CN109385385A - A kind of preparation method and applications of avian mycoplasmas culture medium, avian mycoplasmas bacterium solution - Google Patents
A kind of preparation method and applications of avian mycoplasmas culture medium, avian mycoplasmas bacterium solution Download PDFInfo
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Abstract
The present invention relates to a kind of avian mycoplasmas culture mediums, the preparation method and applications of avian mycoplasmas bacterium solution, belong to field of biotechnology.Avian mycoplasmas culture medium of the invention is made of basal medium and auxiliary ingredients, and the basal medium is conventional avian mycoplasmas fluid nutrient medium, and auxiliary ingredients are gelatin, Tween 80 and calcium chloride；Production is chicken virus mycoplasma or chicken Mycoplasma synoviae with strain, and through bacterium solution inoculation, culture, harvest, avian mycoplasmas vaccine is made in production avian mycoplasmas bacterium solution.Preparation method of the present invention improves the antigenic content of avian mycoplasmas bacterium solution, simplifies production technology, reduces production cost, reduces vaccine immunity side reaction occurrence probability, promotes immune effect of vaccine.
Description
Technical field
The present invention relates to biomedicine technical fields, and in particular to a kind of avian mycoplasmas culture medium, avian mycoplasmas bacterium solution
Preparation method and applications.
Background technique
Avian mycoplasmas disease (Avian Mycoplasmosis, AM) is a kind of infectious disease of the poultry as caused by avian mycoplasmas,
Clinically common cause of disease is chicken virus mycoplasma (Mycoplasma Galliscepticum, MG) and chicken Mycoplasma synoviae
(Mycoplasma Synoviae, MS).Mycoplasma gallisepticum infection can cause the chronic respiratory tract disease of fowl
(Chronicrespiratory Disease, CRD), air bag inflammation and sinusitis；The infection of chicken Mycoplasma synoviae can cause swollen joint
Greatly, bursa synovialis and the symptoms such as tendon inflammation and organa parenchymatosum's enlargement；Effective vaccine immunity is the main means of the prevention and control disease.
Avian mycoplasmas production of vaccine is mainly cultivated using improvement FreyShi culture medium, CM2 culture medium etc. at present, these
Culture medium serum-concentration is higher (generally 10% or more), and the concentration for cultivating bacterium solution is not high by (general 108-10CCU/ml), per secondary
Production need to breed strain again, and production operation is cumbersome, high expensive, and (antigenic content and purity are or not vaccine product unstable quality
It is high), it is easy to appear side reaction.Therefore, it needs to develop efficient one kind, low cost, convenient and fast avian mycoplasmas culture medium, avian mycoplasmas
The preparation method and applications of bacterium solution.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defect of the prior art, provide it is a kind of efficiently it is convenient, low at
The preparation method and applications of avian mycoplasmas culture medium originally, avian mycoplasmas bacterium solution.
In order to solve above-mentioned technical proposal, the present invention provides the following technical scheme that a kind of avian mycoplasmas of the invention is trained
Base is supported, the avian mycoplasmas culture medium is made of avian mycoplasmas basal medium and auxiliary ingredients, the basal medium
For conventional avian mycoplasmas fluid nutrient medium, the auxiliary ingredients are made of gelatin, Tween 80 and calcium chloride.
Further, the basal medium is conventional CM2 fluid nutrient medium, CM culture medium or improvement FreyShi training
Support any one in base.
Further, the auxiliary ingredients dosage is following group of finished product culture medium mass volume ratio (m:V) and is grouped as:
Gelatin 0.2-0.4%,
Tween 80 0.5-1%,
Calcium chloride 0.2-0.4%.
