CN103182076B - Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof - Google Patents
Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of veterinary biological new medicine, and relates to a swine mycoplasma pneumoniae inactivated vaccine and a preparation method thereof. The vaccine adopts a bacterial strain with good immunogenicity, has an immunization persistent period of up to 6 months after intramuscular injection of a single dose of 2.0 ml once, and can effectively prevent the generation of swine mycoplasma pneumoniae diseases. The invention solves the problems of complex operation and difficult popularization of domestic live attenuated vaccines, and fills in the gaps of domestic independent research and development of swine mycoplasma pneumoniae inactivated vaccines.
Description
Technical field
The invention belongs to technical field of veterinary biological new medicine, relate to a kind of mycoplasmal pneumonia of swine inactivated vaccine and preparation method thereof.
Background technology
Mycoplasmal pneumonia of swine, also known as mycoplasma pneumoniae of swine or epidemic swine pneumonia, is a kind of contact chronic respiratory infectious disease caused by mycoplasma hyopneumoniae.The main clinic symptoms of ill pig is cough and asthma, and body temperature is normal, growth retardation, and feed conversion rate is low.When cuing open inspection, based on pulmonary lesion, especially with two lung lobus cardiacuses, middle leaf and sharp leaf occur that pancreas sample becomes and carnification is its feature.This disease is present in all over the world, although its mortality rate is not high, due to popular popularity, chronicity and expendable, feed conversion rate can be made to reduce, and cause the multiple intercurrent disease of pig, is one of most important disease causing pig industry economic loss.
At present, the control of this disease is improved rearing conditions, Drug therapy and vaccine immunity 3 kinds of modes.Improve the generation that rearing conditions and Drug therapy all can not eradicate this disease, and antibiotic therapy exists drug resistance problems, vaccine immunity is first-selected, effective control mode.The vaccine of domestic independent research only has attenuated live vaccines, comprise the rabbit lung freeze-dried vaccine of China Veterinery Drug Inspection Office's development, newborn rabbit muscle freeze-dried vaccine and chick embryo yolk sac freeze-dried vaccine, the mycoplasmal pneumonia of swine 168 strain live vaccine that sky, Nanjing nation and academy of agricultural sciences of Jiangsu Province develop jointly.Attenuated live vaccines due to needs pleural inoculation, so be not easy to popularize; Have Duo Jia veterinary drug company development & production mycoplasmal pneumonia of swine inactivated vaccine abroad, as Merial Limited, Schering Plough animal health company of the U.S. and Fu Dao company of the U.S. etc., all inoculated by intramuscular injection, be easy to operation, but its price is higher.
Summary of the invention
The object of the present invention is to provide a kind of mycoplasmal pneumonia of swine inactivated vaccine, this immune effect of vaccine is remarkable, mycoplasma hyopneumoniae disease effectively can be prevented to occur, filled up domestic independent research porcine mycoplasmal inactivated vaccine blank, the problem solve attenuated live vaccines complicated operation, not easily promoting.
Another object of the present invention is to provide the preparation method of this mycoplasmal pneumonia of swine inactivated vaccine.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of mycoplasmal pneumonia of swine inactivated vaccine, this vaccine strain used is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) DJ-166 strain, the preserving number of strain is CGMCC No.4545, above-mentioned strain after deactivation as aqueous phase, adjuvant is as oil phase, and aqueous phase and oil phase form by proportioning emulsifying.
Wherein, described adjuvant is mineral oil adjuvant.
Further, mineral oil adjuvant is any one in white oil, 206 adjuvants, 50V adjuvant (i.e. MontanideISA 50V adjuvant).
In mycoplasmal pneumonia of swine inactivated vaccine of the present invention, aqueous phase and oil phase carry out emulsifying by 1: 1 ~ 1: 3 volume proportions.
Further, aqueous phase and oil phase carry out emulsifying by 1: 1 volume proportion.
Vaccine every part vaccine antigen content prepared by the present invention is not less than 100ug.
Further, every part vaccine antigen content is 100 ~ 500ug.
Further, every part vaccine antigen content is 300ug.
Immunity inoculation mode of the present invention is every pig 1 intramuscular injection 2.0 milliliters, and duration of immunity is 6 months; Plant swinery repeated inoculation every half a year once.
