CN106434502A - Swine mycoplasma hyopneumoniae culture medium and preparation method and application thereof - Google Patents
Swine mycoplasma hyopneumoniae culture medium and preparation method and application thereof Download PDFInfo
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- CN106434502A CN106434502A CN201611209512.4A CN201611209512A CN106434502A CN 106434502 A CN106434502 A CN 106434502A CN 201611209512 A CN201611209512 A CN 201611209512A CN 106434502 A CN106434502 A CN 106434502A
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- 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
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Abstract
The invention provides a swine mycoplasma hyopneumoniae culture medium and a preparation method and application thereof and belongs to the technical field of bioengineering. The swine mycoplasma hyopneumoniae culture medium is prepared from a basal culture medium and an auxiliary culture medium, which are mainly prepared from ingredients such as MEM, beef extract powder, yeast leachate powder, lactoalbumin hydrolysate, gastric mucin, an arginine solution, pig blood serum and chicken blood serum. The swine mycoplasma hyopneumoniae culture medium is prepared through subjecting the basal culture medium and the auxiliary culture medium to sterile treatment, then, carrying out volume determination by using injection water, and adjusting the pH value of the solution. The culture medium provided by the invention is low in blood serum content and is applied to the preparation of vaccine antigens, the growth speed of swine mycoplasma hyopneumoniae is high, the culture cycle is short, the fungus content of a semi-finished product fungus solution is high, the production cost is low, and the prepared vaccines are good in immunization effect and low in side reaction occurrence probability, so that the culture medium is suitable for being industrially produced on a large scale.
Description
Technical field
The present invention relates to a kind of culture medium and its preparation method and application, more particularly, to a kind of mycoplasma hyopneumoniae culture medium
And its preparation method and application, belong to technical field of bioengineering.
Background technology
Mycoplasmal pneumonia of swine is also known as pig endemic conditions pneumonia (Swine enzootic hyopneumoniae), China's custom
Claim mycoplasma pneumoniae of swine, be that the one kind being caused by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) is chronic, contact, exhale
Inhale road transmission disease.Main clinical symptom is cough and pants, and often has other pathogenic bacterias (as pasteurella multocida, the secondary bloodthirsty bar of pig
Bacterium, actinobacillus pleuropneumoniae etc.) secondary infection.Primary disease is distributed widely in all over the world, on the pig farm in the many areas of China
There is generation.After pig infection would generally retarded growth, production performance is remarkably decreased, thus leading to feed conversion rate to drop
Low, Drug therapy cost increases, and easy secondary virus or bacterium infection, causes pig mortality rate to raise, leads to economic loss heavy.
From domestic and international clinical experience, vaccine immunity is the prevention most important means of primary disease.
At present the culture medium of culture mycoplasma hyopneumoniae mainly has a Friis culture medium, KM2 culture medium, A26 culture medium and specially
Sharp formula culture medium (Patent publication No：103060220A).It is still desirable to enter one in all of above culture medium and training method
Step reduces the using dosage of serum, or even the training method of serum-free medium, improves vaccine antigen content simultaneously, can be only achieved
Current Production requirement.
Content of the invention
Problem to be solved by this invention is to provide a kind of mycoplasma hyopneumoniae culture medium and its preparation method and application, uses
To reduce amount of serum needed for culture mycoplasma hyopneumoniae, improve i (mycoplasma hyopneumoniae) vaccine antigen valence.Specific as follows：
An aspect of of the present present invention, provides a kind of mycoplasma hyopneumoniae culture medium, and it is by basal medium and auxiliary culture medium
After aseptic process, made with injection water constant volume, the component of wherein basal medium and auxiliary culture medium is respectively：
Basal medium：
Auxiliary culture medium：
Mycoplasma hyopneumoniae culture medium as above, contains porcine blood serum and chicken serum 50- in every 1000mL culture medium
80mL, the volume ratio that is, in overall broth, serum adds is 5-8%.
Another aspect of the present invention, provides a kind of preparation method of mycoplasma hyopneumoniae culture medium, it comprises the following steps：
(1) prepare basal medium：(1)-(8) composition proportion is taken to dissolve in one by one in 200-400mL injection water, at 115 DEG C
Sterilizing 20min, standby after cooling；
(2) preparation auxiliary culture medium：Take (9)-(14) composition filtration sterilization, (15) and (16) composition is through inactivation, at irradiation
Reason, dissolves in one by one in the injection water of 200-400mL sterilizing, obtains final product auxiliary culture medium；
(3) by basal medium and auxiliary culture medium mixing, 1000ml is made with sterile water for injection constant volume, with aseptic
1mol/L NaOH adjusts pH value 7.6-7.8, and subpackage is standby.