A kind of preparation method of avian mycoplasmas bacterium solution of the invention, production are chicken virus mycoplasma or chicken bursa synovialis branch with strain
Substance, bacterium solution include the following steps:
(1) by qualified avian mycoplasmas production seed, (V:V) 10% is inoculated in avian mycoplasmas culture medium by volume,
Rear 36-37 DEG C of stationary culture is mixed well, is cultivated 6-8 hours；
(2) cultivation temperature is improved to 38-39 DEG C, 50-100r/min stir culture 8-12 hours, and the pH to culture medium declines
Stop stirring to 7.2, reduces cultivation temperature to 22 DEG C, then stand 2-4 hours, harvest lower layer's culture solution, harvest yield is culture solution
The 1/3-1/2 of total volume；
(3) it in Xiang Shangshu remaining culture liq, adds the 2-3 times of avian mycoplasmas culture medium being concentrated, repeats step (2) after resuming
It is commissioned to train feeding, harvests lower layer's culture solution of total volume 1/3-1/2；According to this method continuous fed-batch cultivation 4-6 generation, avian mycoplasmas bacterium is made
Liquid.
Avian mycoplasmas culture medium of the present invention is preparing the application in avian mycoplasmas vaccine.
Avian mycoplasmas bacterium solution of the present invention is preparing the application in avian mycoplasmas vaccine.
The utility model has the advantages that preparation method of the present invention improves the antigenic content of avian mycoplasmas bacterium solution, simplify production technology, reduces
Production cost reduces vaccine immunity side reaction occurrence probability, promotes immune effect of vaccine.
Compared with prior art, the present invention has the advantage that
(1) present invention adjusts using novel culture medium and during the cultivation process cultivation temperature, significantly improves avian mycoplasmas
The antigenic content of bacterium solution, bacterium solution titre are up to 1013-14CCU/ml, higher than the quality standard of existing vaccine semi-finished product bacterium solution.
(2) present invention uses fed-batch cultivation mode, and an avian mycoplasmas production seed is only prepared before culture for the first time, saves
The slightly later period is respectively prepared for production with seed, is simplified production technology, is saved manpower, shorten the production cycle 20 days or more.
(3) avian mycoplasmas bacterium solution of the present invention prepares vaccine after can diluting, and reduces production cost；And finished product vaccine antigen is pure
Degree is high, and the impurity such as haemocyanin and metabolite are greatly reduced, and reduces immune side reaction occurrence probability, promotes vaccine immunity effect
Fruit.
Specific embodiment
By following embodiment, present invention be described in more detail, but should be noted that the scope of the present invention is not implemented by these
Any restrictions of example.
Embodiment 1
A kind of avian mycoplasmas culture medium of the invention, the avian mycoplasmas culture medium by avian mycoplasmas basal medium and
Auxiliary ingredients composition, the basal medium are conventional avian mycoplasmas fluid nutrient medium, the auxiliary ingredients by gelatin,
Tween 80 and calcium chloride composition.
The basal medium is improvement FreyShi culture medium.
The auxiliary ingredients dosage is that following group of finished product culture medium mass volume ratio (m:V) is grouped as:
Gelatin 0.2-0.4%,
Tween 80 0.5-1%,
Calcium chloride 0.2-0.4%.
A kind of preparation method of avian mycoplasmas bacterium solution of the invention, production are chicken virus mycoplasma or chicken bursa synovialis branch with strain
Substance, bacterium solution include the following steps:
(1) by qualified avian mycoplasmas production seed, (V:V) 10% is inoculated in avian mycoplasmas culture medium by volume,
36 DEG C of stationary cultures after mixing well are cultivated 8 hours；
(2) cultivation temperature is improved to 39 DEG C, and 100r/min stir culture 8-12 hours, the pH to culture medium dropped to 7.2
Stop stirring, reduce cultivation temperature to 22 DEG C, then stand 4 hours, harvest lower layer's culture solution, harvest yield is culture solution total volume
1/2；
(3) in Xiang Shangshu remaining culture liq, the avian mycoplasmas culture medium of 2 times of concentrations is added, step (2) is repeated and continues to pass on
Culture harvests lower layer's culture solution of total volume 1/2；According to this method continuous 4 generation of fed-batch cultivation, avian mycoplasmas bacterium solution is made.
Avian mycoplasmas culture medium of the present invention is preparing the application in avian mycoplasmas vaccine.
Avian mycoplasmas bacterium solution of the present invention is preparing the application in avian mycoplasmas vaccine.