Present invention also offers the preparation method of this mycoplasmal pneumonia of swine inactivated vaccine, its preparation process is as follows:
(1) prepare mycoplasma hyopneumoniae DJ-166 strain bacterium liquid, be cultured to bacterium liquid pH value and be down to 6.5 ~ 7.0;
A. bacterium liquid is cultivated
By 8% ~ 10% inoculation bacterium liquid cultivating base unit weight, put 37 ± 1 DEG C and cultivate 5 ~ 9, treat culture medium its colour changed into yellow, in slight haziness, pH value gathers in the crops bacterium liquid between being down to 6.50 ~ 7.00.
B. purely check
Purely check by existing " Chinese veterinary pharmacopoeia " annex, should be pure.
C.CCU measures
Bacterium liquid inoculation fluid medium will be gathered in the crops, and carry out 10 times of serial dilutions to 10
-12, put 37 ± 1 DEG C and cultivate 14, viable bacteria titre answers>=10
8cCU/mL.
(2) the bacterium liquid that step (1) obtains is carried out concentrate, purification, and carry out deactivation;
A. concentrated, purification
By bacterium liquid with 10000r/min centrifugal 30 minutes, discard supernatant, precipitation thalline PBS (pH7.2 ~ 7.4) buffer suspended, and preparation becomes stock culture volume 1/100 bacteria suspension ,-40 DEG C of preservations.
B. deactivation
The thimerosal solution adding 1.0% through concentrated bacterium liquid makes its final concentration be 0.01%, mix homogeneously, places 12 ~ 24 hours for 2 ~ 8 DEG C, period jolting 1 ~ 3 time, then ultrasonic treatment 4 ~ 6 times on ice-water bath, each 1 minute, power 250 ~ 300W, ultrasonic 5 ~ 8 seconds, interval 5 ~ 8 seconds.
(3) the bacterium liquid after deactivation is carried out the inspection of semifinished product, comprising: steriling test, deactivation inspection and determination of protein concentration;
A. deactivation inspection
Get 5.0ml inactivated bacterial liquid and be inoculated in 45ml fluid medium, put 37 DEG C and cultivate 14, observe the change of culture medium color.Set non-inactivated bacterial liquid as positive control, fluid medium is negative control simultaneously.Cultivate 5 ~ 7, take out 0.2ml and inoculate solid medium, put 5%CO
2environment, cultivates 10 for 37 DEG C.Fluid medium should change without color, and solid medium should be that deactivation is complete without colony growth.
B. steriling test
Test by existing " Chinese veterinary pharmacopoeia " annex, should without antibacterial, fungus growth.
C. determination of protein concentration
By the absorption value of UV spectrophotometer measuring sample under 260nm and 280nm wavelength, calculate: protein concentration (mg/mL)=1.45 × OD
280-0.74 × OD
260, OD
260/ OD
280< 1.4.
(4) the bacterium liquid after deactivation step (3) obtained and adjuvant proportioning emulsifying in proportion, obtain mycoplasmal pneumonia of swine inactivated vaccine.
A. aqueous phase preparation
By inactivated bacterial liquid PBS (pH7.2 ~ 7.4) buffer dilution, every part vaccine protein antigenic content should be not less than 100 μ g.
B. oil phase preparation
Mineral oil adjuvant is loaded in sterilizing bottle, 121 DEG C of sterilizings 30 minutes, for subsequent use after cooling.
C. emulsifying
Aqueous phase and oil phase are pressed the volume ratio proportioning emulsifying of 1: 1 ~ 1: 3.First oil phase is added in cutter, slowly add aqueous phase while stirring at low speed after, with 10000r/min stir 5 ~ 8 minutes, milky oil emulsion inactivated vaccine.
Beneficial effect: vaccine of the present invention adopts 1 single dose 2.0ml intramuscular injection, easy and simple to handle; This vaccine strain immunogenicity used is good, and immune duration can reach 6 months, can effectively prevent mycoplasma hyopneumoniae disease to occur.The problem that vaccine of the present invention solves domestic attenuated live vaccines complicated operation, not easily promotes, has filled up the blank of domestic independent research mycoplasmal pneumonia of swine inactivated vaccine.
Vaccine of the present invention strain mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae) DJ-166 strain used obtains from the dirty separate tissue of the sick Pulmonis Sus domestica of pig farm, Shanxi 35 age in days two-way cross for applicant, and its immunogenicity is good.Mycoplasma hyopneumoniae of the present invention (Mycoplasma hyopneumoniae) DJ-166 strain applicant was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 19th, 2011, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, be called for short CGMCC, deposit number is: CGMCC No.4545.