Another aspect of the invention, provides application in the preparation of i (mycoplasma hyopneumoniae) vaccine antigen for the above-mentioned culture medium.
Mycoplasma hyopneumoniae culture medium of the present invention has the characteristics that：
(1) in culture medium of the present invention, serum content is low, reduces production cost, alleviates the allergy to pig body for the serum simultaneously
Stress, reduces mycoplasma hyopneumoniae finished product vaccine rate of side effects；
(2) culture medium of the present invention is applied to prepare vaccine antigen, mycoplasma hyopneumoniae fast growth, incubation time is only 2-
3 days, semi-finished product bacterium solution bacteria containing amount up to 1.0 × 109-10CCU/mL, far above Industry code requirements, suitable industrialized great production；
(3) i (mycoplasma hyopneumoniae) vaccine of application the inventive method preparation, antigenic content is high, good immune effect, immunity
Strong, mycoplasma hyopneumoniae attack malicious by force can be resisted.
Specific embodiment
It is described further with reference to specific embodiment, following examples are merely to illustrate, rather than limit
The protection domain of this patent.
Embodiment 1 prepares mycoplasma hyopneumoniae culture medium
1st, prepare basal medium：
1 × Han Keshi balanced salt solution compound method：NaCl 80.0g, CaCl21.4g, KCl 4g, MgCl2·6H2O
1.0g, KH2PO40.6g, MgSO4 7H2O 1.0g, Na2HPO4·12H2O 1.52g, phenol red 0.4g, filling jetting is extremely
1000mL.
Above-mentioned (1)-(8) composition proportion is taken to dissolve in 300mL injection water one by one, 115 DEG C of sterilizing 20min, after cooling
Standby；
2nd, preparation auxiliary culture medium：
Take (9)-(14) composition filtration sterilization, (15) and (16) composition, through inactivation, radiation treatment, dissolves in 300mL one by one and goes out
In the injection water of bacterium, obtain final product auxiliary culture medium；
3rd, by basal medium and auxiliary culture medium mixing, 1000ml is made with sterile water for injection constant volume, uses aseptic 1mol/
L NaOH adjusts pH value 7.8, and subpackage is standby.
Embodiment 2 prepares mycoplasma hyopneumoniae culture medium
1st, prepare basal medium：
Take above-mentioned (1)-(8) composition proportion to dissolve in one by one in 300mL injection water, 115 DEG C sterilize 20min, to be cooled
Standby afterwards；
2nd, preparation auxiliary culture medium：
Take (9)-(14) composition filtration sterilization, (15) and (16) composition, through inactivation, radiation treatment, dissolves in 300mL one by one and goes out
In the injection water of bacterium, obtain final product auxiliary culture medium；
3rd, by basal medium and auxiliary culture medium mixing, 1000ml is made with sterile water for injection constant volume, uses aseptic 1mol/
L NaOH adjusts pH value 7.8, and subpackage is standby.
Embodiment 3 application mycoplasma hyopneumoniae (lacks part to become with incomplete culture medium by culture medium of the present invention respectively
Point) it is compared enrichment culture：
1st, prepare culture medium
By mycoplasma hyopneumoniae J strain (freeze-drying lactobacillus are provided by production department of Ruipu (Baoding) Biological Pharmaceutical Co., Ltd.) respectively
It is inoculated in the culture medium 1 (with embodiment 1) of the present invention and culture medium 2 (requires to be configured according to embodiment 1, but is not added with stomach
Mucoitin, arginine solution, tyrosine solution, serine solution, choline chloride and chicken serum).
2nd, connect bacterium culture
After mycoplasma hyopneumoniae J strain seed subculture rejuvenation, inoculate respectively according to 10% (V/V), 37 DEG C of cultures, work as culture medium
When its colour changed into yellow, pH value are down to 6.8 by 7.8, take out culture.