Embodiment 2
Embodiment 2 the difference from embodiment 1 is that: a kind of avian mycoplasmas culture medium of the invention, the avian mycoplasmas
Culture medium is made of avian mycoplasmas basal medium and auxiliary ingredients, and the basal medium is conventional avian mycoplasmas liquid
Culture medium, the auxiliary ingredients are made of gelatin, Tween 80 and calcium chloride.
The basal medium is CM culture medium.
The auxiliary ingredients dosage is that following group of finished product culture medium mass volume ratio (m:V) is grouped as:
Gelatin 0.2-0.4%,
Tween 80 0.5-1%,
Calcium chloride 0.2-0.4%.
A kind of preparation method of avian mycoplasmas bacterium solution of the invention, production are chicken virus mycoplasma or chicken bursa synovialis branch with strain
Substance, bacterium solution include the following steps:
(1) by qualified avian mycoplasmas production seed, (V:V) 10% is inoculated in avian mycoplasmas culture medium by volume,
36.5 DEG C of stationary cultures after mixing well are cultivated 6 hours；
(2) cultivation temperature is improved to 38.5 DEG C, and 50r/min stir culture 8-12 hours, the pH to culture medium dropped to 7.2
Stop stirring, reduce cultivation temperature to 22 DEG C, then stand 2 hours, harvest lower layer's culture solution, harvest yield is culture solution total volume
1/3；
(3) in Xiang Shangshu remaining culture liq, the avian mycoplasmas culture medium of 3 times of concentrations is added, step (2) is repeated and continues to pass on
Culture harvests lower layer's culture solution of total volume 1/3；According to this method continuous 6 generation of fed-batch cultivation, avian mycoplasmas bacterium solution is made.
Embodiment 3
Embodiment 3 the difference from embodiment 1 is that: a kind of avian mycoplasmas culture medium of the invention, the avian mycoplasmas
Culture medium is made of avian mycoplasmas basal medium and auxiliary ingredients, and the basal medium is conventional avian mycoplasmas liquid
Culture medium, the auxiliary ingredients are made of gelatin, Tween 80 and calcium chloride.
The basal medium is conventional CM2 fluid nutrient medium.
The auxiliary ingredients dosage is that following group of finished product culture medium mass volume ratio (m:V) is grouped as:
Gelatin 0.2-0.4%,
Tween 80 0.5-1%,
Calcium chloride 0.2-0.4%.
A kind of preparation method of avian mycoplasmas bacterium solution of the invention, production are chicken virus mycoplasma or chicken bursa synovialis branch with strain
Substance, bacterium solution include the following steps:
(1) by qualified avian mycoplasmas production seed, (V:V) 10% is inoculated in avian mycoplasmas culture medium by volume,
37 DEG C of stationary cultures after mixing well are cultivated 7 hours；
(2) cultivation temperature is improved to 38 DEG C, and 80r/min stir culture 8-12 hours, the pH to culture medium dropped to 7.2 and stops
It only stirs, reduces cultivation temperature to 22 DEG C, then stand 3 hours, harvest lower layer's culture solution, harvest yield is the 1/ of culture solution total volume
3；
(3) in Xiang Shangshu remaining culture liq, the avian mycoplasmas culture medium of 3 times of concentrations is added, step (2) is repeated and continues to pass on
Culture harvests lower layer's culture solution of total volume 1/3；According to this method continuous 5 generation of fed-batch cultivation, avian mycoplasmas bacterium solution is made.
Test 1
1. avian mycoplasmas culture medium is prepared
The raw material market for preparing culture medium is on sale；Strain: F36 plants of chicken virus mycoplasma, CR plants of chicken virus mycoplasma, synovia branch
Substance (CVCC385 plants) and R plants of chicken virus mycoplasma are purchased from China Veterinery Drug Inspection Office, MS-H plants of chicken Mycoplasma synoviae from
It is obtained in the vaccine strain of market, is only used for experimental verification the method for the present invention.
