CN108392628A - A kind of porcine mycoplasmal pneumonia inactivated vaccine and preparation method thereof - Google Patents
A kind of porcine mycoplasmal pneumonia inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of production methods of porcine mycoplasmal pneumonia inactivated vaccine.The present invention is to make production bacterial strain with being separated to one plant and exempt from ZY plants of the mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae Mhp) that virulence is strong, epidemic focus is good, using efficient culture medium, suitable cultural method, inactivation technology and suitable immunologic adjuvant, mycoplasma hyopneumoniae inactivated vaccine is prepared.Inactivation antigen content CCU is inactivated in vaccine prepared by the present invention is not less than 1.5 × 108/ head part vaccine is safe to use, to not occurring the locally or systemically adverse reaction caused by vaccine, and all strong work after the susceptible piglet inoculation of 2~3 week old health；Compared with the control group, the pneumonia lesion slip of immune group test pig is not less than 75% to the efficacy test result of vaccine Immunization method；The vaccine preserves still safely and effectively vaccine potency inspection of the present invention in 27 months at 2~8 DEG C and the detection rabbit anteserum mycoplasma hyopneumoniae antibody titer progress of IHA methods can also be used, this brings great benefit to the efficacy test of vaccine.
Description
Technical field
The present invention relates to a kind of porcine mycoplasmal pneumonia inactivated vaccines and preparation method thereof, belong to veterinary preparations field.
Background technology
Mycoplasma pneumonia of swine (Mycoplasma Pneumonia of Swine, MPS) is also known as epidemic swine pneumonia
(Enzooticpneumonia of pig) is caused by mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp)
A kind of contact of pig, chronic respiratory infectious disease.It is mainly shown as that cough and asthma, the state of an illness breathe syndrome characterized by dyspnea when aggravating, open one's mouth
Breathing.Lesion characteristic is warm property Bronchopneumonia, before sharp leaf, lobus cardiacus, intermediate leaf and lobus diaphragmaticus one third in " meat sample ' or
" pancreas sample " substantially becomes.The disease is in worldwide Distribution, and there are the generation of mycoplasma pneumonia of swine, the growth of illness swinery in nearly all country
Speed reduces by 12~15%, and feed conversion rate reduces by 10%, and fattening period extends 1 month, with other cause of diseases such as pleuropneumonia unwrapping wire
The mixing senses such as bacterium (APP), porcine reproductive and respiratory syndrome (PRRSV), pneumonia Pasteurella (Pneumonic Pasteurella)
When dye, caused by economic loss it is more serious.
This disease is detected in pig, and the pig of all ages and classes, gender and kind can infect.Cardinal symptom is cough, has difficulty in breathing,
The sharp leaf of visible lungs, lobus cardiacus, intermediate leaf and lobus diaphragmaticus leading edge are in " carnification " or " change of pancreas sample " after sick pig dissection, and incidence is high,
The death rate is low.Morbid pig by cough, sneeze or breathe to external discharge of bacteria, pathogen forms aerosol and floats in air,
Health pig sucks the aerosol to carry disease germs and infects.Cause of disease can survive for a long time in vivo, disappear half a year even in clinical symptoms
It remains to carry disease germs in pig body within 1 year, and outside discharge of bacteria can be continued.Once the therefore incoming swinery of this disease, energy long-term existence, very
Hardly possible is eradicated.
Mycoplasma pneumonia of swine is considered as occurring most frequently the swine disease one of great with economic implications for a long time.Foreign countries' report morbidity
Rate generally 50% or so, Pointon etc. (1990) to Minnesota State slaughter pig carry out naked eyes lesion investigation the result shows that,
125 swinerys 100% have typical mycoplasma pneumonia of swine lesion, 75% pig infected.Guerrero etc. (1990) slaughters 7 countries
The investigation that butcher pig carries out shows that pneumonia Epidemic Scope is 38%~100%.Carino etc. (1999) also reports nineteen ninety-five and is entrusting
Mycoplasma pneumonia of swine is all infected in interior auspicious 27 pig farms for drawing in row investigation.Domestic statistics show China's mycoplasma pneumonia of swine
Incidence is 30%~50%, and to be then up to 75% or more, Huang Jianhua (2001) big-and-middle to 5000, Guangdong Province or more for infection rate
The survey showed that on type pig farm, and mycoplasma pneumonia of swine incidence is 84.17%, and illness pig long term development is bad, growth rate drop
Low 12%, efficiency of feed utilization reduces by 20%.Pointon etc. is found by experiment that for 1985 to be contacted with the breathe sow of disease of infection
Pig growth rate reduces by 15.9%, and efficiency of feed utilization reduces by 13.8%, and the loss of every pig of estimation is about 2.8 dollars.The U.S. by
It is infected in mycoplasma pneumonia of swine, at least loses 1,800,000,000 dollars every year, therefore for Compact Develop, the harm of mycoplasma pneumonia of swine and its institute
Caused by economic loss be obvious.According to related research both domestic and external, simple mycoplasma hyopneumoniae infection will not draw
The mortality for playing pig, is often as secondary other pathogenic bacteria such as Pasteurella, pneumococcus, pleuropneumonia actinomyces, sand
The infection of door Salmonella and various suppurative bacteriums etc. causes the aggravation of swine disease feelings and sick pig mortality.
Mycoplasma hyopneumoniae rehabilitation pig can generate immune protective efficiency, and Thacker etc. (2000) passes through intramuscular injection pig pneumonia
Mycoplasma evaluates part and systemic immunity, it is believed that mucus immunity and cellular immunity are in control mycoplasma hyopneumoniae infection
In play an important role.Many scholars have carried out many researchs to mycoplasma pneumonia of swine attenuated vaccine and inactivated vaccine, and answer clinic
Effect is evaluated, the results showed that vaccine use can reduce pig pneumonia lesion, growth performance be improved, in mycoplasma pneumonia of swine
There is important role in control and purification.
From early 20th century finds this disease, the researcher of countries in the world has carried out largely mycoplasma pneumonia of swine and its cause of disease
Research.Safely and effectively vaccine be control livestock and poultry infectious disease important channel, due to porcine mycoplasmal pneumonia be difficult to it is curative,
Vaccine, which is also expected to become, prevents the most effective preferred approach of the disease.China Veterinery Drug Inspection Office has carried out weak the end of the fifties
Mycoplasma strains are connected in rabbit body and pass generation more than 700 by the research of malicious vaccine, make one plant of velogen strain mycoplasma hyopneumoniae attenuation finally
It causes weak, has cultivated that one plant of virulence is low, the good vaccine of immunogenicity manufactures strain, and the bacterial strain is used in combination to produce three types
Vaccine, i.e.,：Newborn rabbit tissue freeze dried vaccine, chick embryo yolk sac freeze-drying attenuated vaccine and culture medium live vaccine.It raises academy of agricultural sciences of Jiangsu Province
It herds veterinary institute and is engaged in mycoplasma pneumonia of swine research more than 30 years, in the side such as the separation of mycoplasma hyopneumoniae, identification, diagnosis, prevention
Face has obtained multinomial achievement, successfully develops mycoplasma pneumonia of swine attenuated live vaccines (168 plants), had in recent years in the vaccine of Chinese approved
Porcine mycoplasmal pneumonia inactivated vaccine (DJ-166 plants), porcine mycoplasmal pneumonia inactivated vaccine (J plants).Foreign countries breathe disease vaccine also import
To China.Although the appearance of these vaccines plays certain effect to the prevention of mycoplasma pneumonia of swine, due to the poison of mycoplasma hyopneumoniae
Power is closely related with immunogenicity, and under normal circumstances, the virulence of bacterial strain is stronger, then immunogenicity is better.It was passed in culture medium
Cheng Zhong, often virulence attenuation of, immunogenicity also die down.Different strain virulence is also not exclusively the same.This just gives vaccine
Development bring prodigious difficulty.