3rd, viable bacteria titre (CCU) measures
Take 6 groups of sterile test tube, a kind of every 3 groups parallel repetition developmental tubes as culture fluid, measure and take 13 10mL control
Vial, every bottle of subpackage culture medium 1.8mL to be checked, to first bottle, vibration is mixed for the microbial strain culture after the inoculation continuous rejuvenation of 0.2mL
Even, draw 0.2mL and inoculate to the 2nd bottle, take turns doing 10 times and be serially diluted to the 12nd bottle, the 13rd bottle sets the culture being not added with bacterium solution
Base is negative control.37 DEG C of cultures of standing, daily observed and recorded culture medium color change situation, result of determination on the 14th, such as train
Foster base control tube invariant color, liquid culture has color change successively by its dilution factor, then test is set up.Every group of pipe-produced glass bottle
The highest dilution of middle liquid culture variable color is the CCU titre of this culture, records 3 groups of parallel laboratory test results.
4th, result
Measured from CCU, the result that culture medium 1 prepared by the present invention records 3 parallel groups is respectively 109CCU/mL,
109CCU/mL, 1010CCU/mL, result is converted into 4 × 109CCU/mL；And the result that culture medium 2 records 3 parallel groups is respectively
107CCU/mL, 107CCU/mL, 108CCU/mL, result is converted into 4 × 107CCU/mL.
It is not added with stomach mucoitin, arginine solution, tyrosine solution, the training of serine solution, choline chloride and chicken serum
Foster base 2 and the culture medium 1 adding above-mentioned composition, culture effect significant difference, about 100 times of gap, illustrate in the present invention
Related compounds have certain effect to mycoplasma hyopneumoniae culture effect.
Embodiment 4 prepares mycoplasma hyopneumoniae culture medium
Select mycoplasma culture medium of the present invention (serum content is 5%), A26 culture medium (serum content is 20%) and KM2
Culture medium (serum content is 20%) is compared enrichment culture to mycoplasma hyopneumoniae J strain：
1st, the culture of mycoplasma hyopneumoniae bacterium solution and the preparation of seedling bacterium solution：
Mycoplasma hyopneumoniae (J strain) production seed is inoculated in above 3 kinds of culture medium respectively by 10% (V/V)
(200mL) in, 37 DEG C of cultures, pH value is reduced to and harvests during 6.7-6.8；By the bacterium solution after above-mentioned passing on by 10% (V/V) amount
Being inoculated in above 3 kinds of culture medium (2000mL) same methods to pass on again, until harvesting bacterium solution amount to be extended to 200000mL, taking suitable
Measure above-mentioned semi-finished product bacterium solution and carry out viable count mensure, measure 3 times altogether.
2nd, viable bacteria titre (CCU) measures：
The mycoplasma hyopneumoniae J strain seedling bacterium solution of above 3 kinds of culture medium preparation is taken to be serially diluted to for 10 times successively respectively
12 pipes, then set culture medium and make negative control, put culture in 37 DEG C of constant incubators of temperature, daily observed and recorded culture medium color becomes
Change and turbidity change, result of determination on the 14th, such as culture medium control tube invariant color, then test is set up.Color is finally occurred to become
The dilution factor changed is the CCU titre of this culture, and 3 kinds of bacterium solution are each to be measured 3 times, incubation time (cultivation cycle) and bacteria containing amount
Measurement result is shown in Table 1：
The result that 3 kinds of culture medium prepare seedling bacterium solution is inoculated in table 1 mycoplasma hyopneumoniae J strain
3 kinds of culture medium mycoplasma hyopneumoniae incubation time obvious differences：The training that mycoplasma hyopneumoniae J strain is prepared in the present invention
Grow the fastest in foster base, be 2-3 day；KM2 culture medium growth time is 3-5 day, and A26 culture medium is 4-5 day.
The bacteria containing amount measurement result of 3 kinds of culture medium mycoplasma hyopneumoniae semi-finished product bacterium solution：Culture medium of the present invention, KM2 culture
The seedling bacterium solution bacteria containing amount of base and the preparation of A26 culture medium reduces successively, and reaches the time of final bacteria containing amount and extend successively, contains
Bacterium amount (and reaching the minute of final bacteria containing amount) is respectively 4.0 × 109CCU/ml (10 days), 1.0 × 108CCU/ml(13
Day), 7.0 × 107CCU/ml (14 days), illustrates that culture medium of the present invention prepares the life of mycoplasma hyopneumoniae J strain semi-finished product bacteria liquid has
Long rapid, the high feature of bacteria containing amount.
Embodiment 5 prepares mycoplasma hyopneumoniae culture medium
With applying mycoplasma hyopneumoniae (J strain) the semi-finished product bacterium solution of culture medium culturing of the present invention to prepare Pulmonis Sus domestica in embodiment 3
Scorching mycoplasma inactivated vaccine, and carry out safety verification and the efficacy test of vaccine.
