CN103182076A - Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof - Google Patents
Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN103182076A CN103182076A CN2011104521536A CN201110452153A CN103182076A CN 103182076 A CN103182076 A CN 103182076A CN 2011104521536 A CN2011104521536 A CN 2011104521536A CN 201110452153 A CN201110452153 A CN 201110452153A CN 103182076 A CN103182076 A CN 103182076A
- Authority
- CN
- China
- Prior art keywords
- vaccine
- mycoplasmal pneumonia
- swine
- weigh
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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 the biological new medicine technical field of veterinary, relate to a kind of mycoplasmal pneumonia of swine inactivated vaccine and preparation method thereof.
Background technology
Mycoplasmal pneumonia of swine claims mycoplasma pneumoniae of swine or epidemic swine pneumonia again, is a kind of contact chronic respiratory infectious disease that is caused by mycoplasma hyopneumoniae.The main clinic symptoms of ill pig is cough and asthma, and body temperature is normal substantially, growth retardation, and feed conversion rate is low.When cuing open inspection, based on pulmonary lesion, especially with two lung lobus cardiacuses, the change of pancreas sample appears in middle leaf and sharp leaf and carnification is its feature.This disease is present in all over the world, though its mortality rate is not high, because popular popularity, chronicity and expendable can make feed conversion rate reduce, and cause the multiple intercurrent disease of pig, is one of most important disease that causes the pig industry economic loss.
At present, the control of this disease has the raising of improvement condition, Drug therapy and 3 kinds of modes of vaccine immunity.Improve raising condition and Drug therapy and all can not eradicate the generation of this disease, and there is the drug resistance problem in antibiotic therapy, vaccine immunity is first-selected, effective control mode.The vaccine of domestic independent research has only attenuated live vaccines, the rabbit lung freeze-dried vaccine, newborn rabbit muscle freeze-dried vaccine and the chick embryo yolk sac freeze-dried vaccine that comprise China Veterinery Drug Inspection Office's development, the mycoplasmal pneumonia of swine 168 strain live vaccine of the common development in sky, Nanjing nation and academy of agricultural sciences, Jiangsu Province.Attenuated live vaccines is owing to needing pleural inoculation, so be not easy to popularize; External existing how tame veterinary drug company researches and develops and has produced the mycoplasmal pneumonia of swine inactivated vaccine, as Merial Limited, U.S. Schering Plough animal health company and U.S. Fu Dao company etc., all inoculates by intramuscular injection, and easy operating, 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, can effectively prevent the mycoplasma hyopneumoniae disease to take place, fill up domestic independent research porcine mycoplasmal inactivated vaccine blank, solve attenuated live vaccines complicated operation, the difficult problem of promoting.
Another object of the present invention provides the preparation method of this mycoplasmal pneumonia of swine inactivated vaccine.
The objective of the invention is to be achieved through the following technical solutions:
The invention provides a kind of mycoplasmal pneumonia of swine inactivated vaccine, the used strain of this vaccine is mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae) DJ-166 strain, the preserving number of strain is CGMCC No.4545, above-mentioned strain after deactivation as water, adjuvant is as oil phase, and water and oil phase form by proportioning emulsifying.
Wherein, described adjuvant is the mineral oil adjuvant.
Further, the mineral oil adjuvant is any in white oil, 206 adjuvants, the 50V adjuvant (being MontanideISA 50V adjuvant).
Water and oil phase carry out emulsifying by 1: 1～1: 3 volume proportion in the mycoplasmal pneumonia of swine inactivated vaccine of the present invention.
Further, water and oil phase carry out emulsifying by 1: 1 volume proportion.
Every part vaccine antigen of vaccine content of the present invention's preparation 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 2.0 milliliters of 1 intramuscular injection of every pig, and duration of immunity is 6 months; Plant swinery repeated inoculation every half a year once.
The present invention also provides the preparation method of this mycoplasmal pneumonia of swine inactivated vaccine, and its preparation process is as follows:
(1) preparation mycoplasma hyopneumoniae DJ-166 strain bacterium liquid is cultured to bacterium liquid pH value and is down to 6.5～7.0;
A. bacterium liquid is cultivated
By 8%～10% inoculation bacterium liquid of cultivating base unit weight, put 37 ± 1 ℃ and cultivated 5～9, treat the culture medium its colour changed into yellow, be slight muddiness, results bacterium liquid between pH value is down to 6.50～7.00.