Accompanying drawing explanation
Fig. 1 antibody variation curve chart
Detailed description of the invention
The preparation of embodiment 1 mycoplasmal pneumonia of swine inactivated vaccine
(1) bacterium (CGMCC No.4545) liquid preparation
A. bacterium liquid is cultivated
In cultivation base unit weight 10% ratio Pigs Inoculated mycoplasma pneumoniae CGMCC No.4545 bacterium liquid, put 37 ± 1 DEG C and cultivate 5, culture medium its colour changed into yellow, in slight haziness, pH value is down to 7.00 results bacterium liquid.
B. purely check
Purely check by existing " Chinese veterinary pharmacopoeia " annex, should be pure.
C.CCU measures
Get the bacterium liquid inoculation fluid medium obtained, and carry out 10 times of serial dilutions to 10
-10, put 37 ± 1 DEG C and cultivate 14, viable bacteria titre is 10
10cCU/ml.
D. concentrated, purification
By bacterium liquid with 10000r/min centrifugal 30 minutes, discard supernatant, precipitation thalline PBS (pH7.2 ~ 7.4) buffer suspended, and preparation becomes stock culture volume 1/100 bacteria suspension ,-40 DEG C of preservations.
E. deactivation
The thimerosal solution adding 1.0% through concentrated bacterium liquid makes its final concentration be 0.01%, mix homogeneously, places 12 hours for 2 ~ 8 DEG C, period jolting 1 time, then ultrasonic treatment 4 times on ice-water bath, each 1 minute (power 250 ~ 300W, ultrasonic 5 seconds, interval 5 seconds).
(2) inspection of semifinished product
A. deactivation inspection
Get 5.0ml inactivated bacterial liquid and be inoculated in 45ml fluid medium, put 37 DEG C and cultivate 14, observe the change of culture medium color.Set non-inactivated bacterial liquid as positive control, fluid medium is negative control simultaneously.Cultivate 5 ~ 7, take out 0.2ml and inoculate solid medium, put 5%CO
2environment, cultivates 10 for 37 DEG C.Fluid medium should change without color, and solid medium should be that deactivation is complete without colony growth.
B. steriling test
Test, without antibacterial, fungus growth by existing " Chinese veterinary pharmacopoeia " annex.
C. determination of protein concentration
By the absorption value of UV spectrophotometer measuring sample under 260nm and 280nm wavelength, calculate: protein concentration (mg/mL)=1.45 × OD
280-0.74 × OD
260, OD
260/ OD
280< 1.4.
(3) preparation of oil emulsion inactivated vaccine
A. aqueous phase preparation
Inactivated bacterial liquid PBS (pH7.2 ~ 7.4) buffer is diluted to protein concentration 300 μ g/ml.
B. oil phase preparation
Injection white oil is loaded in sterilizing bottle, 121 DEG C of sterilizings 30 minutes, for subsequent use after cooling.
C. emulsifying
Water-phase component and oil-phase component are pressed the volume ratio proportioning emulsifying of 1: 1.First oil phase is added in cutter, slowly add aqueous phase while stirring at low speed after, with 10000r/min stir 5 ~ 8 minutes, milky oil emulsion inactivated vaccine.
The preparation of embodiment 2 mycoplasmal pneumonia of swine inactivated vaccine
(1) bacterium solution preparation
A. bacterium liquid is cultivated
By the 8% Pigs Inoculated mycoplasma pneumoniae CGMCC No.4545 bacterium liquid cultivating base unit weight, put 37 ± 1 DEG C and cultivate 9, treat culture medium its colour changed into yellow, in slight haziness, pH value is down to 6.50 results bacterium liquid.
B. purely check
Purely check by existing " Chinese veterinary pharmacopoeia " annex, should be pure.
C.CCU measures
Get the bacterium liquid inoculation fluid medium obtained, and carry out 10 times of serial dilutions to 10
-10, put 37 ± 1 DEG C and cultivate 14, viable bacteria titre is 10
10cCU/ml.
D. concentrate
By bacterium liquid with 10000r/min centrifugal 30 minutes, discard supernatant, precipitation thalline PBS (pH7.2 ~ 7.4) buffer suspended, and preparation becomes the bacteria suspension of stock culture volume 1/100 ,-40 DEG C of preservations.
E. deactivation
The thimerosal solution adding 1.0% through bacterium liquid that is concentrated, purification makes its final concentration be 0.01%, and mix homogeneously is placed 24 hours for 2 ~ 8 DEG C, period jolting 3 times, then ultrasonic treatment 6 times on ice-water bath, each 1 minute (power 250 ~ 300W, ultrasonic 8 seconds, interval 8 seconds).
