CN106754376B - A kind of microorganism low-temperature preservation protective agent - Google Patents
A kind of microorganism low-temperature preservation protective agent Download PDFInfo
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- CN106754376B CN106754376B CN201611120538.1A CN201611120538A CN106754376B CN 106754376 B CN106754376 B CN 106754376B CN 201611120538 A CN201611120538 A CN 201611120538A CN 106754376 B CN106754376 B CN 106754376B
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- bcg vaccine
- viable bacteria
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
Abstract
The present invention relates to a kind of microorganism low-temperature preservation protective agent, and its preparation method and application.The ingredient of microorganism low-temperature preservation protective agent is：Nonreducing sugar, glycerine, nonionic surface active agent, sylvite, glutamate and water.The present invention has also set up the technical method of BCG vaccine low temperature long-term preservation, and the correlative study to carry out BCG vaccine in a deep going way provides technical support.
Description
Technical field
The present invention relates to a kind of microorganism low-temperature preservation protective agent, and its preparation method and application.
Background technology
Freeze-drying is a kind of common method (CarcobaR, 2000) for realizing the microorganism long shelf-life, is regarded extensively
To be one of most suitable bacterium dehydration, its object is to obtain stable final reagent (Carvalho AS, 2004),
And one of the most common process for storing microbial cell culture.Cell survival rate after freeze-drying reflects cell
Resist fast freezing and dry influence (Miyamoto-ShinoharaY, 2008).It is generally believed that most of unshielded thin
Bacterium can all be killed after freeze-drying, and those of survival is dead rapidly when being stored.To obtain ideal freezing
And effect, in addition to technique, screening and the use of suitable cryoprotector is also highly important.Suitable freeze drying protectant
Microorganism can be made to reduce the death rate during freeze-drying and defrosting, caused bacterium when mitigating freeze-drying and thawing
Bulk damage.Thus, type, concentration and the configuration method of freeze drying protectant have apparent influence to the preservation effect of microorganism.
Harrison (1963) recommends freeze drying protectant that should have the following conditions：1) protective agent should contain the substance that can form skeleton；2)
It should contain the buffer substance of restricted residual moisture；3) content of electrolyte should be lacked and wait.
A variety of trials are had been carried out to increase the quantity of bacterium survived after freeze-drying and storage, but only obtain it is limited at
Work(.
It is to increase microorganism especially bacterial micro-organism be lyophilized to select dried medium appropriate/cryoprotection agent composition
With the key factor of survival rate during subsequent storage (Carvalho AS, 2004).In the prior art, CN101108164A is reported
Several researchs of the BCG polysaccharide nucleic acid in freeze-drying are accused.Castro et al. has evaluated 5% trehalose to bulgarian milk
The advantageous effect of bacillus (Lactobacillusbulgaricus) survival rate after freeze-drying, has been compared in individual water
About 1% retention ratio, they show 25% retention ratio.2003, Carvalho et al. demonstrated sodium glutamate to freeze-drying
With the stabilization of the survival during 3-6 months subsequent storages, but the Microbial survival rate reported is still very low.
BCG vaccine (Bacillus Calmette-Gu é rin, BCG), which is originated from, has pathogenic bacillus tuberculosis bovis through 230
The attenuated strain formed after generation continuous passage.It is main that China is related to biological products in the listing or clinical research of BCG vaccine at present
Have intracutaneous injection BCG vaccine, BCG polyose nuclear acid injection (BCG-PSN), Purified Protein Derivative Bacillus Calmette-guerin (BCG-PPD),
Therapeutic BCG vaccine, BCG vaccine CpG-DNA etc..These products all refer to harvest and the application of BCG vaccine viable bacteria, therefore current edition
Pharmacopeia requires its seed lot passage generation, it is therefore an objective to reduce the risk of bacterial dissociation.Particularly with the bacterium of viable bacteria product
Kind passage generation requires to be essentially：From working seed lots breakdown to microorganism collection, passage should be no more than for 12 generations.However domestic card is situated between
The main reason for generation majority of seedling product was 12 generations, and the generation of external BCG vaccine product is generally 3-5 generations, this difference is bacterium
Viable bacteria amount is few after kind is recovered for the first time, can only cultivate generation by increase to meet production needs.Solve the problems, such as this primary key
One of be the viable bacteria content for how improving BCG vaccine bacteria and recovering for the first time.Under normal circumstances, the viable bacteria content of inoculation is more, strain
Time needed for recovery is shorter, and the lawn of formation or the quality of mycoderm are higher, conversely, viable bacteria content is lower, required recovery
Time is longer, further influences subsequent passage and harvest.The preservation shape of number of viable and strain contained by BCG vaccine strain
Formula is related.There are two types of the preservation forms of strain, and one kind preserves for freeze-drying, and one kind is that liquid freezing preserves.Freeze-drying lactobacillus is that card is situated between
Bacterium culture is prepared after adding freeze drying protectant by low temperature vacuum freezedrying, and viable bacteria is relatively stablized after freeze-drying, can be at 2-8 DEG C
Preservation and transport, can also keep relative stability at room temperature；But strain viable bacteria content declines after being lyophilized, and average is only liquid before being lyophilized
The 16%-20% or so of body strain, and the recovery time is also longer after drying for bacterial strain.Another preserving type of strain is liquid
Freezen protective strain after that is, BCG vaccine fresh cultured object adds Cord blood protective agent, refrigerates, the party in -20 DEG C and following temperature
Formula has the characteristics that viable bacteria content height is kept to be easy recovery.