B. check purely
Check purely by existing " Chinese veterinary drug allusion quotation " appendix, should be pure.
C.CCU measures
To gather in the crops bacterium liquid inoculation fluid medium, and carry out 10 times of serial dilutions to 10
-12, to put 37 ± 1 ℃ and cultivated 14, the viable bacteria titre answers 〉=10
8CCU/mL.
(2) the bacterium liquid that step (1) is obtained concentrate, purification, and carry out deactivation;
A. concentrate, purification
With bacterium liquid with 10000r/min centrifugal 30 minutes, discard supernatant, the precipitation thalline suspends with PBS (pH7.2～7.4) buffer, and preparation becomes stock culture volume 1/100 bacteria suspension ,-40 ℃ of preservations.
B. deactivation
It is 0.01% that the thimerosal solution of the bacterium liquid adding 1.0% through concentrating makes its final concentration, and mix homogeneously was placed 12～24 hours for 2～8 ℃, jolting during this time 1～3 time, ultrasonic treatment 4～6 times on ice-water bath then, each 1 minute, power 250～300W, ultrasonic 5～8 seconds, intermittently 5～8 seconds.
(3) the bacterium liquid after the deactivation is carried out the inspection of semifinished product, comprising: steriling test, deactivation check and determination of protein concentration;
A. deactivation check
Get the 5.0ml inactivated bacterial liquid and be inoculated in the 45ml fluid medium, put 37 ℃ and cultivated 14, observe the culture medium change color.Establish the not positive contrast of inactivated bacterial liquid simultaneously, the negative contrast of fluid medium.Cultivated 5～7, and took out 0.2ml inoculation solid medium, put 5%CO
2Environment was cultivated 10 for 37 ℃.Fluid medium should not have change color, should asepticly fall to being grown to deactivation complete on the solid medium.
B. steriling test
Test by existing " Chinese veterinary drug allusion quotation " appendix, should not have antibacterial, fungus growth.
C. determination of protein concentration
With 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) bacterium liquid and the adjuvant proportioning emulsifying in proportion after the deactivation that step (3) is obtained obtains the mycoplasmal pneumonia of swine inactivated vaccine.
A. water preparation
Inactivated bacterial liquid is diluted with PBS (pH7.2～7.4) buffer, and every part vaccine protein antigenic content should be not less than 100 μ g.
B. oil phase preparation
The mineral oil adjuvant is packed in the sterilization bottle, and 121 ℃ of sterilizations 30 minutes, the cooling back was standby.
C. emulsifying
With the volume ratio proportioning emulsifying by 1: 1～1: 3 of water and oil phase.Earlier oil phase is added in the cutter, in stirring at low speed, slowly add water after, stirred 5～8 minutes with 10000r/min, the milky oil emulsion inactivated vaccine.
Beneficial effect: vaccine of the present invention adopts 1 single dose 2.0ml intramuscular injection, and is easy and simple to handle; The used strain immunogenicity of this vaccine is good, and immune duration can reach 6 months, can effectively prevent the mycoplasma hyopneumoniae disease to take place.Vaccine of the present invention has solved domestic attenuated live vaccines complicated operation, has been difficult for the problem of popularization, has filled up the blank of domestic independent research mycoplasmal pneumonia of swine inactivated vaccine.
The dirty separate tissue of the sick Pulmonis Sus domestica of 35 age in days two-way cross obtains used strain mycoplasma hyopneumoniae (Mycoplasmahyopneumoniae) the DJ-166 strain of vaccine of the present invention from the pig farm, Shanxi for the applicant, and its immunogenicity is good.Mycoplasma hyopneumoniae of the present invention (Mycoplasma hyopneumoniae) DJ-166 strain applicant has been preserved in China Committee for Culture Collection of Microorganisms 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.
Description of drawings
Fig. 1 antibody its growth figure
The specific embodiment
The preparation of embodiment 1 mycoplasmal pneumonia of swine inactivated vaccine
(1) bacterium (CGMCC No.4545) liquid preparation
A. bacterium liquid is cultivated
In cultivating base unit weight 10% ratio inoculation mycoplasma hyopneumoniae CGMCC No.4545 bacterium liquid, put 37 ± 1 ℃ and cultivated 5, the culture medium its colour changed into yellow, be slight muddiness, pH value is down to 7.00 results bacterium liquid.