But it is found from the application observation of clinical test and vaccine, can reach very ideal pre- still without a kind of vaccine so far
The height of anti-effect, the specific protective rate of these vaccines is difficult estimation, and general report is that inactivated vaccine can be used for suckling pig, bosom
Pregnant sow and herd boar, intramuscular inoculation, immunity inoculation is twice, without side-effects to pig safety, compared with nonimmune control pig, exempts from
The pneumonia morbidity program of epidemic disease pig can decline 50%~60%, immune duration 6 months.
At present the efficacy test of the product of some producers oneself is formulated with reference to vaccine, judges immune exempt from according to reference to seedling
Whether epidemic disease effect is qualified.And the standard formulation determined when with reference to vaccine generally by production of vaccine enterprise oneself according to experimental study
(Ning Yibao is edited, live vaccine, Beijing：Chinese agriculture publishing house, 2008, p443).Therefore, prevent at present and control pig breathes heavily
Gas disease is largely to rely on comprehensive preventive health measures, by the use of drug and vaccine, introduces the pig without disease of breathing, eliminate
It carries disease germs pig and stringent feeding management measure, clinically prevents and control mycoplasma pneumonia of swine to reach.
The evaluation method of domestic vaccine potency：
It is tested with the pig of no mycoplasma hyopneumoniae infection, is immunized latter 50 days or so to immune swine and nonimmune control pig
It is attacked with the strong poisons tracheae of mycoplasma hyopneumoniae.Clinical symptoms are observed, and pig was cutd open in 25 days after attacking poison and kills, observe lung
Lesion, compared with nonimmune control pig, the pneumonia morbidity program of immune swine declines 60% or more vaccine and is judged to qualification.
External vaccine potency evaluation method：It is determined when with reference to vaccine generally by production of vaccine enterprise oneself according to experimental study
Standard formulation.It is tested with the pig of no mycoplasma hyopneumoniae infection, 50 or so days are to the nonimmune control of immune swine after being immunized
Pig is attacked with strong poison.Clinical symptoms are observed, and pig was cutd open in 25 days after attacking poison and kills, pulmonary lesion are observed, using statistics
Analysis, immune group and nonimmune control group pig pulmonary lesion (P when there were significant differences<0.05) vaccine is judged to qualification.
Invention content
Due to mycoplasma hyopneumoniae be difficult to it is radical, although current vaccine is using more ineffective.Therefore, it develops
Efficiently, safety, vaccine easy to use have a very important significance for preventing porcine mycoplasmal pneumonia.The purpose of the present invention is
Using the mycoplasma hyopneumoniae that we detach, pass through the identifying of bacterial strain, the foundation of seed lot at different levels, culture and inactivation technology, reality
Test the campaigns research such as room trial-production, vaccine safety, immune efficacy and duration of immunity, storage life, to market provide it is a kind of more
Add safely and effectively, the inactivated vaccine of the generation of the longer prevention porcine mycoplasmal pneumonia disease of storage life.
Technical scheme of the present invention
1. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the vaccine contains mycoplasma hyopneumoniae
(M.hyopneumoniae, Mhp) strain is ZY plants, this plant of mycoplasma hyopneumoniae delivers Beijing on May 08th, 2018
No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1 are common
The preservation of microorganism center, deposit number are CGMCC No.15244；In vaccine the content of inactivation antigen be CCU not less than 1.5 ×
108/ head part.
2. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the vaccine with the susceptible piglet 5 of 2~3 week old health,
Each intramuscular injection vaccine 4.0ml is observed 14, the locally or systemically adverse reaction caused by vaccine should not occurs, and all strong
It is living.
3. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the efficacy test result of vaccine Immunization method
Compared with the control group, the pneumonia lesion slip of immune group test pig is not less than 75%.
4. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the vaccine preserves 27 months at 2~8 DEG C.
5. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine of the present invention, it is characterised in that preparation method
It is as follows：
(1) production seed is prepared：Freeze-drying lactobacillus is broken seal, strain is inoculated in fluid nutrient medium by 10% inoculum concentration
In, in 37~37.5 DEG C cultivate 3~7 days, and Medium's PH Value be 6.8 when, obtain level-one production seed；Two level is taken to give birth to legal system
Produce seed；
(2) bacterium solution culture：It is packed into improvement GoodwinShi fluid nutrient mediums, 121 DEG C of sterilizings by the 60% of fermenter volume
20min is added 20% healthy Swine serum and 250 units/ml penicillin, stirs evenly when culture medium temperature is cooled to 37 DEG C,
Seed is produced by 10% inoculation two level of culture medium total amount, 100r/min stir cultures 3~7 days, wait for culture medium under the conditions of 37 DEG C
Colour changed into yellow harvests bacterium solution when being down to 6.5 or so by 7.5 in slight muddy, pH value, and the viable bacteria titre in bacterium solution should be not less than 1
×108CCU/ml；
(3) it inactivates and blocks：The 0.2mol/L BEI solution of sterilizing is added in fermentation tank, keeps inactivator final concentration of
5% (V/V), stirs evenly, and then squeezes into fermentation tank bacterium solution and is heated up in 37 DEG C of inactivation tank in advance, 37 DEG C of inactivation stirrings 24
Hour；After inactivation stops, sampling carries out inactivation inspection, while final concentration of 1.5%~2.0% sulphur being added in inactivated bacterial liquid
Sodium thiosulfate solution, 22~25 DEG C are stirred continuously 2 hours, neutralize remaining BEI；
(4) it concentrates：Harvest bacterium solution is concentrated into the 1/10 of original volume through 200Ku molecular weight ultrafiltration membrane packets；
(5) match seedling：1 part of Montanide GEL 01RP water-soluble adjuvants ratio is added to carry out matching seedling in 9 parts of bacterium solutions, low speed stirs
It mixes, stirs 1% thimerosal solution of preceding addition terminating, its final concentration is made to be no more than 0.01%.
6. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine of the present invention, which is characterized in that in step (1)
The culture medium is improvement GoodwinShi A26 fluid nutrient mediums.
7. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine of the present invention, it is characterised in that the improvement
The group of GoodwinShi A26 fluid nutrient mediums becomes：PPLO powder 20.0g, 25% yeast leachate 100.0ml, glucose
5.0g, L- cysteine hydrochloride 0.1g, Sodium Pyruvate 2g, MEM 20g, ampicillin 0.25g, phenol red 0.02g, water for injection
Add to 1000ml.