(1) avian mycoplasmas culture medium 1 is prepared
Basal medium is CM2 fluid nutrient medium, prepares culture medium according to " agriculture and animal husbandry letter (1998) 38 "；Preparing CM2
When the basic ingredient of fluid nutrient medium, while auxiliary ingredients are added, according to finished product culture medium mass volume ratio (m:V) gelatin 0.2-
0.4%, Tween 80 0.5-1% and calcium chloride 0.2-0.4% addition, 115 DEG C of high pressure 20min, sterile addition pig blood after cooling
Clearly, sterile 25% yeast leachate and penicillin are configured to avian mycoplasmas training with 1.0mol/L NaOH tune pH value to 7.6-8.0
Support base 1.It is spare after steriling test is qualified.
(2) avian mycoplasmas culture medium 2 is prepared
Basal medium is CM fluid nutrient medium, prepares culture medium according to " Ministry of Agriculture announces No. 297 "；Preparing CM liquid
When the basic ingredient of culture medium, while auxiliary ingredients are added, according to finished product culture medium mass volume ratio (m:V) gelatin 0.2-
0.4%, Tween 80 0.5-1% and calcium chloride 0.2-0.4% addition, 115 DEG C of high pressure 20min, sterile addition pig blood after cooling
Clearly, 25% yeast leachate, penicillin are configured to avian mycoplasmas culture medium 2 with sodium hydroxide tune PH to 7.6-8.0.Without bacterial examination
It tests spare after qualification.
(3) avian mycoplasmas culture medium 3 is prepared
Basal medium is improvement Frey fluid nutrient medium, according to Republic of China Veterinary Pharmacopoeia (2015 editions) annex system
Standby culture medium；When preparing the basic ingredient of improvement Frey fluid nutrient medium, while auxiliary ingredients are added, according to finished product culture medium
Mass volume ratio (m:V) gelatin 0.2-0.4%, Tween 80 0.5-1% and calcium chloride 0.2-0.4% addition, 115 DEG C of high pressures
20min, sterile addition Swine serum, 2% cozymase, 1%L- cysteine solution, penicillin after cooling, extremely with sodium hydroxide tune pH
7.6-8.0 being configured to avian mycoplasmas culture medium 3.It is spare after steriling test is qualified.
Chicken virus mycoplasma 2. (F36 plants) bacterium solution culture
(1) by chicken virus mycoplasma (F36 plants) production seed, (V:V) 10% is inoculated in avian mycoplasmas culture medium by volume
In 1,37 DEG C of stationary cultures after mixing well are cultivated 7 hours, are then improved cultivation temperature to 39 DEG C, 80r/min stir culture
The pH of 10.5 hours culture mediums drops to 7.2, stops stirring, reduces cultivation temperature to 22 DEG C, then stand 3 hours, harvests lower layer
Culture solution, harvest yield is the 1/2 of culture solution total volume, and carries out bacterial concentration CCU measurement.Control group is set up simultaneously, by existing
Culture process produces (agriculture and animal husbandry letter (1998) 38).
(2) in Xiang Shangshu remaining culture liq, the avian mycoplasmas culture medium 1 of 2 times of concentrations is added to original volume, then 39 DEG C,
80r/min stir culture 8-12 hours, the pH to culture medium dropped to 7.2 stoppings stirring, reduced cultivation temperature to 22 DEG C, then quiet
It sets 3 hours, harvests lower layer's culture solution, harvest yield is the 1/2 of culture solution total volume, and carries out bacterial concentration CCU measurement；According to this
In method continuous 5 generation of fed-batch cultivation, harvest bacterium solution, which is used as, prepares vaccine semi-finished product.