B. check purely
Check purely by existing " Chinese veterinary drug allusion quotation " appendix, should be pure.
C.CCU measures
Get the bacterium liquid inoculation fluid medium that obtains, and carry out 10 times of serial dilutions to 10
-10, to put 37 ± 1 ℃ and cultivated 14, the viable bacteria titre is 10
10CCU/ml.
D. concentrate, purification
With bacterium liquid with 10000r/min centrifugal 30 minutes, discard supernatant, the precipitation thalline suspends with PBS (pH7.2～7.4) buffer, and preparation becomes stock culture volume 1/100 bacteria suspension ,-40 ℃ of preservations.
E. deactivation
It is 0.01% that the thimerosal solution of the bacterium liquid adding 1.0% through concentrating makes its final concentration, and mix homogeneously was placed 12 hours for 2～8 ℃, jolting during this time 1 time, ultrasonic treatment 4 times on ice-water bath then, each 1 minute (power 250～300W, ultrasonic 5 seconds, intermittently 5 seconds).
(2) inspection of semifinished product
A. deactivation check
Get the 5.0ml inactivated bacterial liquid and be inoculated in the 45ml fluid medium, put 37 ℃ and cultivated 14, observe the culture medium change color.Establish the not positive contrast of inactivated bacterial liquid simultaneously, the negative contrast of fluid medium.Cultivated 5～7, and took out 0.2ml inoculation solid medium, put 5%CO
2Environment was cultivated 10 for 37 ℃.Fluid medium should not have change color, should asepticly fall to being grown to deactivation complete on the solid medium.
B. steriling test
Test no antibacterial, fungus growth by existing " Chinese veterinary drug allusion quotation " appendix.
C. determination of protein concentration
With 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. water preparation
Inactivated bacterial liquid is diluted to protein concentration 300 μ g/ml with PBS (pH7.2～7.4) buffer.
B. oil phase preparation
The injection white oil is packed in the sterilization bottle, and 121 ℃ of sterilizations 30 minutes, the cooling back was standby.
C. emulsifying
With the volume ratio proportioning emulsifying by 1: 1 of water-phase component and oil-phase component.Earlier oil phase is added in the cutter, in stirring at low speed, slowly add water after, stirred 5～8 minutes with 10000r/min, the milky oil emulsion inactivated vaccine.
The preparation of embodiment 2 mycoplasmal pneumonia of swine inactivated vaccines
(1) bacterium liquid preparation
A. bacterium liquid is cultivated
By the 8% inoculation mycoplasma hyopneumoniae CGMCC No.4545 bacterium liquid of cultivating base unit weight, put 37 ± 1 ℃ and cultivated 9, to treat the culture medium its colour changed into yellow, be slight muddiness, pH value is down to 6.50 results bacterium liquid.
B. check purely
Check purely by existing " Chinese veterinary drug allusion quotation " appendix, should be pure.
C.CCU measures
Get the bacterium liquid inoculation fluid medium that obtains, and carry out 10 times of serial dilutions to 10
-10, to put 37 ± 1 ℃ and cultivated 14, the viable bacteria titre is 10
10CCU/ml.
D. concentrate
With bacterium liquid with 10000r/min centrifugal 30 minutes, discard supernatant, the precipitation thalline suspends with PBS (pH7.2～7.4) buffer, and preparation becomes the bacteria suspension of stock culture volume 1/100 ,-40 ℃ of preservations.
E. deactivation
Through concentrate, the bacterium liquid of purification adds 1.0% thimerosal solution to make its final concentration is 0.01%, mix homogeneously was placed 24 hours for 2～8 ℃, jolting during this time 3 times, ultrasonic treatment 6 times on ice-water bath then, each 1 minute (power 250～300W, ultrasonic 8 seconds, intermittently 8 seconds).
(2) inspection of semifinished product
A. deactivation check
Get the 5.0ml inactivated bacterial liquid and be inoculated in the 45ml fluid medium, put 37 ℃ and cultivated 14, observe the culture medium change color.Establish the not positive contrast of inactivated bacterial liquid simultaneously, the negative contrast of fluid medium.Cultivated 5～7, and took out 0.2ml inoculation solid medium, put 5%CO
2Environment was cultivated 10 for 37 ℃.Fluid medium should not have change color, should asepticly fall to being grown to deactivation complete on the solid medium.