(2) inspection of semifinished product
A. deactivation inspection
Get 5.0ml inactivated bacterial liquid and be inoculated in 45ml fluid medium, put 37 DEG C and cultivate 14, observe the change of culture medium color.Set non-inactivated bacterial liquid as positive control, fluid medium is negative control simultaneously.Cultivate 5 ~ 7, take out 0.2ml and inoculate solid medium, put 5%CO
2environment, cultivates 10 for 37 DEG C.Fluid medium should change without color, and solid medium should be that deactivation is complete without colony growth.
B. steriling test
Test by existing " Chinese veterinary pharmacopoeia " annex, should without antibacterial, fungus growth.
C. determination of protein concentration
By the absorption value of UV spectrophotometer measuring sample under 260nm and 280nm wavelength, calculate: protein concentration (mg/ml)=1.45 × OD
280-0.74 × OD
260, OD
260/ OD
280< 1.4.
(3) preparation of oil emulsion inactivated vaccine
A. aqueous phase preparation
Inactivated bacterial liquid PBS (pH7.2 ~ 7.4) buffer is diluted to protein concentration 750 μ g/ml.
B. oil phase preparation
50V adjuvant is loaded in sterilizing bottle, 121 DEG C of sterilizings 30 minutes, for subsequent use after cooling.
C. emulsifying
Water-phase component and oil-phase component are pressed the volume ratio proportioning emulsifying of 1: 2.First oil phase is added in cutter, slowly add aqueous phase while stirring at low speed after, with 10000r/min stir 5 ~ 8 minutes, milky oil emulsion inactivated vaccine.
Prepared by embodiment 3 mycoplasmal pneumonia of swine inactivated vaccine
(1) seedling bacterium solution preparation
A. bacterium liquid is cultivated
By cultivating 9% Pigs Inoculated mycoplasma pneumoniae CGMCC No.4545 bacterium liquid of base unit weight, putting 37 ± 1 DEG C and cultivating 7, culture medium its colour changed into yellow, in slight haziness, pH value is down to 6.82 results bacterium liquid.
B. purely check
Purely check by existing " Chinese veterinary pharmacopoeia " annex, should be pure.
C.CCU measures
Get the bacterium liquid inoculation fluid medium obtained, and carry out 10 times of serial dilutions to 10
-10, put 37 ± 1 DEG C and cultivate 14, viable bacteria titre is 10
11cCU/ml.
D. concentrate
By bacterium liquid with 10000r/min centrifugal 30 minutes, remove supernatant, precipitation thalline PBS (pH7.2 ~ 7.4) buffer suspended, and preparation becomes stock culture volume 1/100 bacteria suspension ,-40 DEG C of preservations.
E. deactivation
The thimerosal solution adding 1.0% through concentrated bacterium liquid makes its final concentration be 0.01%, mix homogeneously, places 18 hours for 2 ~ 8 DEG C, period jolting 2 times, then ultrasonic treatment 5 times on ice-water bath, each 1 minute (power 250 ~ 300W, ultrasonic 6 seconds, interval 6 seconds).
(2) inspection of semifinished product
A. deactivation inspection
Get 5.0ml inactivated bacterial liquid and be inoculated in 45ml fluid medium, put 37 DEG C and cultivate 14, observe the change of culture medium color.Set non-inactivated bacterial liquid as positive control, fluid medium is negative control simultaneously.Cultivate 5 ~ 7, take out 0.2ml and inoculate solid medium, put 5%CO
2environment, cultivates 10 for 37 DEG C.Fluid medium should change without color, and solid medium should be that deactivation is complete without colony growth.
B. steriling test
Test, without antibacterial, fungus growth by existing " Chinese veterinary pharmacopoeia " annex.
C. determination of protein concentration
By the absorption value of UV spectrophotometer measuring sample under 260nm and 280nm wavelength, calculate: protein concentration (mg/ml)=1.45 × OD
280-0.74 × OD
260, OD
260/ OD
280< 1.4.
(3) preparation of oil emulsion inactivated vaccine
A. aqueous phase preparation
Inactivated bacterial liquid PBS (pH7.2 ~ 7.4) buffer is diluted to protein concentration 200 μ g/ml.
B. oil phase preparation
206 adjuvants are loaded in sterilizing bottle, 121 DEG C of sterilizings 30 minutes, for subsequent use after cooling.