Currently, in the prior art in the research of BCG vaccine, spininess studies the preservation of BCG polysaccharide nucleic acid, and
It is less to the fungi preservation research of BCG vaccine viable bacteria, meanwhile, in the preservation of strain, it is substantially and is preserved using freeze-drying, existed
Freeze-drying lactobacillus recovery is slow, preserves the problems such as inefficient.
In order to overcome disadvantage existing in the prior art, the present inventor to invent a kind of microorganism by long-term research
Cord blood protective agent, the liquid freezing which can effectively improve microorganism preserve efficiency, can especially improve card
Viable bacteria content after Jie bacterium viable bacteria sample deepfreeze or freezing, and then improve BCG vaccine production viable bacteria strain recovery matter for the first time
Amount, solve the above problem in the prior art, compensate for meet production need cutting passage generation more than defect.
Invention content
The present invention provides a kind of microorganism low-temperature preservation protective agent, it can be used for but be not limited to BCG vaccine bacteria, card is situated between
The deepfreeze of the BCG vaccines viable bacterias such as bacterium viable bacteria sample or other bacteriums, microorganism, to improve microorganism, such as BCG vaccine life
Production strain, recovery quality, solves freeze-drying lactobacillus recovery equal technical barriers slowly for the first time.In order to improve microorganism to the maximum extent,
Such as Cord blood protective agent is added to microculture by BCG vaccine production strain, freeze survival rate before Cord blood
In object.
The present invention relates to a kind of microorganism low-temperature preservation protective agents, by nonreducing sugar, glycerine, non-ionic surfactant
Agent, sylvite, glutamate and water composition；Wherein, the microorganism low-temperature preservation protective agent each component content is that 1 unit concentration contains
Amount；Also, the amount of nonreducing sugar is 4% to 16% (w/v), preferably 5% to 15% (w/v) in 1 unit concentration content；Glycerine
Amount be 0.9% to 11% (v/v), preferably 1% to 10% (v/v)；The amount of nonionic surface active agent arrives for 0.4%
2.5% (v/v), preferably 0.5% to 2% (v/v)；The amount of sylvite is 0.5% to 2% (w/v)；The amount of the glutamate is
0.15% to 1% (w/v), preferably 0.2% to 0.8% (w/v).
In one embodiment of the invention, the amount of nonreducing sugar is 5% to 15% (w/v) in 1 unit concentration content；
The amount of glycerine is 1% to 10% (v/v)；The amount of nonionic surface active agent is 0.5% to 2% (v/v)；The amount of sylvite is
0.5% to 1% (w/v)；The amount of the glutamate is 0.2% to 0.4% (w/v).
In another embodiment of the present invention, the amount of nonreducing sugar is 5% to 15% (w/ in 1 unit concentration content
v)；The amount of glycerine is 1% to 10% (v/v)；The amount of nonionic surface active agent is 0.5% to 2% (v/v)；The amount of sylvite
For 1% to 2% (w/v)；The amount of the glutamate is 0.4% to 0.8% (w/v).
In one embodiment of the invention, a kind of microorganism low-temperature preservation protective agent stoste is further related to, each component contains
Amount is the multiple of the protectant 1 unit concentration content of mentioned microorganism Cord blood.
Preferably, each component content of microorganism low-temperature preservation protective agent stoste is 1.5 times~the 10 of 1 unit concentration content
Times, further preferably 2 times~5 times, such as 1.5 times, 2 times, 2.5 times or 3 times etc..