1st, mycoplasma hyopneumoniae (J strain) inactivated vaccine preparation
(1) inactivate：Mass concentration (m is added in mycoplasma hyopneumoniae bacterium solution：V) 37% formalin inactivation, formaldehyde
Solution：Mycoplasma hyopneumoniae bacterium solution=1.5：1000(V：V), 37 DEG C of inactivation 24h, every 1h shake up once.
(2) emulsifying：5 parts of phase of water intaking is added in magnetic agitation tank, while stirring at low speed, is slowly added to 1 part of oil phase,
Stirred 30 minutes with 300r/min, that is, mycoplasma hyopneumoniae (J strain) inactivated vaccine is obtained.
2nd, safety verification：Take 14 age in days sodium selenite 5, musculi colli vaccinates, Continuous Observation 14 days is simultaneously periodically surveyed
Determine body temperature.Result shows, injection site absorbs good, note Seedling piglet no body temperature rising phenomenon, and diet and the mental status are without exception,
Safety verification is qualified.
3rd, efficacy test：Take 30 age in days piglet 10, attack poison by force after vaccine immunity, scored according to pulmonary lesion, according to
The pulmonary lesion slip of standard immunoassay pig should be not less than 60%；The vaccine immunity hyopneumoniae pathological changes slip that this produces is average
Higher than 80%, efficacy test is qualified.
Claims (4)
1. a kind of mycoplasma hyopneumoniae culture medium it is characterised in that by basal medium, auxiliary culture medium and injection water form, its
The component of middle basal medium and auxiliary culture medium is respectively：
Basal medium：
Auxiliary culture medium：
2. mycoplasma hyopneumoniae culture medium as claimed in claim 1 is it is characterised in that contain Sanguis sus domestica in every 1000mL culture medium
Cleer and peaceful chicken serum 50-80mL, the volume ratio that is, in overall broth, serum adds is 5-8%.
3. a kind of preparation method of mycoplasma hyopneumoniae culture medium as claimed in claim 1 or 2 is it is characterised in that include following
Step：
(1) prepare basal medium：(1)-(8) as claimed in claim 1 composition proportion is taken to dissolve in 200-400mL injection one by one
In water, 115 DEG C sterilize 20min, standby after cooling；
(2) preparation auxiliary culture medium：Take (9)-(14) composition filtration sterilization as claimed in claim 1, (15) and (16) composition
Through inactivation, radiation treatment, dissolve in one by one in the injection water of 200-400mL sterilizing, obtain final product auxiliary culture medium；
(3) by basal medium and auxiliary culture medium mixing, 1000ml is made with sterile water for injection constant volume, uses aseptic 1mol/L
NaOH adjusts pH value 7.6-7.8, and subpackage is standby.
4. application in the preparation of i (mycoplasma hyopneumoniae) vaccine antigen for the culture medium as claimed in claim 1 or 2.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468248A (en) * | 2018-12-14 | 2019-03-15 | 南京天邦生物科技有限公司 | A kind of porcine mycoplasmal pneumonia antigen high-efficient culture method |
| CN110607260A (en) * | 2019-09-23 | 2019-12-24 | 山东甲骨文生物科技有限公司 | Culture medium and method for low-serum culture of mycoplasma hyopneumoniae |
| CN110804563A (en) * | 2019-11-13 | 2020-02-18 | 山东滨州沃华生物工程有限公司 | Culture medium for low-serum culture of mycoplasma hyopneumoniae |
| CN115058354A (en) * | 2022-04-28 | 2022-09-16 | 四川诺顺科技有限公司 | Mycoplasma hyopneumoniae culture medium with definite chemical components and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109468248A (en) * | 2018-12-14 | 2019-03-15 | 南京天邦生物科技有限公司 | A kind of porcine mycoplasmal pneumonia antigen high-efficient culture method |
| CN110607260A (en) * | 2019-09-23 | 2019-12-24 | 山东甲骨文生物科技有限公司 | Culture medium and method for low-serum culture of mycoplasma hyopneumoniae |
| CN110804563A (en) * | 2019-11-13 | 2020-02-18 | 山东滨州沃华生物工程有限公司 | Culture medium for low-serum culture of mycoplasma hyopneumoniae |
| CN115058354A (en) * | 2022-04-28 | 2022-09-16 | 四川诺顺科技有限公司 | Mycoplasma hyopneumoniae culture medium with definite chemical components and preparation method thereof |
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