C. emulsifying
Water-phase component and oil-phase component are pressed the volume ratio proportioning emulsifying of 1: 3.First oil phase is added in cutter, slowly add aqueous phase while stirring at low speed after, with 10000r/min stir 5 ~ 8 minutes, milky oil emulsion inactivated vaccine.
The product inspection of embodiment 4 vaccine
3 batches of vaccines of preparation, lot number is 20100901 (prepared by embodiment 1), 20101005 (prepared by embodiment 2) and 20101106 (prepared by embodiment 3), carries out character inspection, steriling test, safety examination, efficacy test and the inspection of thimerosal residual quantity.
(1) character inspection
Vaccine outward appearance is Water-In-Oil (O/W) type milky Emulsion, and being long placed in rear upper strata has a small amount of oil, in even emulsion after jolting.With 3000r/min centrifugal 15 minutes, separate out without aqueous phase bottom pipe, vaccine stability was good.
(2) steriling test
3 batches of vaccines of preparation are tested, all without antibacterial, fungus growth by existing " Chinese veterinary pharmacopoeia " annex.
(3) safety examination
A. white mice safety testing
3 batches of vaccines each 8 of subcutaneous injection 18 ~ 22g white mice respectively of preparation, every 0.5ml, establishes matched group white mice 8 simultaneously, every subcutaneous injection normal saline 0.5ml, in observation period on the 14th, white mice is all strong to live, and vaccine is to non-target animals white mice use safety.The results are shown in Table 1.
Table 13 batches of vaccine injection white mice safety testings
B. piglet safety testing
3 batches of vaccines of preparation inject healthy each 5 of susceptible pig in 2 week age respectively with single dose (2.0ml), overdose (4.0ml) and duplicate injection dosage (2.0ml) respectively, establish normal healthy controls group pig 5 simultaneously, raise with under condition.In observation period on the 14th, matched group pig and immune group are showed no abnormal response; Body temperature is all within normal range, and the mental status is good, and searching for food and drinking water is showed no exception, and injection site has no red and swollen, scleroma, cuts open inspection injection site, immune group visible broomcorn millet grain sample tuberosity, parenchymal viscera no abnormality seen.Vaccine to target animals pig use safety, in table 2.
Table 23 batches of vaccine injection piglet clinical symptoms and injection site
Note: "-" represents no abnormality seen.
(4) efficacy test
3 batches of vaccines each 10 of intramuscular injection mycoplasma hyopneumoniae serum antibody negative healthy susceptible pig respectively of preparation, every 2.0ml, separately establishes 10 pigs in contrast, with isolated rearing under condition.Inject latter 28 days, carry out counteracting toxic substances together with condition identical contrast pig, and within 28 days after counteracting toxic substances, cut open and kill, by 28 points of scoring method record hyopneumoniae pathological changes marks, by following formulae discovery pneumonia pathological changes slip, the results are shown in Table 3.
Table 33 batches of vaccine immunity counteracting toxic substances protection results
Note: "/" represents that no this item calculates.
As can be seen from Table 3, immune group pneumonia disease to become point significantly lower than counteracting toxic substances matched group, and average pneumonia disease variability reduces 84.2%, and vaccine potency is qualified.
(5) thimerosal residual quantity inspection
3 batches of vaccines of preparation, often criticize random sampling observation 5 bottles of vaccines, and test by present " Chinese veterinary pharmacopoeia " annex, thimerosal residual quantity is all less than 0.01%, meet veterinary biologics general rule regulation.
Embodiment 5 immune duration is tested
3 batches of vaccines musculi colli injection healthy each 10 of susceptible pig in 2 week age respectively of preparation, every 2.0ml, meanwhile, if counteracting toxic substances contrast pig 10.7th, 14,28,42,2 months, 3 months, 4 months, 5 months and blood sampling in 6 months after injection, mycoplasma hyopneumoniae antibody horizontal (table 4 and Fig. 1) in serum is detected by ELISA method, and within latter 6 months, carrying out counteracting toxic substances in immunity, counteracting toxic substances the results are shown in Table 5.
Different time mean antibody levels (S/P value) after table 4 immunity
Table 5 immunity latter 6 months counteracting toxic substances results
Note: "/" represents that no this item calculates.