In terms of the preparation of microorganism low-temperature preservation protective agent stoste, those skilled in the art can be according to real work need
Be adjusted, as preservative agent stoste can be 1 unit concentration 1 times of (including but not limited to), 1.1 times, 1.2 times, 1.3 times,
1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times,
2.7 times, 2.8 times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4
Times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times, 5 times, 5.1 times, 5.2 times, 5.3
Times, 5.4 times, 5.5 times, 5.6 times, 5.7 times, 5.8 times, 5.9 times, 6 times, 6.1 times, 6.2 times, 6.3 times, 6.4 times, 6.5 times, 6.6
Times, 6.7 times, 6.8 times, 6.9 times, 7 times, 7.1 times, 7.2 times, 7.3 times, 7.4 times, 7.5 times, 7.6 times, 7.7 times, 7.8 times, 7.9
Times, 8 times, 8.1 times, 8.2 times, 8.3 times, 8.4 times, 8.5 times, 8.6 times, 8.7 times, 8.8 times, 8.9 times, 9 times, 9.1 times, 9.2 times,
9.3 times, 9.4 times, 9.5 times, 9.6 times, 9.7 times, 9.8 times, 9.9 times, 10 times etc..
When in use, those skilled in the art can will preserve liquid stoste and be diluted according to the needs of real work, example
10 times, 9 times, 8 times, 7 times, 5 times, 4 times, 3 times, 2 times, 1 times are such as diluted, makes finally to preserve a concentration of above-mentioned micro- when liquid uses
The biological protectant 1 unit concentration content of Cord blood.
It is furthermore preferred that in the microorganism low-temperature preservation protective agent stoste, the amount of nonreducing sugar is 10% to 30% (w/
v)；The amount of glycerine is 2% to 20% (v/v)；The amount of nonionic surface active agent is 1% to 4% (v/v)；The amount of sylvite is
1% to 4% (w/v)；And the amount of glutamate is 0.4% to 1.6% (w/v).
In one embodiment of the invention, in the microorganism low-temperature preservation protective agent stoste, the amount of nonreducing sugar
For 10% to 30% (w/v)；The amount of glycerine is 2% to 20% (v/v)；The amount of nonionic surface active agent is 1% to 4%
(v/v)；The amount of sylvite is 1% to 2% (w/v)；And the amount of glutamate is 0.4% to 0.8% (w/v).
In another embodiment of the present invention, in the microorganism low-temperature preservation protective agent stoste, nonreducing sugar
Amount is 10% to 30% (w/v)；The amount of glycerine is 2% to 20% (v/v)；The amount of nonionic surface active agent is 1% to 4%
(v/v)；The amount of sylvite is 2% to 4% (w/v)；And the amount of glutamate is 0.8% to 1.6% (w/v).
" nonreducing sugar " used herein can be trehalose, raffinose, sucrose etc..In the preferred reality of the present invention
It applies in scheme, nonreducing sugar is trehalose.
" nonionic surface active agent " used herein can be polysorbate 20, polysorbate 40, poly- sorb
The combination of one or more of ester 60 and polyoxyethylene sorbitan monoleate.In a preferred embodiment of the invention, non-ionic surface is lived
Property agent is polyoxyethylene sorbitan monoleate.
" sylvite " used herein can be potassium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium sulfate and potassium chloride
One or more of combination.In a preferred embodiment of the invention, sylvite is potassium chloride or dipotassium hydrogen phosphate.
" glutamate " used herein typically refers to the water soluble salt of edible glutamic acid.This kind of " edible
" salt is to be approved in human food and is those of food-grade, such as the sodium salt of glutamic acid, the sylvite of glutamic acid.At this
In the preferred embodiment of invention, glutamate is sodium glutamate.
It is related to a kind of microorganism low-temperature preservation protective agent or its stoste in another embodiment of the present invention, wherein non-go back
Originality sugar is trehalose；The nonionic surface active agent is polyoxyethylene sorbitan monoleate；The sylvite is dipotassium hydrogen phosphate and chlorination
At least one of potassium；And/or glutamate is sodium glutamate.
Preferred microorganism low-temperature preservation protective agent contains in every 100 milliliter of 1 unit concentration：Trehalose：4-16g, it is excellent
Select 5-15g；Glycerine：0.9-11mL, preferably 1-10mL；Polyoxyethylene sorbitan monoleate：0.4-2.5mL, preferably 0.5-2.0mL；Potassium chloride or
Dipotassium hydrogen phosphate：0.5‐2g；Sodium glutamate：0.15-1g, preferably 0.2-0.8g.