8. a kind of efficacy test of mycoplasma hyopneumoniae inactivated vaccine of the present invention, it is characterised in that following method is appointed
Select one：
(1) IHA methods：With the susceptible rabbit 5 of 1.5~2.0kg health, each leg muscle vaccinates 0.5ml, is pressed after 14 days
Same dose and approach carry out two and exempt from；Other 5 are not inoculated with as a contrast, and two exempt from 30 days afterwards, together with control rabbit blood sampling, use IHA
Method detects rabbit anteserum mycoplasma hyopneumoniae antibody titer, and control group Serum Antibodies of Rabbits answers all feminine genders, immunizing rabbit to answer
At least 4 Serum Antibodies of Rabbits potency are not less than 1:16；
(2) Immunization method：With the susceptible piglet 5 of 2~3 week old health, each musculi colli vaccinates 2.0ml, after 3 weeks
It carries out two with dosage in the same way to exempt from, two exempt from 35 days afterwards, and together with control pig 5, each intratracheal injection mycoplasma hyopneumoniae is strong
Malicious ZY lung tissues containing viral disease suspension 5.0ml is observed 28, and mycoplasma hyopneumoniae infection classical symptom should occur in control pig, test
At the end of cut open and kill total Test pig, score the lung of each test pig disease lesion of breathing by Goodwin55 minute marks point-score, with
Control group is compared, and the pneumonia lesion slip of immune group test pig reaches 79.8%~83.8%.
The specific implementation mode of the present invention
1. the separation of strain is identified
ZY plants of mycoplasma hyopneumoniae is to fall ill from the doubtful porcine mycoplasmal pneumonia of Sichuan Ziyang pig farm inspection for 2006
The pulmonary lesion tissue of pig carries out bacterium and is separately cultured, and inhibits experiment, generation through Bacteria Culture characteristic, biochemical test, Serum Growth
It thanks to the identification methods such as inhibition experiment, PCR identifications and animal Orthogonal Rotational Regressive Tests, the bacterial strain being separated to is accredited as mycoplasma hyopneumoniae,
And it is named as ZY plants of mycoplasma hyopneumoniae, this plant of mycoplasma hyopneumoniae has delivered Chaoyang District, Beijing City north on May 08th, 2018
In the institute 3 of occasion West Road 1 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms
Heart preservation, deposit number are CGMCC No.15244.
By the identification of foundation and each biological characteristics to ZY plants of seed lots of mycoplasma hyopneumoniae, by mycoplasma hyopneumoniae
The ZY plants of production strains as porcine mycoplasmal pneumonia inactivated vaccine are used ZY plants of lung-tissue virus of mycoplasma hyopneumoniae as inspection
Poison.
2. ZY plants of strain properties of mycoplasma hyopneumoniae
(1) production strain
1) form and biochemical characteristic：With microbial strain culture smear, dyed through Ji's nurse Sa, microscopy, spherical in shape, annular, rod-shaped,
The variforms such as pyriform.Biochemical characteristic should meet the feature of mycoplasma in systematic bacteriology.
2) cultural character：Strain (note 1) in the improvement GoodwinShi A26 fluid nutrient mediums containing phenolic red indicator, 37
DEG C culture 3~7 days, is in uniform turbid growth, pH value drops to 6.8~7.0 by 7.5, and fluid nutrient medium is in golden yellow.Containing
On the improvement GoodwinShi A26 agar plates of 30% Swine serum, 5%CO237 DEG C of cultures in wet environment, the tiny circle of formation,
Neat in edge, the pan-fried poached egg sample petite slightly swelled like dew drop-wise, intermediate densification.
3) serological characteristic：
1. growth inhibition test：On the GoodwinShi A26 agar plates of strain streak inoculation improvement, pig pneumonia will be contained
The sterilizing filter paper piece of mycoplasma hyper-immune serum is attached on the GoodwinShi A26 agar plates of the improvement of inoculum culture,
5%CO237 DEG C of constant temperature incubations in wet environment, the inhibition zone for answering around the scraps of paper sterile long or bacterium colony of being born to significantly reduce.
2. metabolic inhibition test：Strain is inoculated in the GoodwinShi A26 liquid of the improvement containing glucose and phenolic red indicator
In culture medium, suitable mycoplasma hyopneumoniae hyper-immune serum, 37 DEG C of cultures is added, significant change should not occur in culture medium color.
4) count plate：Assay is carried out by CCU method of counting, every milliliter of viable count should be not less than 1.0 × 108CCU。
5) virulence：10~11 week old are taken, (see note 2, the same below) 5, each intratracheal injection mycoplasma hyopneumoniae is malicious by force
The ZY suspensions of lung tissue containing viral disease 5.0ml (containing about 0.5g diseases lung tissue) is observed 28, and the pigs such as cough, asthma should occur in Pigs Inoculated
Eaton agent pneumonia clinical symptoms, dissect lung should be observed that the interstitial pneumonia disease of apparent " carnification " or " change of pancreas sample "
Become.
6) immunogenicity：Inactivated vaccine is prepared by the present invention.Take the susceptible pig 10 of 2~3 week old health, 5 each neck fleshes
Meat vaccinates 2.0ml, and by same dose and mode booster shot 1 time, in addition 5 are not inoculated with as a contrast within 21 days after inoculation,
It is raised under the conditions of.Two exempt from after 35 days, together with control pig 5, each intratracheal injection mycoplasma hyopneumoniae poison ZY lungs containing viral disease by force
Tissue suspension 5.0ml (containing about 0.5g diseases lung tissue) is observed 28, is cutd open and kill total Test pig.By Goodwin55 minute mark point-scores pair
The lung of each test pig disease lesion of breathing scores, and compared with the control group, the pneumonia lesion of Computation immunity group test pig is reduced
Rate (see note 3).
7) it purely examines：By Republic of China Veterinary Pharmacopoeia (the Chinese veterinary pharmacopoeia committee, People's Republic of China's veterinary drug
Allusion quotation, two 〇 First Five-Year Plans year version three, Chinese agriculture publishing house, 2016, hereinafter referred to as《Chinese veterinary pharmacopoeia》) annex tests, strain
It should be pure.
8) specific：Strain culture extraction DNA is subjected to PCR detections, by PCR product into row agarose gel electrophoresis,
The purpose band of 427bp sizes should occur.
9) basic bacteria generation：3rd~9 on behalf of basic bacteria.
(2) effect inspection ZY plants of lung-tissue virus of strong poison
1) pathogenic：The susceptible piglet 5 of 10~11 week old health is taken, poison ZY contains each intratracheal injection mycoplasma hyopneumoniae by force
Viral disease lung tissue suspension 5.0ml (containing about 0.5g diseases lung tissue) is observed 28, and it is former that the pigs branch such as cough, asthma should occur in Pigs Inoculated
Body pneumonia clinical symptoms, dissect lung should be observed that the interstitial pneumonia lesion of apparent " carnification " or " change of pancreas sample ".
2) pure property：It presses《Chinese veterinary pharmacopoeia》) annex tests, it should be without bacterium, mould and external source mycoplasma and virus
Pollution.
3) specific：Strain culture extraction DNA is subjected to PCR detections, by PCR product into row agarose gel electrophoresis,
The purpose band of 427bp sizes should occur.
(3) fungi preservation：For freeze-drying lactobacillus in -70 DEG C or less preservations, storage life is 48 months.