B. steriling test
Test by existing " Chinese veterinary drug allusion quotation " appendix, should not have antibacterial, fungus growth.
C. determination of protein concentration
With 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. water preparation
Inactivated bacterial liquid is diluted to protein concentration 750 μ g/ml with PBS (pH7.2～7.4) buffer.
B. oil phase preparation
The 50V adjuvant is packed in the sterilization bottle, and 121 ℃ of sterilizations 30 minutes, the cooling back was standby.
C. emulsifying
With the volume ratio proportioning emulsifying by 1: 2 of water-phase component and oil-phase component.Earlier oil phase is added in the cutter, in stirring at low speed, slowly add water after, stirred 5～8 minutes with 10000r/min, the milky oil emulsion inactivated vaccine.
The preparation of embodiment 3 mycoplasmal pneumonia of swine inactivated vaccines
(1) seedling prepares with bacterium liquid
A. bacterium liquid is cultivated
By the 9% inoculation mycoplasma hyopneumoniae CGMCC No.4545 bacterium liquid of cultivating base unit weight, put 37 ± 1 ℃ and cultivated 7, the culture medium its colour changed into yellow, be slight muddiness, pH value is down to 6.82 results bacterium liquid.
B. check purely
Check purely by existing " Chinese veterinary drug allusion quotation " appendix, should be pure.
C.CCU measures
Get the bacterium liquid inoculation fluid medium that obtains, and carry out 10 times of serial dilutions to 10
-10, to put 37 ± 1 ℃ and cultivated 14, the viable bacteria titre is 10
11CCU/ml.
D. concentrate
With bacterium liquid with 10000r/min centrifugal 30 minutes, remove supernatant, the precipitation thalline suspends with PBS (pH7.2～7.4) buffer, and preparation becomes stock culture volume 1/100 bacteria suspension ,-40 ℃ of preservations.
E. deactivation
It is 0.01% that the thimerosal solution of the bacterium liquid adding 1.0% through concentrating makes its final concentration, and mix homogeneously was placed 18 hours for 2～8 ℃, jolting during this time 2 times, ultrasonic treatment 5 times on ice-water bath then, each 1 minute (power 250～300W, ultrasonic 6 seconds, intermittently 6 seconds).
(2) inspection of semifinished product
A. deactivation check
Get the 5.0ml inactivated bacterial liquid and be inoculated in the 45ml fluid medium, put 37 ℃ and cultivated 14, observe the culture medium change color.Establish the not positive contrast of inactivated bacterial liquid simultaneously, the negative contrast of fluid medium.Cultivated 5～7, and took out 0.2ml inoculation solid medium, put 5%CO
2Environment was cultivated 10 for 37 ℃.Fluid medium should not have change color, should asepticly fall to being grown to deactivation complete on the solid medium.
B. steriling test
Test no antibacterial, fungus growth by existing " Chinese veterinary drug allusion quotation " appendix.
C. determination of protein concentration
With 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. water preparation
Inactivated bacterial liquid is diluted to protein concentration 200 μ g/ml with PBS (pH7.2～7.4) buffer.
B. oil phase preparation
206 adjuvants are packed in the sterilization bottle, and 121 ℃ of sterilizations 30 minutes, the cooling back was standby.
C. emulsifying
With the volume ratio proportioning emulsifying by 1: 3 of water-phase component and oil-phase component.Earlier oil phase is added in the cutter, in stirring at low speed, slowly add water after, stirred 5～8 minutes with 10000r/min, the milky oil emulsion inactivated vaccine.
Embodiment 4 vaccine product inspections
3 batches of vaccines of preparation, lot number are 20100901 (embodiment 1 preparations), 20101005 (embodiment 2 preparations) and 20101106 (embodiment 3 preparations), carry out character check, steriling test, safety examination, efficacy test and the check of thimerosal residual quantity.
(1) character check
The vaccine outward appearance is Water-In-Oil (O/W) type milky Emulsion, and there is a small amount of oil on the postpone upper strata for a long time, is even emulsion after the jolting.With 3000r/min centrifugal 15 minutes, the no water in pipe bottom was separated out, and vaccine stability is good.
(2) steriling test
3 batches of vaccines of preparation are tested by existing " Chinese veterinary drug allusion quotation " appendix, all do not have antibacterial, fungus growth.