It is furthermore preferred that containing trehalose 5-15g in every 100 milliliter of 1 unit concentration, glycerine 9-11mL, preferably 10mL, gather
Sorb ester 80 0.4-0.6mL, preferably 0.5mL, potassium chloride or dipotassium hydrogen phosphate 1-2g, sodium glutamate 0.4-0.8g；Alternatively, every
Contain trehalose 5-10g, glycerine 0.9-2mL, preferably 1mL, polyoxyethylene sorbitan monoleate 1.5-2.5mL in 100 milliliter of 1 unit concentration,
It is preferred that 2mL, potassium chloride or dipotassium hydrogen phosphate 1-2g, sodium glutamate 0.4-0.8g；
In another embodiment of the present invention, in every 100 milliliter of 1 unit concentration contain trehalose 5g, glycerine 10mL,
Polyoxyethylene sorbitan monoleate 0.5mL, potassium chloride 1g, sodium glutamate 0.4g；
In another embodiment of the present invention, in every 100 milliliter of 1 unit concentration contain trehalose 10g, glycerine 1mL,
Polyoxyethylene sorbitan monoleate 2mL, potassium chloride 1g, sodium glutamate 0.4g；
In another embodiment of the present invention, trehalose 5g is contained in every 100 milliliter of 1 unit concentration, glycerine 1mL, is gathered
80 2mL of sorb ester, potassium chloride 1g, sodium glutamate 0.4g.
In another embodiment of the present invention, in every 100 milliliter of 1 unit concentration contain trehalose 10g, glycerine 5mL,
Polyoxyethylene sorbitan monoleate 1mL, potassium chloride 1g, sodium glutamate 0.4g.
In another embodiment of the present invention, in every 100 milliliter of 1 unit concentration contain trehalose 10g, glycerine 1mL,
Polyoxyethylene sorbitan monoleate 2mL, dipotassium hydrogen phosphate 1g, sodium glutamate 0.4g.
In another embodiment of the present invention, in every 100 milliliter of 1 unit concentration contain trehalose 10g, glycerine 1mL,
Polyoxyethylene sorbitan monoleate 2mL, potassium chloride 2g, sodium glutamate 0.4g.
In another embodiment of the present invention, in every 100 milliliter of 1 unit concentration contain trehalose 10g, glycerine 1mL,
Polyoxyethylene sorbitan monoleate 2mL, potassium chloride 1g, sodium glutamate 0.8g.
In one embodiment of the invention, a kind of Cord blood protective agent stoste is also disclosed, described in every 100 milliliters
Contain in protective agent stoste：Trehalose：8-32g, preferably 10-30g；Glycerine：1.8-22mL preferably 2-20mL；Polyoxyethylene sorbitan monoleate：
0.8-5mL, preferably 1-4mL；Potassium chloride or dipotassium hydrogen phosphate：1‐4g；And sodium glutamate：0.3-2g, preferably 0.4-1.6g.
Further preferably, trehalose 10-30g, glycerine 18-22mL are contained in every 100 milliliters of protective agent stoste, preferably
20mL, polyoxyethylene sorbitan monoleate 0.8-1.2mL, preferably 1mL, potassium chloride or dipotassium hydrogen phosphate 2-4g, sodium glutamate 0.8-1.6g；Or
Person contains trehalose 10-20g, glycerine 1.8-4mL, preferably 2mL, polyoxyethylene sorbitan monoleate 3- in every 100 milliliters of protective agent stoste
5mL, preferably 4mL, potassium chloride or dipotassium hydrogen phosphate 2-4g, sodium glutamate 0.8-1.6g.
It is furthermore preferred that in every 100 milliliters of protective agent stoste contain trehalose 10g, glycerine 20mL, polyoxyethylene sorbitan monoleate 1mL,
Potassium chloride 2g, sodium glutamate 0.8g；Alternatively, containing trehalose 20g, glycerine 2mL, poly- sorb in every 100 milliliters of protective agent stoste
80 4mL of ester, potassium chloride 2g, sodium glutamate 0.8g；Alternatively, containing trehalose 10g, glycerine in every 100 milliliters of protective agent stoste
2mL, polyoxyethylene sorbitan monoleate 4mL, potassium chloride 2g, sodium glutamate 0.8g；Alternatively, containing seaweed in every 100 milliliters of protective agent stoste
Sugared 20g, glycerine 10mL, polyoxyethylene sorbitan monoleate 2mL, potassium chloride 2g, sodium glutamate 0.8g；Alternatively, every 100 milliliters of protective agent stoste
In contain trehalose 20g, glycerine 2mL, polyoxyethylene sorbitan monoleate 4mL, dipotassium hydrogen phosphate 2g, sodium glutamate 0.8g；Alternatively, every 100
Contain trehalose 20g, glycerine 2mL, polyoxyethylene sorbitan monoleate 4mL, potassium chloride 4g, sodium glutamate 0.8g in milliliter protective agent stoste；Or
Person contains trehalose 20g, glycerine 2mL, polyoxyethylene sorbitan monoleate 4mL, potassium chloride 2g, glutamic acid in every 100 milliliters of protective agent stoste
Sodium 1.6g.
Preferably, the pH value of protective agent of the invention or its stoste is 7-8, such as 7.0,7.1,7.2,7.3,7.4,7.5,
7.6,7.7,7.7,7.8,7.9,8.0.The pH regulating measures that this field routine can be used adjust protectant pH value to required model
It encloses, such as hydrochloric acid and/or sodium hydroxide, potassium hydroxide solution can be used to adjust.