(4) seed lot of strain is established：By the way that the passage of ZY plants of strain primordial seeds of mycoplasma hyopneumoniae is studied and is planted
The foundation of son batch, and by the comprehensive identification of strain basic bacteria, determine ZY plants of F1~F2 generations of mycoplasma hyopneumoniae as former
Beginning seed, basic bacteria batch generation range are limited to F3~F9 generations, and production strain is limited within F14 generations, and seed subculture is answered
No more than 5 generations.
3. vaccine preparation
(1) production seed is prepared：Freeze-drying lactobacillus is broken seal, strain is inoculated in fluid nutrient medium by 10% inoculum concentration
In, in 37~37.5 DEG C cultivate 3~7 days, and Medium's PH Value be 6.8 when, obtain level-one production seed；Two level is taken to give birth to legal system
Produce seed；
(2) bacterium solution culture：It is packed into improvement GoodwinShi fluid nutrient mediums, 121 DEG C of sterilizings by the 60% of fermenter volume
20min is added 20% healthy Swine serum and 250 units/ml penicillin, stirs evenly when culture medium temperature is cooled to 37 DEG C,
Seed is produced by 10% inoculation two level of culture medium total amount, 100r/min stir cultures 3~7 days, wait for culture medium under the conditions of 37 DEG C
Colour changed into yellow harvests bacterium solution when being down to 6.5 or so by 7.5 in slight muddy, pH value, and the viable bacteria titre in bacterium solution should be not less than 1
×108CCU/ml；
(3) it inactivates and blocks：The 0.2mol/L BEI solution of sterilizing is added in fermentation tank, keeps inactivator final concentration of
5% (V/V), stirs evenly, and then squeezes into fermentation tank bacterium solution and is heated up in 37 DEG C of inactivation tank in advance, 37 DEG C of inactivation stirrings 24
Hour；After inactivation stops, sampling carries out inactivation inspection, while final concentration of 1.5%~2.0% sulphur being added in inactivated bacterial liquid
Sodium thiosulfate solution, 22~25 DEG C are stirred continuously 2 hours, neutralize remaining BEI；
(4) it concentrates：Harvest bacterium solution is concentrated into the 1/10 of original volume through 200Ku molecular weight ultrafiltration membrane packets；
(5) match seedling：1 part of Montanide GEL 01RP water-soluble adjuvants ratio is added to carry out matching seedling in 9 parts of bacterium solutions, low speed stirs
It mixes, stirs 1% thimerosal solution of preceding addition terminating, its final concentration is made to be no more than 0.01%.
4. product inspection
(1) physical behavior：Rose pink homogenous suspension.
(2) steriling test：It presses《Chinese veterinary pharmacopoeia》Annex is tested, and asepsis growth is answered.
(3) loading quantity inspection：It presses《Chinese veterinary pharmacopoeia》Annex is tested, and regulation should be met.
(4) the antiseptic mercurials determination of residual amount：By existing《Chinese veterinary pharmacopoeia》Annex carries out, and should meet regulation.
(5) safety verification：With the susceptible piglet 5 of 2~3 week old health, each intramuscular injection vaccine 4.0ml is observed 14, is answered
Do not occur the locally or systemically adverse reaction caused by vaccine, and all strong work.
(6) efficacy test：The following optional one of method.
1) IHA methods：With the susceptible rabbit (see note 2) 5 of 1.5~2.0kg health, each leg muscle vaccinates 0.5ml,
Two are carried out after 14 days by same dose and approach to exempt from；Other 5 are not inoculated with as a contrast.Two exempt from 30 days afterwards, are adopted together with control rabbit
Blood detects rabbit anteserum mycoplasma hyopneumoniae antibody titer with IHA methods, and control group Serum Antibodies of Rabbits is answered all feminine genders, exempted from
Epidemic disease rabbit at least 4 Serum Antibodies of Rabbits potency should be not less than 1:16.
2) Immunization method：With the susceptible piglet 5 of 2~3 week old health, each musculi colli vaccinates 2.0ml, after 3 weeks
It carries out two with dosage in the same way to exempt from, two exempt from 35 days afterwards, and together with control pig 5, each intratracheal injection mycoplasma hyopneumoniae is strong
Malicious ZY lung tissues containing viral disease suspension 5.0ml (containing about 0.5g diseases lung tissue) is observed 28, and mycoplasma hyopneumoniae should occur in control pig
Classical symptom (such as abdominal respiration or cough) is infected, when off-test, which is cutd open, kills total Test pig.By Goodwin55 minute mark point-scores pair
The lung of each test pig disease lesion of breathing is scored (see note 3).
Microbial resources information of the present invention
ZY plants of mycoplasma hyopneumoniae is to fall ill from the doubtful porcine mycoplasmal pneumonia of Sichuan Ziyang pig farm inspection for 2006
The pulmonary lesion tissue of pig carries out bacterium and is separately cultured, and inhibits experiment, generation through Bacteria Culture characteristic, biochemical test, Serum Growth
It thanks to the identification methods such as inhibition experiment, PCR identifications and animal Orthogonal Rotational Regressive Tests, the bacterial strain being separated to is accredited as mycoplasma hyopneumoniae,
And be named as ZY plants of mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae Mhp), this plant of mycoplasma hyopneumoniae in
On May 08th, 2018 delivers the micro- life of China of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
The common micro-organisms center preservation of object culture presevation administration committee, deposit number are CGMCC No.15244.
The positive effect of the present invention
The present invention relates to a kind of production methods of porcine mycoplasmal pneumonia inactivated vaccine.The present invention is to exempt from poison with being separated to one plant
ZY plants of the mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae Mhp) that power is strong, epidemic focus is good makees production bacterial strain, uses
Efficient culture medium, suitable cultural method, inactivation technology and suitable immunologic adjuvant, prepare mycoplasma hyopneumoniae inactivated vaccine.
Inactivation antigen content CCU is inactivated in vaccine prepared by the present invention is not less than 1.5 × 108/ head part vaccine is safe to use, to 2~3
Do not occur the locally or systemically adverse reaction caused by vaccine, and all strong work after the susceptible piglet inoculation of week old health；The vaccine
Compared with the control group with the efficacy test result of Immunization method, pneumonia lesion slip >=75% of immune group test pig；Exempt from
The epidemic disease duration is 180 days；Vaccine preserves 27 months at 2~8 DEG C still safely and effectively；Vaccine potency inspection of the present invention can also be used
IHA methods detect rabbit anteserum mycoplasma hyopneumoniae antibody titer and carry out, this brings great benefit to the efficacy test of vaccine.
Embodiment
Following embodiment is the technical solution further illustrated the present invention, is not limited the invention.
Embodiment 1 --- vaccine preparation
1. production is prepared with seed
After taking freeze-drying lactobacillus to break seal, after being dissolved with culture medium, inoculation improvement GoodwinShi A26 fluid nutrient mediums, 37 DEG C of trainings
Support 3~7, when culture medium colour changed into yellow, be down to 6.8 or so in slight muddy, bacterium solution pH value when, resume 1 by 10% inoculum concentration
In generation, 37 DEG C are cultivated 3~7, when culture medium colour changed into yellow, be down to 6.8 or so in slight muddy, bacterium solution pH value when harvest, through pure
It is pure after the assay was approved, as first order seed.First order seed bacterium solution is taken to be inoculated in improvement GoodwinShi A26 liquid by 10% inoculum concentration
In body culture medium.37 DEG C of shaken cultivations 3~7 days wait for culture medium colour changed into yellow, are down to 6.8 or so in slight muddy, bacterium solution pH value
When harvest, through purely after the assay was approved, seed is produced as two level, pH value sets -20 to secondary seed to after 7.2 or so after the adjustment
DEG C preserve, validity period be no more than 14 days.
2. the preparation of seedling bacterium solution
(1) bacterium solution culture：Improvement GoodwinShi A26 fluid nutrient mediums are packed by the 60% of fermenter volume, 121 DEG C go out
Bacterium 20min is added 20% Swine serum and 250 units/ml mould ropes, stirs evenly, press when culture medium temperature is cooled to 37 DEG C
10% inoculation two level of culture medium total amount produces seed, and 100r/min shaken cultivations 3~7 days, wait for pH value by 7.5 under the conditions of 37 DEG C
Bacterium solution is harvested when being down to 6.5 or so.
(2) it purely examines：Sampling inoculation is by improvement GoodwinShi A26 fluid nutrient mediums and solid medium, by existing
《Chinese veterinary pharmacopoeia》Annex is tested, and sees whether sheer growth (being shown in Table 1).
(3) viable bacteria titer determination：Sampling carries out CCU assays before inactivation, and the viable bacteria titre in bacterium solution answers >=1 ×
108CCU/ml (is shown in Table 1).
The preparation of 1 mycoplasma hyopneumoniae antigen liquid of table and pure inspection result
| Bacterium solution lot number | Harvest yield | 10 times of concentrations | Viable bacteria titre before concentration | Purely examine |
| 001 | 10400ml | 1010ml | 1.0×109CCU/ml | Without bacterium, fungus growth, purely |
| 002 | 10280ml | 1000ml | 5.5×108CCU/ml | Without bacterium, fungus growth, purely |
| 003 | 10320ml | 1010ml | 1.0×108CCU/ml | Without bacterium, fungus growth, purely |
(4) it inactivates
1) cyclisation of BEA (2- bromine ethamine hydrobromic acid)：BEA powder is added to the sodium hydroxide solution of the 0.2mol/L newly configured
In, water-bath at 37 DEG C is set, is vibrated 1 time every 10min, cyclisation is terminated after 60min, the BEI (two of final concentration of 0.2mol/L is made
Aziridine), after aseptic filtration, set 2-8 DEG C it is spare.
2) bacterium solution inactivates：The 0.1M BEI solution of sterilizing is added in fermentation tank, makes inactivator final concentration of 5% (V/V),
It stirs evenly, then fermentation tank bacterium solution is squeezed into and is heated up in 37 DEG C of inactivation tank in advance, 37 DEG C of inactivations are stirred 24 hours.It is cooling
Inactivated bacterial liquid, 2~8 DEG C of preservations.
3) it blocks：After inactivation stops, sampling carries out inactivation inspection, while sterilized thio sulphur being added in inactivated bacterial liquid
Acid sodium solution makes its volume final concentration of 1.5%~2.0%, room temperature (22~25 DEG C) be stirred continuously 2h, neutralize remaining BEI.
(5) inactivation is examined：Inactivation inspection is carried out with following method, answers asepsis growth.
1ml inactivated bacterial liquids to be checked are taken to be inoculated in the GoodwinShi A26 fluid nutrient mediums of 9ml improvement, 37 DEG C of cultures 12
Day, observe culture medium color change；0.2ml objects to be checked are taken to be inoculated in improvement GoodwinShi A26 Agar Platings simultaneously,
5%CO237 DEG C of constant temperature incubations 12 days in wet environment, whether there is or not the growths of mycoplasma bacterium colony for observation.Non- inactivated bacterial liquid is taken to carry out simultaneously
Same inoculation, as positive control.If the fluid nutrient medium and agar medium that are inoculated with object to be checked have growth, show not completely
Inactivation；If be inoculated with object to be checked fluid nutrient medium and agar medium without growth, transplanting 1 time must be carried out again, take Liquid Culture
Culture 1ml in base is inoculated in 9ml fluid nutrient mediums, and 37 DEG C are cultivated 12, while 0.2ml cultures being taken to be inoculated in improvement
GoodwinShi A26 Agar Platings, 5%CO237 DEG C of constant temperature incubations 12 days in wet environment observe and record test tube and flat
Growing state on plate, positive control are similarly inoculated with.
Result judgement：If color change in fluid nutrient medium in positive control passage for the first time and subculture, i.e., by red change
Huang indicates that pH becomes 6.5 or so and pass on twice that bacterium colony presence can be observed on agar from 7.4, then it represents that positive control is set up.
Bacterium solution inspection result is that in passage for the first time and subculture fluid nutrient medium remains red without color change
(pH 7.4 or so) and also sterile length of being born on agar plate is passed on twice；The fluid nutrient medium colour changed into yellow of positive control,
There is the growth of mycoplasma bacterium colony on Agar Plating, it is determined that all object complete inactivations to be checked.
(6) it concentrates：Harvest bacterium solution is concentrated into the 1/10 of original volume through 200Ku molecular weight ultrafiltration membrane packets, sets 2~8 DEG C of guarantors
It deposits.
3. matching seedling：According to seedling ratio, the amount of required PBS, bacterium solution and adjuvant is calculated, is added in emulsion tank, by 9 parts of bacterium
Liquid adds 1 part of Montanide GEL 01RP water-soluble adjuvants ratio to carry out matching seedling, and low speed (2000r/min) stirs 15~20min,
It is sufficiently mixed emulsification, is stirred evenly, 1% thimerosal solution of preceding addition is stirred terminating, so that its final concentration is no more than 0.01%, together
When make every ml vaccines containing inactivation before bacterium number be at least 1.5 × l08CCU。
4. the inspection of semifinished product：Sampling is pressed after mixing《Chinese veterinary pharmacopoeia》Annex method carries out, the equal asepsis growth of bacterium solution.
5. packing：After steriling test qualification, quantitative separating is carried out, when packing should stir at any time, it is made to be uniformly mixed.Add
Lid sealing, and adhesive label.Prepare 3 batches of products altogether with method, lot number is respectively 001,002,003.
The safety testing of embodiment 2 --- vaccine
It using 3 batches of mycoplasma hyopneumoniae bacterium solutions are prepared for, is concentrated again after BEI is inactivated, Montanide GEL is added
3 batches of porcine mycoplasmal pneumonia inactivated vaccines are made in 01RP water-soluble adjuvants, and lot number is respectively 001,002 and 003.Product is carried out
Safety testing.Prepare 3 batches of vaccines, to minimum using age in days piglet single dose inoculation, to piglet single dose repeated inoculation, to son
Overdose inoculation of pig uses day to pregnant animal single dose repeated inoculation, to pregnant animal overdose repeated inoculation, to non-
The safety of overdose of herd boar inoculation is tested in the inoculation of age animal single dose, the results showed that：Within the observation period,
The spirit of experimental animal, feeding are normal, and body temperature is normal after inoculation, and without changing by a relatively large margin, farrowing sow is not flowed
Production, stillborn foetus, the mummification of fetus or weak son.Touch test pig injection site, the anomalous variations such as no swelling and lump, tubercle, test pig
Without porcine mycoplasmal pneumonia disease classical symptom.Test result shows that the vaccine has good safety, stress be small to test pig,
Without other adverse reactions, vaccine safety is good.
The immuning effect test of embodiment 3 --- vaccine
Following immuning effect test is carried out with 3 batches of vaccines of preparation：Porcine mycoplasmal pneumonia inactivated vaccine is to target animals piglet
Immuning effect test；Immuning effect test of the porcine mycoplasmal pneumonia inactivated vaccine to target animals farrowing sow.Test result is such as
Under：
(1) immuning effect test of 2~3 week old piglet of porcine mycoplasmal pneumonia inactivated vaccine pair, porcine mycoplasmal pneumonia inactivation
Vaccine intramuscular injection is inoculated with 2~3 week old piglets, and 2.0ml/ heads carry out booster immunization after 21 days by same dose and approach, and two exempt from
35 days afterwards, immune group and each intratracheal injection mycoplasma hyopneumoniae of control group by force ZY plants of suspension 5.0ml of lung tissue containing viral disease of poison into
Row attacks poison, observes 28, the clinical manifestation of observation control pig and immune swine, and when off-test, which is cutd open, kills total Test pig.It presses
Goodwin55 minute marks point-score scores to the lung of test group and control group test pig disease lesion of breathing, as a result immune group pig
It is respectively 79.8%, 80.9%, 83.8% that pneumonia disease, which becomes average slip, shows that the porcine mycoplasmal pneumonia inactivated vaccine developed is exempted from
Epidemic disease piglet can resist the attack of the strong poison of mycoplasma hyopneumoniae, play a protective role.
(2) immuning effect test of the porcine mycoplasmal pneumonia inactivated vaccine to farrowing sow：Porcine mycoplasmal pneumonia inactivated vaccine
It is inoculated with pregnant sow in intramuscular injection in first 6 weeks or so of giving a birth, 2.0ml/ heads are carried out reinforcing exempting from after 21 days by same dose and approach
Epidemic disease, after Farrowing produce surviving of son, piglet sucks breast milk, takes immune sow and compares the piglet that sow produces 14 ages in days or so, respectively
The ZY plants of suspension of lung tissue containing viral disease 5.0ml of poison carry out attacking poison intratracheal injection mycoplasma hyopneumoniae by force, observe 28, poison is attacked in observation
The clinical manifestation of piglet afterwards, when off-test, which is cutd open, kills total Test pig.By Goodwin55 minute mark point-scores to test group and control group
The pulmonary lesion of experiment piglet scores, and as a result the pneumonia lesion slip of 3 batches of vaccine immunity sow litters is respectively
85.5%, 84.5%, 87.6%, show that farrowing sow is immunized in the porcine mycoplasmal pneumonia inactivated vaccine developed, produces 14 ages in days son
Pig can resist the attack of the strong poison of mycoplasma hyopneumoniae, play a protective role.
Embodiment 4 --- the correlation that experimental animal serology efficacy test is protected with Immunization
Porcine mycoplasmal pneumonia inactivated vaccine various dose is immunized piglet and rabbit respectively, piglet after 21 days by same dose and
Approach carries out two and exempts from, and two exempt from then to carry out within latter 35 days attacking poison, and rabbit carries out two by same dose and approach after 14 days and exempts from, after two exempt from
Blood sampling on the 30th surveys antibody with IHA methods, as a result：After piglet attacks poison, 0.5ml dose vaccine immune groups be averaged pneumonia disease become score 9.8
Point, pneumonia disease becomes average slip 44.9%；1.0ml dose vaccine immune groups be averaged pneumonia disease become score 5.0 divide, pneumonia lesion
Average slip 71.9%；The 1.5ml dose vaccine immune groups pneumonia disease that is averaged becomes score 3.2 and divides, and pneumonia disease becomes average slip
82.0%；The 2.0ml dose vaccine immune groups pneumonia disease that is averaged becomes score 2.6 and divides, and pneumonia disease becomes average slip 85.3%；Attack poison
Control group be averaged pneumonia disease change be scored at 17.8 points, as a result 1.0ml or more dose vaccines be immunized piglet after, can make immune swine lung
Scorching lesion slip plays immanoprotection action up to 71.9% or more.Measure rabbit anteserum, 0.2ml vaccine immunity group rabbit blood
Clear antibody titer 1:4~1:16,2/5 protections；0.5ml vaccine immunity groups serum antibody titer 1:8~1:32,4/5 protections；0.7、
1.0ml vaccine immunities group serum antibody titer equal 1:16~1:32, equal 5/5 protection.
Embodiment 5 --- the measurement of vaccine immunity duration
Porcine mycoplasmal pneumonia inactivated vaccine intramuscular injection is inoculated with 2~3 week old piglets, and 2.0ml/ heads press identical dose after 21 days
Amount and approach carry out booster immunization：
(1) respectively at head exempt from first 1 day, head exempt from after 7 days, 14 days, 21 days, two exempt from after 35 days, 90 days, 180 days take a blood sample, measure
Antibody (the results are shown in Table 2)：
The serum mean antibody levels (S/P values) of different time after 2 vaccine immunity pig of table
| Blood sampling time | Immune group | Control group |
| Head exempts from first 1 day | 0.01±0.02 | 0.03±0.01 |
| Head exempts from 7 days latter | 0.14±0.04 | 0.01±0.02 |
| Head exempts from 14 days latter | 1.12±0.20 | -0.01±0.02 |
| Head exempts from 21 days latter | 1.35±0.24 | -0.01±0.03 |
| Two exempt from 30 days afterwards | 2.12±0.28 | -0.01±0.02 |
| Two exempt from 60 days afterwards | 2.41±0.22 | -0.02±0.02 |
| Two exempt from 90 days afterwards | 2.53±0.19 | -0.01±0.02 |
| Two exempt from 120 days afterwards | 1.98±0.21 | -0.01±0.01 |
| Two exempt from 150 days afterwards | 1.62±0.18 | -0.02±0.01 |
| Two exempt from 180 days afterwards | 1.18±0.23 | -0.02±0.02 |
According to 2 result of table it is found that before vaccine injection, piglet serum antibody S/P values average out to 0.01, serum antibody is the moon
Property, after vaccine injection, piglet serum antibody S/P values average out to 0.14 on the 7th after head exempts from, test pig serum antibody is feminine gender, first
Piglet serum antibody S/P values average out to 1.12 on the 14th after exempting from, serum 12/15 are the positive, piglet serum antibody S/P on the 21st after head exempts from
It is worth average out to 1.35, serum 15/15 is the positive, and two, which exempt from rear 30~90 serum antibodies, peaks, S/P values average out to 2.12
~2.53, antibody level is on a declining curve later, until serum antibody S/P values average out to 1.18,5/5 is positive at 180 days.