(3) safety examination
A. white mice safety testing
Each 8 of 3 batches of vaccines difference subcutaneous injection 18～22g white mice of preparation, every 0.5ml establishes 8 of matched group white mice simultaneously, every subcutaneous injection normal saline 0.5ml, in observation period on the 14th, white mice is all strong alive, and vaccine uses safety to the non-target animals white mice.The results are shown in Table 1.
3 batches of vaccine injection white mice of table 1 safety testing
B. piglet safety testing
3 batches of vaccines of preparation are injected respectively 5 of healthy susceptible pigs in 2 ages in week respectively with single dose (2.0ml), overdose (4.0ml) and duplicate injection dosage (2.0ml) respectively, establish 5 of normal healthy controls group pigs simultaneously, raise with under the condition.In observation period on the 14th, matched group pig and immune group there is no abnormal response; All in normal range, the mental status is good for body temperature, searches for food and drink water to there is no unusually, and not swollen, the scleroma of show of injection site is cutd open the inspection injection site, the visible broomcorn millet grain of immune group sample tuberosity, parenchymal viscera no abnormality seen.Vaccine uses safety to the target animals pig, sees Table 2.
Table 23 batches of vaccine injection piglets clinical symptoms and injection site
Annotate: "-" expression no abnormality seen.
(4) efficacy test
Each 10 of 3 batches of negative healthy susceptible pigs of vaccines difference intramuscular injection mycoplasma hyopneumoniae serum antibody of preparation, every 2.0ml, other establishes 10 pigs in contrast, with isolated rearing under the condition.Injected back 28 days, and carried out counteracting toxic substances together with the identical contrast of condition pig, and behind counteracting toxic substances, cutd open in 28th and kill, by 28 fens scoring method record hyopneumoniae pathological changes marks, calculate pneumonia pathological changes slip by following formula, the results are shown in Table 3.
3 batches of vaccine immunity counteracting toxic substances protections of table 3 result
Annotate: "/" expression no this item is calculated.
As can be seen from Table 3, the immune group pneumonia disease branch that becomes significantly is lower than the counteracting toxic substances matched group, and average pneumonia disease variability reduces 84.2%, and vaccine potency is qualified.
(5) thimerosal residual quantity check
3 batches of vaccines of preparation are inspected 5 bottles of vaccines by random samples for every batch at random, test by present " Chinese veterinary drug allusion quotation " appendix, and the thimerosal residual quantity meets veterinary biologics general rule regulation all less than 0.01%.
The test of embodiment 5 immune durations
Each 10 of 3 batches of vaccines difference musculi collis injection health susceptible pigs in 2 ages in week of preparation, every 2.0ml simultaneously, establishes counteracting toxic substances and contrasts 10 of pigs.Injection back the 7th, 14,28,42,2 months, 3 months, 4 months, 5 months and blood sampling in 6 months, detect mycoplasma hyopneumoniae antibody horizontal (table 4 and Fig. 1) in the serum with the ELISA method, and carrying out counteracting toxic substances in back 6 months in immunity, counteracting toxic substances the results are shown in Table 5.
Table 4 immunity back different time average antibody level (S/P value)
Back 6 months counteracting toxic substances results of table 5 immunity
Annotate: "/" expression no this item is calculated.
Serum antibody on the 14th is all positive behind the vaccine immunity, mycoplasma hyopneumoniae antibody S/P value is average out to 1.18,1.30 and 1.16 respectively, and immunity peaked in back 42 days～3 months, and antibody is on a declining curve afterwards, antibody is still positive during to 6 months, S/P value average out to 1.32,1.50 and 1.23; Back 6 months counteracting toxic substances of immunity, the average pneumonia disease variability of immune group has reduced 71.9%.Immune group and matched group compare, and the pneumonia pathological changes is significantly improved.Immune effect of vaccine is remarkable, and duration of immunity is 6 months.
Embodiment 6 compares with the like product immune effect
With the import Seedling of selling on the domestic market, musculi colli injection healthy susceptible pig in 2 age in week is each 10 respectively, every 2.0ml, immunity was carried out counteracting toxic substances in back 28 days, and cutd open in 28th behind counteracting toxic substances and kill, by 28 fens scoring method record hyopneumoniae pathological changes marks, by formula calculate pneumonia pathological changes slip, the results are shown in Table 6.