In one embodiment of the invention, the preservation condition of Cord blood protective agent or its stoste is 2-8 DEG C.
Meanwhile the present invention relates to a kind of methods of Cord blood microorganism, including mentioned microorganism Cord blood is used to protect
Protect agent or microorganism low-temperature preservation protective agent stoste.
Preferably, the microbial suspension for waiting for Cord blood is mixed with Cord blood protective agent stoste, or will waited for low
The microorganism that temperature preserves mixes with Cord blood protective agent, obtains the microorganism mixed liquor for waiting for Cord blood, in the mixed liquor
The each component content of microorganism low-temperature preservation protective agent is 1 unit concentration content.
The Cord blood refers to one kind in liquid cryogen refrigeration or liquid freezing preservation.
Following article embodiment explains in detail, and in the presence of the Cord blood is protectant, is obtained after Cord blood
High survivaling cell or bacterial population., it is surprising that the survival rate highest of bacterium or cell can reach 95% left side after Cord blood
It is right.In particular, compared with existing Cryoprotectant, Cord blood protective agent of the invention is particularly suitable for long-term frozen preservation
And the freezen protective of the microorganism under needing multigelation to operate.
In the present invention, the microorganism includes but not limited to bacterium, virus, fungi, actinomyces, Richettsia, clothing original
Body, conveyor screw.
In one embodiment of the invention, the microorganism is bacterium, preferably mycobacteria, including but not limited to：
Mycobacterium tuberculosis, mycobacterium kansasii, Mycobacterium marinum, scrofula mycobacteria, bird-Mycobacterium intracellulare, occasional branching stem
Bacillus, Mycobacterium chelonei, mycobacterium buruli, mycobacterium smegmatis, Mycobacterium leprae.More preferably mycobacterium tuberculosis.
In one particular embodiment of the present invention, the microorganism is BCG vaccine bacteria or BCG vaccine viable bacteria.
" BCG vaccine " used herein or " BCG vaccine " typically refer to a kind of active bacillus tuberculosis bovis of attenuation.
In a preferred embodiment of the invention, BCG vaccine viable bacteria includes but not limited to bacillus tuberculosis bovis-BCG-
Russia (Mycobaxteriumbovis-BCG-Russia), bacillus tuberculosis bovis-BCG-Moreau
(Mycobaxteriumbovis-BCG-Moreau), bacillus tuberculosis bovis-BCG-Japan (Mycobaxteriumbovis-
BCG-Japan), bacillus tuberculosis bovis-BCG-Sweden (Mycobaxteriumbovis-BCG-Sweden), bovine tuberculosis bar
Bacterium-BCG-Birkhaug (Mycobaxteriumbovis-BCG-Birkhaug), bacillus tuberculosis bovis-BCG-Prague
(Mycobaxteriumbovis-BCG-Prague), bacillus tuberculosis bovis-BCG-Glaxo (Mycobaxteriumbovis-
BCG-Glaxo), bacillus tuberculosis bovis-BCG-Denmark (Mycobaxteriumbovis-BCG-Denmark), bovine tuberculosis
Bacillus-BCG-Tice (Mycobaxteriumbovis-BCG-Tice), bacillus tuberculosis bovis-BCG-Frappier
(Mycobaxteriumbovis-BCG-Frappier), bacillus tuberculosis bovis-BCG-Connaught
(Mycobaxteriumbovis-BCG-Connaught), bacillus tuberculosis bovis-BCG-Phipps
(Mycobaxteriumbovis-BCG-Phipps) and/or bacillus tuberculosis bovis-BCG-Pasteur
(Mycobaxteriumbovis-BCG-Pasteur), BCG vaccine D2PB302 (CMCC95050) BCG vaccine BCG NIFDC945SⅢ
(CMCC94103), BCG vaccine BCG NIFDC945SⅡ(CMCC94102)。
In one embodiment of the invention, the step of Cord blood microbial process includes protecting microorganism low-temperature
It deposits protective agent to mix with microorganism to be saved, then mixed material is placed under storage temperature.
In one embodiment of the invention, the method for Cord blood BCG vaccine viable bacteria is to preserve microorganism low-temperature and protect
Shield agent is mixed with BCG vaccine viable bacteria, mixed material is placed 2-24h under the arbitrary temp between 0 DEG C to 5 DEG C, then will mix
2-24h is placed under the arbitrary temp that material transfer after conjunction is placed between subzero 25 DEG C to subzero 15 DEG C, it finally will be mixed
Material is positioned in subzero 50 DEG C or following temperature and preserves.