After vaccine immunity, serum antibody on the 14th is the positive, mycoplasma hyopneumoniae antibody S/P value is average out to 1.18,1.30 and 1.16 respectively, and immunity peaks for latter 42 days ~ 3 months, and antibody is on a declining curve afterwards, be still positive to antibody when 6 months, S/P value average out to 1.32,1.50 and 1.23; Immunity latter 6 months counteracting toxic substances, the average pneumonia disease variability of immune group decreases 71.9%.Immune group compares with matched group, and pneumonia pathological changes is significantly improved.Immune effect of vaccine is remarkable, and duration of immunity is 6 months.
Embodiment 6 compares with like product immune effect
With the import Seedling that domestic market is sold, musculi colli injection healthy each 10 of susceptible pig in 2 week age respectively, every 2.0ml, immunity carries out counteracting toxic substances in latter 28 days, and within 28 days after counteracting toxic substances, cuts open and kill, by 28 points of scoring method record hyopneumoniae pathological changes marks, by formulae discovery pneumonia pathological changes slip, the results are shown in Table 6.
Table 6 immunogenic effect compares
Note: "/" indicates without related data.
As can be seen from Table 6, this product average pneumonia pathological changes slip is respectively 86.2%, 87.1% and 82.8%, all reduces situation higher than the average pneumonia pathological changes of import vaccine.
Claims (10)
1. a mycoplasmal pneumonia of swine inactivated vaccine, it is characterized in that, strain is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) DJ-166 strain, the preserving number of strain is CGMCC No.4545, above-mentioned strain after deactivation as aqueous phase, adjuvant is as oil phase, and aqueous phase and oil phase form by proportioning emulsifying.
2. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 1, it is characterized in that, described adjuvant is mineral oil adjuvant.
3. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 2, it is characterized in that, described mineral oil adjuvant is any one in white oil, 206 adjuvants, 50V adjuvant.
4. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 1, it is characterized in that, aqueous phase and oil phase carry out emulsifying by 1: 1 ~ 1: 3 volume proportions.
5. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 4, it is characterized in that, aqueous phase and oil phase carry out emulsifying by 1: 1 volume proportion.
6. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 1, it is characterized in that, every part vaccine antigen content is not less than 100ug.
7. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 6, it is characterized in that, every part vaccine antigen content is 100 ~ 500ug.
8. mycoplasmal pneumonia of swine inactivated vaccine as claimed in claim 7, it is characterized in that, every part vaccine antigen content is 300ug.
9. a preparation method for the mycoplasmal pneumonia of swine inactivated vaccine described in any one of claim 1-8, it is characterized in that, step is as follows:
(1) prepare mycoplasma hyopneumoniae DJ-166 strain bacterium liquid, be cultured to bacterium liquid pH value and be down to 6.5 ~ 7.0;
(2) the bacterium liquid that step (1) obtains is carried out concentrate, purification, and carry out deactivation;
(3) the bacterium liquid after deactivation is carried out the inspection of semifinished product, comprising: steriling test, deactivation is examined
Test and determination of protein concentration;
(4) the bacterium liquid after deactivation step (3) obtained and adjuvant proportioning emulsifying in proportion, obtain mycoplasmal pneumonia of swine inactivated vaccine.
10. preparation method as claimed in claim 9, it is characterized in that, deactivation mode be concentrate, antigen after purification adds final concentration 0.01% thimerosal solution, place 12 ~ 24 hours for 2 ~ 8 DEG C after mixing, period jolting 1 ~ 3 time, then ultrasonic treatment 4 ~ 6 times on ice-water bath, each 1 minute, power 250 ~ 300W, ultrasonic 5 ~ 8 seconds, interval 5 ~ 8 seconds.
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| CN103585622B (en) * | 2013-11-25 | 2015-09-02 | 江苏省农业科学院 | The application of Vaccine strain for mycoplasma pneumonia of swine |
| CN104324370B (en) * | 2014-09-30 | 2019-12-20 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
| CN104399070B (en) * | 2014-10-31 | 2016-09-07 | 哈药集团生物疫苗有限公司 | Porcine mycoplasmal pneumonia inactivated vaccine and preparation method thereof |
| CN104940918B (en) * | 2015-05-15 | 2018-03-16 | 北京中海生物科技有限公司 | A kind of production method of porcine mycoplasmal pneumonia inactivated vaccine |
| CN104894009B (en) * | 2015-05-15 | 2018-02-09 | 北京中海生物科技有限公司 | One plant of mycoplasma hyopneumoniae strain and its application |
| CN108392628A (en) * | 2018-06-01 | 2018-08-14 | 北京万牧源农业科技有限公司 | A kind of porcine mycoplasmal pneumonia inactivated vaccine and preparation method thereof |
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