Table 6 immunogenicity effect relatively
Annotate: the no related data of "/" expression.
As can be seen from Table 6, the average pneumonia pathological changes of this product slip is respectively 86.2%, 87.1% and 82.8%, all is higher than the average pneumonia pathological changes of import vaccine and reduces situation.
Claims (10)
1. 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 CGMCCNo.4545, above-mentioned strain after deactivation as water, adjuvant is as oil phase, and water and oil phase form by proportioning emulsifying.
2. weigh 1 described mycoplasmal pneumonia of swine inactivated vaccine, it is characterized in that described adjuvant is the mineral oil adjuvant.
3. weigh 2 described mycoplasmal pneumonia of swine inactivated vaccines, it is characterized in that described mineral oil adjuvant is any in white oil, 206 adjuvants, the 50V adjuvant.
4. weigh 1 described mycoplasmal pneumonia of swine inactivated vaccine, it is characterized in that water and oil phase carry out emulsifying by 1: 1～1: 3 volume proportion.
5. weigh 4 described mycoplasmal pneumonia of swine inactivated vaccines, it is characterized in that water and oil phase carry out emulsifying by 1: 1 volume proportion.
6. weigh 1 described mycoplasmal pneumonia of swine inactivated vaccine, it is characterized in that every part vaccine antigen content is not less than 100ug.
7. weigh 6 described mycoplasmal pneumonia of swine inactivated vaccines, it is characterized in that every part vaccine antigen content is 100～500ug.
8. weigh 7 described mycoplasmal pneumonia of swine inactivated vaccines, it is characterized in that every part vaccine antigen content is 300ug.
9. preparation method as each described mycoplasmal pneumonia of swine inactivated vaccine of claim 1-8 is characterized in that step is as follows:
(1) preparation mycoplasma hyopneumoniae DJ-166 strain bacterium liquid is cultured to bacterium liquid pH value and is down to 6.5～7.0;
(2) the bacterium liquid that step (1) is obtained concentrate, purification, and carry out deactivation;
(3) the bacterium liquid after the deactivation is carried out the inspection of semifinished product, comprising: steriling test, deactivation check and determination of protein concentration;
(4) bacterium liquid and the adjuvant proportioning emulsifying in proportion after the deactivation that step (3) is obtained obtains the mycoplasmal pneumonia of swine inactivated vaccine.
10. weigh 9 described preparation methoies, it is characterized in that, the deactivation mode for concentrate, antigen behind the purification adds final concentration 0.01% thimerosal solution, placed 12～24 hours for 2～8 ℃ behind the mixing, during jolting 1～3 time, ultrasonic treatment 4～6 times on ice-water bath then, each 1 minute, power 250～300W, ultrasonic 5～8 seconds, intermittently 5～8 seconds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110452153.6A CN103182076B (en) | 2011-12-29 | 2011-12-29 | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110452153.6A CN103182076B (en) | 2011-12-29 | 2011-12-29 | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103182076A true CN103182076A (en) | 2013-07-03 |
| CN103182076B CN103182076B (en) | 2015-06-17 |
Family
ID=48673690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110452153.6A Active CN103182076B (en) | 2011-12-29 | 2011-12-29 | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103182076B (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103585622A (en) * | 2013-11-25 | 2014-02-19 | 江苏省农业科学院 | Application of swine mycoplasma pneumonia vaccine strain |
| CN103602637A (en) * | 2013-11-25 | 2014-02-26 | 江苏省农业科学院 | Vaccine strain for mycoplasma pneumonia of swine |
| CN104324370A (en) * | 2014-09-30 | 2015-02-04 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
| CN104399070A (en) * | 2014-10-31 | 2015-03-11 | 哈药集团生物疫苗有限公司 | Swine mycoplasma pneumonia inactivated vaccine and preparation method thereof |
| CN104894009A (en) * | 2015-05-15 | 2015-09-09 | 北京中海生物科技有限公司 | Mycoplasma hyopneumoniae strain and application thereof |