(3) above-mentioned 1,2,4,6 generations and control group bacterium solution being subjected to viable count measurement respectively, concentration is respectively 4.0 ×
1014CCU/ml、3.0×1014CCU/ml、6.0×1013CCU/ml、8.0×1014CCU/ml and 4.0 × 109CCU/ml。
Chicken virus mycoplasma 3. (CR plants) bacterium solution culture
Chicken virus mycoplasma CR plants is cultivated according to 2 method of embodiment avian mycoplasmas culture medium 2, and sets up control group (by existing
Have technique production, the Ministry of Agriculture announces No. 297), 1,2,4,6 generations and control group bacterium solution carry out viable count measurement, concentration difference respectively
It is 1.0 × 1013CCU/ml、6.0×1013CCU/ml、5.0×1014CCU/ml、7.0×1014CCU/ml and 3.0 × 109CCU/
ml。
4. chicken virus mycoplasma vaccine preparation
Live vaccine preparation: by chicken virus mycoplasma F36 plants of semi-finished product bacterium solution 0.1M of 1,2,4,6 generation in embodiment 1
PH7.2PBS is diluted to 4.0 × 10 respectively9CCU/ml and control group bacterium solution are manufactured and are examined according to maternal antibodies
It tests Trial Regulation draft (agriculture and animal husbandry letter (1998) 38) and prepares maternal antibodies.It is spraying respectively by 5 groups of vaccines of preparation
20 age in days SPF chickens, 10/group, 1 plumage part/only, and blank control is set up, observed after immunization 14 days, and chicken is used within 28th after immune
Virulent R plants of virus mycoplasma carries out attacking poison, observes 14 after attacking poison, analyses observation lesion situation, and different culture process chicken poison branch are former
See Table 1 for details for the safety of body live vaccine and effect comparison result.
Table 1
As it can be seen from table 1 the maternal antibodies product of original production technology preparation carries out spraying immune and has gently
Micro- immune side reaction, immune protective rate has no immune using vaccine spraying immune prepared by present invention process 9/10 after attacking poison
Side reaction, immune group chicken obtain good protection.
The preparation of inactivated vaccine: by chicken virus mycoplasma CR plants of semi-finished product bacterium solution 0.1M of 1,2,4,6 generation in embodiment 1
PH7.2PBS is diluted to 3.0 × 10 respectively9CCU/ml and control group bacterium solution, according to chicken virus mycoplasma inactivated vaccine manufacture and
Trial Regulation draft (Ministry of Agriculture announces No. 297) is examined to prepare chicken virus mycoplasma inactivated vaccine.By 5 groups of vaccines of preparation, respectively
40 age in days SPF chicken of intramuscular immunity, 10/group, 1 plumage part/only, and blank control is set up, observed after immunization 14 days, and after immune
It carries out attacking poison with virulent R plants of chicken virus mycoplasma within 28th, be observed 14 after attacking poison, analyse observation lesion situation, different culture process
See Table 2 for details for the safety of chicken virus mycoplasma inactivated vaccine and effect comparison result.
Table 2
From table 2 it can be seen that the chicken virus mycoplasma inactivated vaccine product of original production technology preparation has slight immune pair anti-
It answers, immune protective rate has no immune side reaction using vaccine prepared by present invention process, immune group chicken is equal 9/10 after attacking poison
Obtain good protection.
5. chicken Mycoplasma synoviae bacterium solution culture and vaccine preparation
(1) by chicken Mycoplasma synoviae (MS-H plants) production seed, (V:V) 10% is inoculated in avian mycoplasmas by volume
In culture medium 3,36 DEG C of stationary cultures after mixing well are cultivated 8 hours, are then improved cultivation temperature to 38 DEG C, 100r/min
The pH of 9 hours culture mediums of stir culture drops to 7.2 stoppings stirring, reduces cultivation temperature to 22 DEG C, then stand 4 hours, harvests
Lower layer's culture solution, harvest yield is the 1/3 of culture solution total volume, and carries out bacterial concentration CCU measurement.
(2) in Xiang Shangshu remaining culture liq, the avian mycoplasmas culture medium 3 of 3 times of concentrations is added to original volume, then 38 DEG C,
100r/min stir culture 8-12 hours, the pH to culture medium dropped to 7.2 stoppings stirring, reduced cultivation temperature to 22 DEG C, then
4 hours are stood, harvests lower layer's culture solution, harvest yield is the 1/3 of culture solution total volume, and carries out bacterial concentration CCU measurement；According to
In this method continuous 5 generation of fed-batch cultivation, harvest bacterium solution, which is used as, prepares vaccine semi-finished product.
(3) above-mentioned 1,2,4 and 6 are withheld and obtains bacterium solution and carries out viable count measurement, concentration is respectively 3.0 × 1014CCU/ml、
5.0×1014CCU/ml、7.0×1013CCU/ml and 8.0 × 1014CCU/ml。
(4) above-mentioned 1,2,4,6 F36 plants of semi-finished product bacterium solutions of generation chicken Mycoplasma synoviae are distinguished with 0.1M pH7.2PBS dilute
It releases to 4.0 × 109CCU/ml, referring to (1998) No. 38 preparation chicken Mycoplasma synoviae live vaccines of agriculture and animal husbandry letter.By 4 groups of preparation
Vaccine difference 40 age in days SPF chicken of spraying immune, 10/group, 1 plumage part/only, and set up import vaccine control group and blank control
Group, observed after immunization 14 days, and carried out within 28th attacking poison with chicken Mycoplasma synoviae after immune, it observes 14, analyses after attacking poison
Lesion situation is observed, see Table 3 for details for different culture process chicken virus mycoplasma inactivation live vaccine safeties and effect comparison result.
Table 3
There is slight immune pair anti-from table 3 it can be seen that import control chicken Mycoplasma synoviae live vaccine carries out spraying immune
It answers, immune protective rate has no immune side reaction using vaccine spraying immune prepared by present invention process, be immunized 9/10 after attacking poison
Group chicken obtains good protection.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention
Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Claims (6)
1. a kind of avian mycoplasmas culture medium, it is characterised in that: the avian mycoplasmas culture medium is by avian mycoplasmas basal medium
It is formed with auxiliary ingredients, the basal medium is conventional avian mycoplasmas fluid nutrient medium, and the auxiliary ingredients are by bright
Glue, Tween 80 and calcium chloride composition.
2. avian mycoplasmas culture medium according to claim 1, it is characterised in that: the basal medium is conventional
CM2 fluid nutrient medium, CM culture medium or improvement FreyShi culture medium in any one.
3. avian mycoplasmas culture medium according to claim 2, it is characterised in that: the auxiliary ingredients dosage is finished product training
Following group for supporting base mass volume ratio (m:V) is grouped as:
Gelatin 0.2-0.4%,
Tween 80 0.5-1%,
Calcium chloride 0.2-0.4%.
4. a kind of preparation method of avian mycoplasmas bacterium solution, it is characterised in that: production is chicken virus mycoplasma or chicken bursa synovialis with strain
Mycoplasma, bacterium solution include the following steps:
(1) by qualified avian mycoplasmas production seed, (V:V) 10% is inoculated in avian mycoplasmas culture medium by volume, sufficiently
36-37 DEG C of stationary culture after mixing is cultivated 6-8 hours；
(2) cultivation temperature is improved to 38-39 DEG C, and 50-100r/min stir culture 8-12 hours, the pH to culture medium dropped to
7.2 stop stirring, reduce cultivation temperature to 22 DEG C, then stand 2-4 hours, harvest lower layer's culture solution, and harvest yield is that culture solution is total
The 1/3-1/2 of volume；
(3) in Xiang Shangshu remaining culture liq, the 2-3 times of avian mycoplasmas culture medium being concentrated is added, step (2) is repeated and continues passage training
It supports, harvests lower layer's culture solution of total volume 1/3-1/2；According to this method continuous fed-batch cultivation 4-6 generation, avian mycoplasmas bacterium solution is made.
5. a kind of described in any item avian mycoplasmas culture mediums of claims 1 to 3 are preparing the application in avian mycoplasmas vaccine.
6. a kind of avian mycoplasmas bacterium solution as claimed in claim 4 is preparing the application in avian mycoplasmas vaccine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111304279A (en) * | 2020-03-09 | 2020-06-19 | 珠海市银科医学工程股份有限公司 | Mycoplasma identification agar culture medium and preparation method thereof |
| CN117568227A (en) * | 2023-11-29 | 2024-02-20 | 瑞普（保定）生物药业有限公司 | Mycoplasma synoviae vaccine strain, inactivated vaccine and preparation method thereof |
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