In one particular embodiment of the present invention, the step of Cord blood BCG vaccine viable bacteria is：BCG vaccine thalline is harvested,
Measured concentration obtains bacterium stoste after grinding；Bacterium stoste is diluted to required concentration with Cord blood protective agent, for example, per 1ml
Bacteria containing amount 0.1mg obtains semi-finished product；Packing, for example, every cryopreservation tube 0.5ml, 4 DEG C of pre-freezes 12 hours or more, -20 DEG C of pre-freezes 12 are small
When more than, -70 DEG C are stored refrigerated.
Microorganism low-temperature preservation protective agent provided by the invention realizes the microorganism of freezen protective, especially BCG vaccine and lives
Bacterium, the stability of long-term preservation at different temperatures.Thus BCG vaccine viable bacteria is storing 2 months, is storing 4 months, storage 6
It the moon stores 9 months, stores 12 months, storage 1 year and a half, stores 2 years, storage 3 years, storage 4 years, storage 5 years stores 6 years, stores up
It deposits 7 years, stores 8 years, store 9 years, store 10 years, store 11 years, store 12 years, store 13 years, store 14 years, store 15 years,
The survival rate that storage retains after 20 years is more than 10%, is preferably greater than 20%, more preferably above 30%, more preferably above 40%, more
Preferably greater than 50%, more preferably above 60%, more preferably above 70%, more preferably above 80%, more preferably above 90%.
In terms of using the ratio of preservative agent, those skilled in the art can need to be adjusted according to real work, such as
Preservative agent stoste can account for (the including but not limited to) 1% of Cord blood system total volume, and 2%, 3%, 4%, 5%, 6%,
7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,
23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%,
53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 90% etc..
In related fields, the present invention also provides a kind of protectant preparation methods of Cord blood, including weigh each component
And be dissolved in suitable quantity of water, constant volume simultaneously adjusts pH as 7-8, sterilizes.
In one embodiment, when Cord blood protective agent and the expression of the mixture of microorganism made in accordance with the present invention
When antigen, the present invention can also provide vaccine.
" vaccine " this term refers to pharmaceutically acceptable composition, when with effective quantity be administered to animals or humans by
When examination person, the antibody and/or excitation protective immunity for the immunogene for including in vaccine can be induced in subject.
On the other hand, the application the invention further relates to the Cord blood protective agent in Cord blood microorganism.It is described
Microorganism includes but not limited to bacterium, virus, fungi, actinomyces, Richettsia, Chlamydia, conveyor screw.
In one embodiment of the invention, the microorganism is bacterium, preferably mycobacteria, including but not limited to：
Mycobacterium tuberculosis, mycobacterium kansasii, Mycobacterium marinum, scrofula mycobacteria, bird-Mycobacterium intracellulare, occasional branching stem
Bacillus, Mycobacterium chelonei, mycobacterium buruli, mycobacterium smegmatis, Mycobacterium leprae.More preferably mycobacterium tuberculosis.
In one particular embodiment of the present invention, the microorganism is BCG vaccine bacteria or BCG vaccine viable bacteria.
The Cord blood protective agent of the present invention improves microorganism low-temperature and preserves efficiency, especially BCG vaccine viable bacteria low temperature guarantor
Liquid cryogenic protective agent is made using common sucrose, gelatin etc. in microorganism frozen-dried protective, obtained sample in the survival rate deposited
Survival rate is only 20% or so, and uses the Cord blood protective agent sample Viable detection range of the present invention in 33%-99%
Between；Also, compared with other cryoprotectors, Cord blood protective agent of the invention is highly suitable in the case of multigelation
Bacterium preserve.Therefore, it is better under the conditions of deepfreeze to impart BCG vaccine viable bacteria to Cord blood protective agent of the invention
Survival rate improves the viable bacteria content after BCG vaccine viable bacteria refrigeration sample is recovered for the first time, is conducive to improve BCG vaccine viable bacteria sample
Recovery quality for the first time, be conducive to it is subsequent culture and harvest.
Term " containing ", "comprising" and " comprising " used herein are synonyms, are inclusiveness or open,
It is not excluded for composition that is additional, not being quoted, element or method and step.
With the numberical range that endpoint value indicates include all numerical value for being included and score within the scope of this and recited
Endpoint value.
When describing a measurable value, such as parameter, amount, time limit etc., term " about " used herein
Be intended to cover to differ ± 20% or less with designated value, preferably ± 10% or less, more preferably ± 5% or less, it is more preferable ±
1% or less, more preferable ± 0.1% or less variation, such variation is suitable to be used in disclosed invention.
The All Files enumerated in this specification are incorporated herein by reference in its entirety.Specifically, herein especially
The introduction for the All Files mentioned is incorporated herein by reference.