| CN104940918A (en) * | 2015-05-15 | 2015-09-30 | 北京中海生物科技有限公司 | Production method of swine mycoplasma pneumonia inactivated vaccine |
| 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 |
| CN111707822A (en) * | 2020-08-20 | 2020-09-25 | 兆丰华生物科技(南京)有限公司 | Mycoplasma gallisepticum antibody detection reagent and preparation method and application thereof |
| CN112294953A (en) * | 2020-12-31 | 2021-02-02 | 北京科牧丰生物制药有限公司 | PCV2 type baculovirus vector, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine and preparation method thereof |
| CN112316131A (en) * | 2020-12-31 | 2021-02-05 | 北京科牧丰生物制药有限公司 | PCV2 type baculovirus and mycoplasma hyopneumoniae bivalent inactivated vaccine and preparation method thereof |
| CN117305192A (en) * | 2023-12-01 | 2023-12-29 | 北京瑞阳瑞泰生物科技有限公司 | Mycoplasma hyopneumoniae RT02 strain, vaccine composition and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991018627A1 (en) * | 1990-05-29 | 1991-12-12 | Smithkline Beecham Corporation | Swine pneumonia vaccine and method for the preparation thereof |
| WO1992003157A1 (en) * | 1990-08-16 | 1992-03-05 | Solvay Animal Health, Inc. | Inactivated mycoplasma hyopneumoniae bacterin and method of use thereof |
| CN1852735A (en) * | 2003-07-24 | 2006-10-25 | 梅瑞尔有限公司 | Vaccine formulations comprising an oil-in-water emulsion |
| CN101448521A (en) * | 2006-03-03 | 2009-06-03 | 梅瑞尔有限公司 | Mycoplasma hyopneumoniae vaccine |
| CN102258776A (en) * | 2011-07-07 | 2011-11-30 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof |
-
2011
- 2011-12-29 CN CN201110452153.6A patent/CN103182076B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991018627A1 (en) * | 1990-05-29 | 1991-12-12 | Smithkline Beecham Corporation | Swine pneumonia vaccine and method for the preparation thereof |
| WO1992003157A1 (en) * | 1990-08-16 | 1992-03-05 | Solvay Animal Health, Inc. | Inactivated mycoplasma hyopneumoniae bacterin and method of use thereof |
| CN1852735A (en) * | 2003-07-24 | 2006-10-25 | 梅瑞尔有限公司 | Vaccine formulations comprising an oil-in-water emulsion |
| CN101448521A (en) * | 2006-03-03 | 2009-06-03 | 梅瑞尔有限公司 | Mycoplasma hyopneumoniae vaccine |
| CN102258776A (en) * | 2011-07-07 | 2011-11-30 | 普莱柯生物工程股份有限公司 | Combined inactivated vaccine against mycoplasma hyopneumoniae (MHP) and mycoplasma hyorhinis and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| KOBISCH M,ET AL: "The Mycoplasma hyopneumoniae plasma membrane as a vaccine against porcine enzootic pneumonia", 《ANN INST PASTEUR IMMUNOL》 * |
| SELWYN A.WILSON DAVID，ET AL: "Evaluation of Mycoplasma Inactivation during Production of Biologics:Egg-Based Viral Vaccines as a Model", 《APPLED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103585622A (en) * | 2013-11-25 | 2014-02-19 | 江苏省农业科学院 | Application of swine mycoplasma pneumonia vaccine strain |
| CN103602637A (en) * | 2013-11-25 | 2014-02-26 | 江苏省农业科学院 | Vaccine strain for mycoplasma pneumonia of swine |
| 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 |
| CN104324370A (en) * | 2014-09-30 | 2015-02-04 | 普莱柯生物工程股份有限公司 | Vaccine composition, preparation method and application thereof |
| CN104399070A (en) * | 2014-10-31 | 2015-03-11 | 哈药集团生物疫苗有限公司 | Swine mycoplasma pneumonia inactivated vaccine and preparation method 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 |
| CN104940918A (en) * | 2015-05-15 | 2015-09-30 | 北京中海生物科技有限公司 | Production method of swine mycoplasma pneumonia inactivated vaccine |
| CN104894009A (en) * | 2015-05-15 | 2015-09-09 | 北京中海生物科技有限公司 | Mycoplasma hyopneumoniae strain and application thereof |
| 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 |
| CN111707822A (en) * | 2020-08-20 | 2020-09-25 | 兆丰华生物科技(南京)有限公司 | Mycoplasma gallisepticum antibody detection reagent and preparation method and application thereof |
| CN112294953A (en) * | 2020-12-31 | 2021-02-02 | 北京科牧丰生物制药有限公司 | PCV2 type baculovirus vector, mycoplasma hyopneumoniae and haemophilus parasuis triple inactivated vaccine and preparation method thereof |
| CN112316131A (en) * | 2020-12-31 | 2021-02-05 | 北京科牧丰生物制药有限公司 | PCV2 type baculovirus and mycoplasma hyopneumoniae bivalent inactivated vaccine and preparation method thereof |