(2) while after exempting from two it carries out within 35,90,180 attacking poison, immune group and each intratracheal injection pig lung of control group
ZY plants of poison (the suspension 5.0ml of lung tissue containing viral disease) attack poison to scorching mycoplasma by force, observes 28, observation control pig and immune swine
Clinical manifestation, when off-test, which is cutd open, kills total Test pig.By Goodwin55 minute marks point-score to test group and control group test pig
Lung's disease lesion of breathing is scored and (the results are shown in Table 3)：
The lungs pneumonia lesion appraisal result of 35,90 and 180 days challenge test pigs after table 3 two is exempted from
According to 3 result of table it is found that porcine mycoplasmal pneumonia inactivated vaccine (ZY plants) immune swine, two exempt to attack poison, vaccine within 35th afterwards
The immune group test pig pneumonia disease that is averaged becomes score 3.2 and divides, and test pig pneumonia disease becomes average slip 82.9%；Attack malicious control group examination
Test pig be averaged pneumonia disease change be scored at 18.8 points；Immune group test pig pneumonia disease becomes score and attacks malicious control group significant difference；Two
Attack within 90th poison after exempting from, the vaccine immunity group test pig pneumonia disease that be averaged becomes score 3.6 and divides, and test pig pneumonia disease becomes the slip that is averaged
78.0%；Attack malicious control group test pig be averaged pneumonia disease change be scored at 16.4 points；Immune group test pig pneumonia disease becomes score and attacks
Malicious control group significant difference；Two exempt from after attack within 180th poison, the vaccine immunity group test pig pneumonia disease change score 4.6 that be averaged is divided, test pig
Pneumonia disease becomes average slip 70.1%, illustrates the attack that can resist the strong poison of mycoplasma hyopneumoniae, plays a protective role；Attack poison
Control group test pig be averaged pneumonia disease change be scored at 15.4 points；Immune group test pig pneumonia disease becomes score and attacks malicious control group difference
Significantly.
(3) lungs sample P CR testing results after poison are attacked
Different time is attacked malicious lungs sample P CR testing results and is shown in Table after (ZY plants) of porcine mycoplasmal pneumonia inactivated vaccine is immune
4。
4 different time of table attacks the lungs mycoplasma hyopneumoniae PCR testing results of poison
According to 4 result of table it is found that after different time attacks poison, immune group and the PCR inspections of control group pig lungs mycoplasma hyopneumoniae
Survey is the positive, it was demonstrated that it is effective to attack malicious result.
It it is 6 months to the immune duration of piglet by porcine mycoplasmal pneumonia inactivated vaccine (ZY plants) according to test result.
Embodiment 6 --- the measurement of vaccine storage life
The 3 batches of porcine mycoplasmal pneumonia inactivated vaccines (lot number 001,002,003) prepared to the present invention have carried out storage life
Experiment, 3 batches of vaccines are stored in 2~8 DEG C, at 1,6,12,18,24,27 month sampling carry out physical behavior, steriling test,
Safety verification and efficacy test.The results show that this vaccine preserves 27 months at 2~8 DEG C, physical behavior is that rose pink is uniform
Suspension；Steriling test result is asepsis growth.Safety verification and efficacy test, which comply with standard, the results are shown in Table 5, table 6 and table
7.The result shows that this vaccine at least can save 27 months at 2~8 DEG C.
1. safety examination：
5 vaccine of table preserves the safety verification result after different time at 2~8 DEG C
001,002,003 batch of vaccine preserves the safety verification after 1,6,12,18,24,27 month at 2~8 DEG C, and safety check pig is not
There is abnormal response, it is all strong to live.
2. efficacy test：
(1) detection of the pneumonia lesion slip of immune group test pig
The susceptible piglet 5 of 2~3 week old health, each musculi colli is taken to vaccinate 2.0ml, after 3 weeks in the same way and agent
Amount two exempt from, two exempt from after 35 days, together with control pig 5, each intratracheal injection mycoplasma hyopneumoniae ZY plants of lungs containing viral disease of poison by force
Tissue suspension 5.0ml (containing about 0.5g diseases lung tissue) is observed 28, and control pig mycoplasma hyopneumoniae infection symptom should occur (such as
Breathe or cough), when off-test, which is cutd open, kills total Test pig.It breathes disease to the lung of test pig by Goodwin55 minute marks point-score
Lesion scores, the pneumonia lesion slip of Computation immunity group test pig.
Efficacy test after 3 batches of vaccines preserve 12 months, 24 months, 27 months at 2~8 DEG C the results are shown in Table 6 and table 7.
63 batches of vaccines of table preserved 12 months, 24 months, 27 months at 2~8 DEG C after efficacy test result
After vaccine preserves 12 months at 2~8 DEG C, vaccine immunity piglet attacks poison, and 001 batch of vaccine immunity group is averaged pneumonia lesion
Score 3.4 is divided, and pneumonia disease becomes average slip 80.0%；002 batch of vaccine immunity group be averaged pneumonia disease become score 3.6 divide, pneumonia
Lesion is averaged slip 78.8%；003 batch of vaccine immunity group pneumonia disease that is averaged becomes score 2.8 and divides, and pneumonia disease becomes average slip
83.5%；Attack malicious control group be averaged pneumonia disease change be scored at 17.0 points；Immune group pneumonia disease becomes score and attacks malicious control group difference
Significantly.
After vaccine preserves 24 months at 2~8 DEG C, vaccine immunity piglet attacks poison, and 001 batch of vaccine immunity group is averaged pneumonia lesion
Score 4.0 is divided, and pneumonia disease becomes average slip 77.5%；002 batch of vaccine immunity group be averaged pneumonia disease become score 3.6 divide, pneumonia
Lesion is averaged slip 79.7%；003 batch of vaccine immunity group pneumonia disease that is averaged becomes score 4.2 and divides, and pneumonia disease becomes average slip
76.4%；Attack malicious control group be averaged pneumonia disease change be scored at 17.8 points；Immune group pneumonia disease becomes score and attacks malicious control group difference
Significantly.
After vaccine preserves 27 months at 2~8 DEG C, vaccine immunity piglet attacks poison, and 001 batch of vaccine immunity group is averaged pneumonia lesion
Score 3.6 is divided, and pneumonia disease becomes average slip 80.4%；002 batch of vaccine immunity group be averaged pneumonia disease become score 3.8 divide, pneumonia
Lesion is averaged slip 79.3%；003 batch of vaccine immunity group pneumonia disease that is averaged becomes score 4.6 and divides, and pneumonia disease becomes average slip
75.0%；Attack malicious control group be averaged pneumonia disease change be scored at 18.4 points；Immune group pneumonia disease becomes score and attacks malicious control group difference
Significantly.
(2) immunizing rabbit serum mycoplasma hyopneumoniae antibody titer detects
1.5~2.0kg healthy rabbits (see note 2) 10,5 each intramuscular injection vaccine 0.5ml are taken to press for 14 days after injection
Same dose and approach carry out two and exempt from, and another 5 are not inoculated with as a contrast.Two exempt from 30 days afterwards, together with control rabbit blood sampling, detach blood
Clearly, rabbit anteserum mycoplasma hyopneumoniae antibody titer is detected with IHA methods.