Unless otherwise indicated, the meaning and this hair of all terms (including technical and scientific term) for disclosing the present invention
Bright one skilled in the art institute is normally understood identical.By further instructing, definition thereafter is for preferably
Understand the teachings of the present invention.
Specific implementation mode
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.People in the art
Member it should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and form into
Row modifications or substitutions, but these modifications and replacement are each fallen in protection scope of the present invention.
In following each embodiments, BCG vaccine used is D2PB302 (CMCC95050), other reagents way all commercially available from
Diameter obtains.
Embodiment 1 configures trehalose, glycerine, Tween-80, the potassium chloride Cord blood different with sodium glutamate content
Protective agent stoste
By the constituent content in table 1, take respective substance, be dissolved in appropriate ultra-pure water, be settled to 100mL and with hydrochloric acid and
Sodium hydroxide solution adjusts pH value to 7.4；115 DEG C of 30min sterilizings, are made the Cord blood protective agent of the present invention of different ratio
Stoste.
1 Cord blood protective agent stoste of table matches table (per 100ml)
| Trehalose (g) | Glycerine (ml) | Polyoxyethylene sorbitan monoleate (ml) | Potassium chloride (g) | Sodium glutamate (g) | |
| Formula 1 | 10 | 20 | 1 | 2 | 0.8 |
| Formula 2 | 10 | 10 | 2 | 2 | 0.8 |
| Formula 3 | 10 | 2 | 4 | 2 | 0.8 |
| Formula 4 | 20 | 20 | 1 | 2 | 0.8 |
| Formula 5 | 20 | 10 | 2 | 2 | 0.8 |
| Formula 6 | 20 | 2 | 4 | 2 | 0.8 |
| Formula 7 | 30 | 20 | 1 | 2 | 0.8 |
| Formula 8 | 30 | 10 | 2 | 2 | 0.8 |
| Formula 9 | 30 | 2 | 4 | 2 | 0.8 |
Control 1
Weigh following substance：Gelatin 2g, sucrose 2g, potassium chloride 2g, sodium glutamate 0.8g；Above-mentioned substance is dissolved in right amount
Ultra-pure water is settled to 100mL and adjusts pH value to 7.4 with hydrochloric acid and sodium hydroxide solution；115 DEG C of 30min sterilizing to get.
The protectant effect disquisition of 2 Cord blood of embodiment
Harvest is grown on the BCG vaccine mycoderm that media surface is led in liquid Soviet Union, is washed with sterile saline, is claimed after pressing dry
Physiological saline is added according to wet bacterium weight in weight, and it is uniform bacterium solution that bacterium solution is ground in aryballos containing steel ball, takes sample to carry out dense
Degree measures.It is 0.2mg/ml with normal saline dilution bacterial concentration, takes respectively in the bacterium solution of same volume and embodiment 1 and be formulated 1-
9 and control 1 Cord blood protective agent stoste 1：1 is uniformly mixed, and obtains semi-finished product, and a concentration of 0.1mg/ml of BCG vaccine is dispensed, often
Branch cryopreservation tube 0.5ml, 4 DEG C of pre-freezes 12 hours or more, -20 DEG C of pre-freezes 12 hours or more, -70 DEG C of freezings obtain finished product.In addition, setting
One group of unprotect agent group is set, BCG vaccine concentration is adjusted to by 0.1mg/ml using physiological saline, same mode freezes.
Control protective agent BCG vaccine sample is taken, viable count detection is carried out with 10 times of dilution methods, as a result as viable count before freezing
Control.
Viable count detection method is：Sample is diluted to 10‐5mg/ml、10‐6Mg/ml takes 2 dilution samples each respectively
0.1ml is seeded to media surface, and each dilution 5 is managed again, and Colony Forming Unit (CFU) is counted after 37 ± 1 DEG C of culture four weekss
Number is scaled the CFU numbers that every mg bacteriums contain after calculating.
Each sample freezes two weeks at -70 DEG C, recovers after four weeks, wherein anti-again after freezing the recovery of a part of sample after four weeks
Multiple freeze thawing 3 times, carries out viable count measurement respectively, with viable count results contrast before jelly, calculates survival rate, as a result as follows：
Viable count contrast table before and after 2 BCG vaccine freezen protective of table
Note：Survival rate=(viable count before viable count/freezing after freezing) × 100%
Viable count unit：×104CFU/mg
The above results show the BCG vaccine prepared using the Cord blood protective agent stoste freezing for being formulated 1-9 in embodiment 1
Viable bacteria sample, Viable detection is high than compareing protective agent (control 1) after freezing, and formula 1-9 is especially suitable for prolonged low temperature
It preserves, and the death rate of bacterium after multigelation can be reduced.
The protectant prolonged cold of 3 Cord blood of embodiment preserves and the preservation effect of multigelation
According to the preparation method of embodiment 1, with the low temperature for forming content and preparing control frozen stock solution stoste and the present invention of table 3
Preserve protective agent stoste：
3 Cord blood protective agent stoste of table matches table (per 100ml)
According to the preparation method of embodiment 2, the BCG vaccine bacterium solution of same volume and formula 10-15 are taken respectively and compares 2-4
Cord blood protective agent stoste 1：1 is uniformly mixed, and obtains semi-finished product, a concentration of 0.1mg/ml of BCG vaccine, packing, every cryopreservation tube
0.5ml, 4 DEG C of pre-freezes 12 hours or more, -20 DEG C of pre-freezes 12 hours or more, -70 DEG C of freezings obtain finished product.Take 2 protective agent cards of control
Jie's bacterium sample carries out viable count detection with 10 times of dilution methods, as a result as viable count control before freezing.
Each sample is recovered after freezing four weeks at -70 DEG C, wherein freezing repeatedly again after freezing the recovery of a part of sample after four weeks
Melt 3 times, carry out viable count measurement respectively, with viable count results contrast before jelly, calculates survival rate, it is as a result as follows：
Viable count contrast table before and after 4 BCG vaccine freezen protective of table
Note：Survival rate=(viable count before viable count/freezing after freezing) × 100%
Viable count unit：×104CFU/mg
The above results show that BCG vaccine viable bacteria sample prepared by the Cord blood protective agent freezing using formula 10-15 is long
Survival rate is high after time freezing, and the survival rate of bacterium is even more to be apparently higher than control 2-4 after multigelation.
The Cord blood protective agent of the present invention can significantly improve the survival rate of BCG vaccine viable bacteria, in recovery strain and card
It can promote recovery quality in terms of sample itself when Jie's bacterium viable bacteria sample.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of BCG vaccine viable bacteria liquid freezen protective protective agent, it is characterised in that by nonreducing sugar, glycerine, nonionic table
Face activating agent, sylvite, glutamate and water composition；The nonreducing sugar is trehalose；The nonionic surface active agent
For polyoxyethylene sorbitan monoleate；The sylvite is potassium chloride；The glutamate is sodium glutamate；Wherein, the preservation protective agent each group
It is 1 unit concentration content to divide content；Also, in every 100 milliliter of 1 unit concentration：Trehalose 10g, glycerine 1mL, polyoxyethylene sorbitan monoleate
2mL, potassium chloride 1g, sodium glutamate 0.8g, protectant pH value are 7-8.
2. a kind of BCG vaccine viable bacteria liquid freezen protective protective agent stoste, it is characterised in that non-in the preservation protective agent stoste to go back
Originality sugar, glycerine, nonionic surface active agent, sylvite and glutamate each component content are preservation described in claim 1
2 times of each component in protectant 1 unit concentration content~5 times.
3. preserving protective agent stoste as claimed in claim 2, which is characterized in that pH value 7-8.
4. a kind of method that liquid freezing preserves BCG vaccine viable bacteria, it is characterised in that protected using preservation described in claim 1
Agent.
5. method as claimed in claim 4 will wait for that the BCG vaccine viable bacteria that liquid freezing preserves mixes with protective agent is preserved, obtain
Wait for the BCG vaccine viable bacteria mixed liquor that liquid freezing preserves, BCG vaccine viable bacteria liquid freezen protective is protectant each in the mixed liquor
Constituent content is 1 unit concentration content.
6. method as claimed in claim 5 will preserve protective agent and be mixed with BCG vaccine viable bacteria, by mixed material at 0 DEG C
It places 2-24h under arbitrary temp between to 5 DEG C, then the transfer of mixed material is placed in subzero 25 DEG C to subzero between 15 DEG C
Arbitrary temp under place 2-24h, finally mixed material is positioned in subzero 50 DEG C or following temperature and is preserved.
7. BCG vaccine viable bacteria liquid freezen protective protective agent as described in claim 1 is in BCG vaccine viable bacteria liquid freezen protective
Application.
8. a kind of method that liquid freezing preserves BCG vaccine viable bacteria, it is characterised in that protected using the preservation described in Claims 2 or 33
Protect agent stoste.
9. method as claimed in claim 8 will wait for the suspension and preservation protective agent of the BCG vaccine viable bacteria of liquid freezing preservation
Stoste mixes, and obtains waiting for the BCG vaccine viable bacteria mixed liquor that liquid freezing preserves, BCG vaccine viable bacteria liquid freezing in the mixed liquor
It is 1 unit concentration content to preserve protectant each component content.
10. BCG vaccine viable bacteria liquid freezen protective protective agent stoste as claimed in claim 2 or claim 3 is cold in BCG vaccine viable bacteria liquid
Freeze the application in preserving.
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