| CN117305192A (en) * | 2023-12-01 | 2023-12-29 | 北京瑞阳瑞泰生物科技有限公司 | Mycoplasma hyopneumoniae RT02 strain, vaccine composition and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103182076B (en) | 2015-06-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103182076B (en) | Swine mycoplasma pneumoniae inactivated vaccine and preparation method thereof | |
| CN103263666B (en) | Porcine circovirus 2 type, porcine mycoplasmal pneumonia bivalent inactivated vaccine and preparation method thereof | |
| CN102499982B (en) | Method for producing trivalent inactivated vaccine against Haemophilus parasuis infection | |
| CN103585626B (en) | Preparation method for newcastle disease and infectious bursal disease bigeminal composite inactivated vaccine | |
| CN102949714B (en) | Swine Streptococcosis trivalent inactivated vaccine and preparation method thereof | |
| CN111500482B (en) | Clostridium perfringens type A strain of sheep, inactivated vaccine and vaccine preparation method | |
| CN101081866A (en) | Domestic animal and aquatic product causal agent resistant specific IgY or compound IgY and application thereof | |
| CN106497890A (en) | A kind of 1 plant of porcine pseudorabies virus variant XF and preparation method and application | |
| CN102294026A (en) | Milk cow streptococcal mastitis inactivated vaccine and preparation method thereof | |
| CN102125687B (en) | Production method for bivalent inactivated vaccine for infectious serositis of ducks | |
| CN104888209B (en) | A kind of B groups of epidemic meningitises coccus recombinant protein vaccine and preparation method thereof | |
| CN104163858B (en) | Pasteurella multocida acellular antigen, preparation method and applications thereof | |
| CN106834168B (en) | A kind of streptococcus suis 2-type low virulent strain and its application | |
| CN102600463A (en) | Production method of trivalent inactivated vaccine preventing duck infectious serositis | |
| CN106190991B (en) | A kind of avian encephalomyclitis virus, inactivated vaccine and preparation method thereof | |
| CN109554420B (en) | Clostridium perfringens type B exotoxin and preparation method, toxin production medium and application thereof | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN101507816A (en) | Riemerlla anatipestifer bivalent inactivated vaccine and preparation method thereof | |
| CN103495162B (en) | Preparation method of porcine reproductive and respiratory syndrome compound inactivated vaccine | |
| CN103893760B (en) | Anti- garget drug-fast bacteria IgY and compound bacteriophage composition and preparation method thereof and preparation | |
| CN102139104A (en) | Production method for triple inactivated vaccine for newcastle disease, avian influenza (H9 subtype) and infectious bursal disease | |
| CN105713855A (en) | Strains, application of strains, vaccine and preparation method of vaccine | |
| CN103316334B (en) | Infectious bursal disease live vaccine and production method thereof | |
| CN109106946B (en) | Inactivated staphylococcus aureus vaccine for dairy cow mastitis and preparation method thereof | |
| CN104854234B (en) | Leptospira with increased amount of antigen |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 100080 Beijing City, Haidian District Zhongguancun street, No. 27 Zhongguancun building 14 DaBeiNong group Co-patentee after: Zhaofenghua Biotechnology (Fuzhou) Co.,Ltd. Patentee after: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. Address before: 100080 Beijing City, Haidian District Zhongguancun street, No. 27 Zhongguancun building 14 DaBeiNong group Co-patentee before: FUZHOU DA BEI NONG BIOTECH Co.,Ltd. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200820 Address after: 211102, 999 Ting Dong Road, Jiangning economic and Technological Development Zone, Nanjing, Jiangsu Co-patentee after: Zhaofenghua Biotechnology (Fuzhou) Co.,Ltd. Patentee after: Zhaofenghua Biotechnology (Nanjing) Co.,Ltd. Address before: 100080 Beijing City, Haidian District Zhongguancun street, No. 27 Zhongguancun building 14 DaBeiNong group Co-patentee before: Zhaofenghua Biotechnology (Fuzhou) Co.,Ltd. Patentee before: BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |