5ml, and sucrose is 2ml.
The other one side of the present invention, the cryopreservation methods of a kind of blue tongue virus freezing protective agent, comprise the following steps:
Blue tongue virus is mixed by 1:1 with freezing protective agent；
Being packaged in ampulla frozen by mixed blue tongue virus and freezing protective agent, the most frozen temperature is-20 DEG C to-80 DEG C.Thaw at room temperature (25 DEG C) during use and just can use.
A kind of blue tongue virus freezing protective agent of the present invention and cryopreservation methods; wherein a kind of blue tongue virus freezing protective agent in the present invention includes that the raw material of following weight portion forms: minimal medium 80-120ml; PH stabilizer 1-5ml; freezing protective agent 2.5-11ml; serum albumin 0.5-2ml; hundred bacterium remove 0.5-2ml; horse serum 5-10ml; the freezing protective agent of the present invention is after appropriate combination and proportioning; the formula formed can be effectively protected virus, and the vigor during virus is frozen that is allowed to will not decline.
Accompanying drawing explanation
For the technical scheme being illustrated more clearly that in the embodiment of the present invention, the accompanying drawing used required in embodiment will be briefly described below, apparently, accompanying drawing in describing below is only some embodiments of the present invention, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is the schematic diagram using freezing protective agent of the present invention with not using the frozen blue tongue virus of freezing protective agent of the present invention.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, it is clear that described embodiment is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art are obtained under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1; the present invention provides a kind of blue tongue virus freezing protective agent; raw material including following weight portion forms: minimal medium 120ml, PH stabilizer 5ml, freezing protective agent 11ml; serum albumin 2ml; hundred bacterium remove 2ml, horse serum 10ml, wherein minimal medium i.e. MEM minimal medium; basic ingredient include some must aminoacid and buffer agent etc., Gibco company produce；PH stabilizer is Hapers, Solarbio company produce；Serum albumin has the effect of protection virus, Gibco company produce；Hundred bacterium prevent germ contamination except having, Bayer company produce；Horse serum provides the albumen substrate that some are basic, the effect of protected virus, Gibco company produce.
Embodiment 2; the present invention provides a kind of blue tongue virus freezing protective agent; raw material including following weight portion forms: minimal medium 80ml, PH stabilizer 1ml, freezing protective agent 2.5ml; serum albumin 0.5ml; hundred bacterium remove 0.5ml, horse serum 5ml, wherein minimal medium i.e. MEM minimal medium; basic ingredient include some must aminoacid and buffer agent etc., Gibco company produce；PH stabilizer is Hapers, Solarbio company produce；Serum albumin has the effect of protection virus, Gibco company produce；Hundred bacterium prevent germ contamination except having, Bayer company produce；Horse serum provides the albumen substrate that some are basic, the effect of protected virus, Gibco company produce.
Embodiment 3; the present invention provides a kind of blue tongue virus freezing protective agent; raw material including following weight portion forms: minimal medium 100ml, PH stabilizer 3ml, freezing protective agent 7ml; serum albumin 1ml; hundred bacterium remove 1ml, horse serum 7ml, wherein minimal medium i.e. MEM minimal medium; basic ingredient include some must aminoacid and buffer agent etc., Gibco company produce；PH stabilizer is Hapers, Solarbio company produce；Serum albumin has the effect of protection virus, Gibco company produce；Hundred bacterium prevent germ contamination except having, Bayer company produce；Horse serum provides the albumen substrate that some are basic, the effect of protected virus, Gibco company produce.
Embodiment 4; the present invention provides a kind of blue tongue virus freezing protective agent, and the raw material including following weight portion forms: minimal medium 100ml, PH stabilizer 3ml; DMSO5ml; sucrose 2ml, serum albumin 1ml, hundred bacterium remove 1ml; horse serum 7ml; wherein minimal medium i.e. MEM minimal medium, basic ingredient include some must aminoacid and buffer agent etc., Gibco company produce；PH stabilizer is Hapers, Solarbio company produce；Serum albumin has the effect of protection virus, Gibco company produce；Hundred bacterium prevent germ contamination except having, Bayer company produce；Horse serum provides the albumen substrate that some are basic, the effect of protected virus, Gibco company produce；DMSO, for preventing ice crystal from producing one of composition, to virus fanout free region, is produced by USB company；Sucrose, for preventing ice crystal from producing one of composition, the effect of protected virus, is produced by SbaseBio company.
The other one side of the present invention, the cryopreservation methods of a kind of blue tongue virus freezing protective agent, comprise the following steps:
Step S1: mixed by 1:1 with freezing protective agent by blue tongue virus, wherein freezing protective agent includes the raw material composition of following weight portion: minimal medium 100ml, PH stabilizer 3ml, DMSO5ml, sucrose 2ml, serum albumin 1ml, hundred bacterium remove 1ml, horse serum 7ml.
Step S2: be packaged in ampulla frozen by mixed blue tongue virus and freezing protective agent, the most frozen temperature is-20 DEG C to-80 DEG C, and thawing at room temperature (25 DEG C) during use just can use.
As it is shown in figure 1, for using freezing protective agent of the present invention and not using the schematic diagram of the frozen blue tongue virus of freezing protective agent of the present invention.Fig. 1 shows to use restrovirus vigor of the present invention without being decreased obviously during frozen, and in Fig. 1, viral viability represents with international index viral titer TCID50, namely the dilution antilogarithm of virus half infection cell.Blue tongue virus preserving type is divided into two parts, and every part comprises 100 and is respectively and carries out frozen equipped with 1ML virus ampulla without the present invention, and the interpolation present invention carries out frozen.Then at interval of a period of time, different frozen modes is respectively taken out 6 and is carried out the poison valency mensuration Random Effect with eliminating poison valency detection, also allows for calculating standard error.Result is shown in Fig. 1, and the one group of poison valency adding protection substantially remains in about TCID50=7, and is not added with one group of poison valency of protective agent and began to decline at 2 days, until dropping to about TCID50=3.0.
In the present invention to illustrate that be used alone conventional freezing protective agent such as DMSO, sucrose etc. also have certain viral protected effect; but there is no other composition and the collocation of other material and proportions; effect can be far short of what is expected; after various composition proportion, viral viability can keep constant during detecting substantially.
In sum; one blue tongue virus freezing protective agent of the present invention and cryopreservation methods; wherein a kind of blue tongue virus freezing protective agent in the present invention includes that the raw material of following weight portion forms: minimal medium 80-120ml; PH stabilizer 1-5ml; freezing protective agent 2.5-11ml; serum albumin 0.5-2ml; hundred bacterium remove 0.5-2ml; horse serum 5-10ml; the freezing protective agent of the present invention is after appropriate combination and proportioning; the formula formed can be effectively protected virus, and the vigor during virus is frozen that is allowed to will not decline.
The above; being only the detailed description of the invention of the present invention, but protection scope of the present invention is not limited thereto, any those skilled in the art is in technical scope disclosed by the invention; the change that can readily occur in or replacement, all should contain within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with described scope of the claims.
Claims (5)
1. a blue tongue virus freezing protective agent, it is characterised in that include that the raw material of following weight portion forms: minimal medium 80-120ml; PH stabilizer 1-5ml, freezing protective agent 2.5-11ml, serum albumin 0.5-2ml; hundred bacterium remove 0.5-2ml, horse serum 5-10ml.
A kind of blue tongue virus freezing protective agent the most according to claim 1, it is characterised in that include the raw material composition of following weight portion: minimal medium 100ml, PH stabilizer 3ml, freezing protective agent 7ml, serum albumin 1ml, hundred bacterium remove 1ml, horse serum 7ml.
3. according to the arbitrary described a kind of blue tongue virus freezing protective agent of claim 1-2, it is characterised in that described freezing protective agent is at least one in DMSO, sucrose.
A kind of blue tongue virus freezing protective agent the most according to claim 3, it is characterised in that described freezing protective agent is made up of DMSO and sucrose, and wherein said DMSO is 5ml, and sucrose is 2ml.
5. the cryopreservation methods of a blue tongue virus freezing protective agent, it is characterised in that comprise the following steps:
Blue tongue virus is mixed by 1:1 with freezing protective agent；
Being packaged in ampulla frozen by mixed blue tongue virus and freezing protective agent, the most frozen temperature is-20 DEG C to-80 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201610314546.3A CN105925541A (en) | 2016-05-13 | 2016-05-13 | Blue tongue virus cryoprotective agent and cryopreservation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610314546.3A CN105925541A (en) | 2016-05-13 | 2016-05-13 | Blue tongue virus cryoprotective agent and cryopreservation method |
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| CN105925541A true CN105925541A (en) | 2016-09-07 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556216A (en) * | 2004-01-02 | 2004-12-22 | 武汉大学 | Detecting method of blue tongue virus and reagent box |
| CN1884497A (en) * | 2006-06-06 | 2006-12-27 | 瑞普（保定）生物药业有限公司 | Reovirus vaccine and its preparing method |
| CN101679954A (en) * | 2007-04-06 | 2010-03-24 | 伊维拉根公司 | The method and composition that is used for attenuated virus alive |
| CN105194680A (en) * | 2015-09-15 | 2015-12-30 | 天士力金纳生物技术（天津）有限公司 | Influenza virus subunit vaccine protection agent and application thereof |
-
2016
- 2016-05-13 CN CN201610314546.3A patent/CN105925541A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556216A (en) * | 2004-01-02 | 2004-12-22 | 武汉大学 | Detecting method of blue tongue virus and reagent box |
| CN1884497A (en) * | 2006-06-06 | 2006-12-27 | 瑞普（保定）生物药业有限公司 | Reovirus vaccine and its preparing method |
| CN101679954A (en) * | 2007-04-06 | 2010-03-24 | 伊维拉根公司 | The method and composition that is used for attenuated virus alive |
| CN105194680A (en) * | 2015-09-15 | 2015-12-30 | 天士力金纳生物技术（天津）有限公司 | Influenza virus subunit vaccine protection agent and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 张智明，李华春: "不同温度下蓝舌病病毒的毒价变化研究", 《安徽农业科学》 * |
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