7 vaccine of table, 2~8 DEG C of Serum Antibody Detection results for preserving 12,24 and 27 months immunizing rabbits
After vaccine preserves 12 months at 2~8 DEG C, 001 batch of vaccine immunity rabbit, serum antibody titer 1:8~1:32,4/5
Protection, 002 batch of vaccine immunity rabbit, serum antibody titer 1:8~1:32,4/5 protections；003 batch of vaccine immunity rabbit, serum are anti-
Body potency 1:16~1:32,5/5 protections.After vaccine preserves 24 months at 2~8 DEG C, 001 batch of vaccine immunity rabbit, serum antibody
Potency 1:6~1:32,5/5 protections, 002 batch of vaccine immunity rabbit, serum antibody titer 1:8~1:32,4/5 protections；003 batch of epidemic disease
Seedling immunizing rabbit, serum antibody titer 1:16~1:32,5/5 protections.After vaccine preserves 27 months at 2~8 DEG C, 001 batch of vaccine
Immunizing rabbit, serum antibody titer 1:8~1:32,4/5 protections, 002 batch of vaccine immunity rabbit, serum antibody titer 1:4~1:
32,4/5 protections；003 batch of vaccine immunity rabbit, serum antibody titer 1:8~1:32,4/5 protections.
Note:
Note 1：Porcine mycoplasmal pneumonia inactivated vaccine production fluid nutrient medium (the GoodwinShi A26 Liquid Cultures of improvement
Base) preparation：
(1) culture medium prescription：PPLO powder 20.0g, 25% yeast leachate 100.0ml, glucose 5.0g, L- hydrochloric acid are partly
Cystine 0.1g, Sodium Pyruvate 2g, MEM 20g, ampicillin 0.25g, phenol red 0.02g, water for injection add to 1000ml.
(2) preparation method：
1) basal medium：Take PPLO powder, phenol red, 25% yeast leachate, after adding water for injection to be completely dissolved, use
NaOH tune pH value 7.4~7.6,15Pa high pressure sterilizations 30min, 2~8 DEG C of preservations.
2) filtering component：Glucose, L- cysteine hydrochlorides, ampicillin, Sodium Pyruvate, MEM are taken, water for injection is added
After dissolving, using 0.2um filter filtration sterilizations, 2~8 DEG C of preservations.
3) filtering component is added in basal medium when using, while the sterile Swine serum of 60 DEG C of inactivation 30min is added
100.0ml is uniformly mixed.
4) when preparing GoodwinShi A26 solid mediums, 1.5% agar is added in liquid medium.
5) preparation of 25% yeast leachate：Yeast cake 500g is taken, adds deionized water or distilled water 2000mL, stirring molten
Solution；With hydrochloric acid (concentrated hydrochloric acid:Water=1:1) pH value 4.5~5.0 is adjusted, 80 DEG C of water-bath 30min are set；It is centrifuged after cooling, with 3000r/
Min centrifuges 20min, abandons precipitation, takes supernatant；Supernatant 2N sodium hydroxide tune pH value 7.8~8.0, room temperature is placed after boiling,
It is filtered with double-layer filter paper, it is yeast leachate to take light brown transparence filtrate.9 pounds of 15min high pressure sterilizations, -20 DEG C or less cold
Jelly saves backup.
Note 2：The standard of the standard and the susceptible rabbit of health of the present invention experiment susceptible pig of health used
1. the standard of the susceptible pig of health
(1) it tests piglet and porcine mycoplasmal pneumonia vaccine was not immunized for sow, clinically spirit, diet are normal for piglet,
No cough, expiratory dyspnea, diarrhea etc. are breathed disease symptoms.
(2) IDEXX companies porcine mycoplasmal pneumonia detection kit detection experiment Swine serum, mycoplasma hyopneumoniae serum are used
Antibody is feminine gender.
(3) experiment Swine serum is taken to be detected through PCR, porcine reproductive and respiratory syndrome virus and 2 type circoviras antigen of pig are equal
It should be negative.
1) primer
2) PCR reaction systems and condition：
System：DNA Template 2.0μl；P1 1.0μl；P2 1.0μl；2×PCR Master Mix 12.5μl；
8.5 μ l of DNase-free water, 25 μ l of total reaction volume.
Reaction condition：94 DEG C preheating 5min, (94 DEG C denaturation 30s, 55 DEG C annealing 30s, 72 DEG C extension 30s) × 30 follow
Ring, 72 DEG C of extension 10min, 4 DEG C of preservations.
3) RT-PCR reaction systems and condition：
System：4 μ l, Forward GSP (20uM) of RNA Template, 0.5 μ l, Reverse GSP (20uM) 0.5 μ l, 2
12.5 μ l, EasyScriptTM One-step Enzyme Mix of × One-step Reaction Mix 0.5 μ l, RNase-
free water 7μl。
Reaction condition：45 DEG C of reverse transcription 45min, 94 DEG C of preheating 5min, (94 DEG C of denaturation 5s, 52 DEG C of annealing 30s, 72 DEG C are prolonged
Stretch 30s) × 35 cycles, 72 DEG C of extension 10min.
2. the standard of the susceptible rabbit of health
1.5~2.0Kg healthy rabbits kinds are that big ear is white, are measured through indirect hemagglutination test IHA methods, measure rabbit anteserum
Mycoplasma pneumoniae antibody is feminine gender (≤1:4).
Note 3：The pneumonia lesion of challenge test pig judges and standards of grading
Each group pig is cutd open after the onset of attacking poison and kills, and is scored by 55 point-scores of GoodwinShi every Tou Zhu lungs pneumonia lesion.
55 point-scores of GoodwinShi are scored：The method is to see lesion according to the typical lung eye of porcine mycoplasmal pneumonia, to feelings of falling ill
Condition carries out quantization judgement, and pig apex pulmonis leaf, lobus cardiacus, the preceding one third of lobus diaphragmaticus and the intermediate leaf of affected pig Eaton agent pneumonia go out reality
Matter sample lesion, such as " carnification ", " change of pancreas sample ".
1) lesion score standard is：Each point leaf, lobus cardiacus full marks are respectively 10 points, and the preceding one third full marks of lobus diaphragmaticus are 5 points,
Intermediate leaf full marks are 5 points.It when lesion score, scores respectively each lobe of the lung, according to the lobe of the lung area that substantive lesion occurs
The ratio for accounting for the lobe of the lung is given a mark, and has 3/10 area generation lobe of the lung if lesion that realizes to beat if full marks are 10 points of the lobe of the lung
3 points, there is 3/10 area generation lobe of the lung if lesion that realizes to make 1.5 scores if full marks are 5 points of the lobe of the lung.Only to the lobe of the lung when scoring
Score on one side, if lobe of the lung tow sides have lesion, score on one side so that lesion area is big.Each lobe of the lung marking
Summation is that the pneumonia disease of the morbid pig becomes score.
2) after scoring respectively immune group pig and control group pig pneumonia lesion, it is immune to calculate each group according to following formula
The pneumonia lesion slip of pig.
Pneumonia lesion slip=(control pig pneumonia lesion score geometrical mean-immune swine pneumonia lesion score geometry
Average value)/control pig pneumonia lesion score geometrical mean × 100%.
Sequence table
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<400> 4
cctacgtggt ctacatttcc 20
Claims (8)
1. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the vaccine contains mycoplasma hyopneumoniae
(M.hyopneumoniae, Mhp) strain is ZY plants, this plant of mycoplasma hyopneumoniae delivers Beijing on May 08th, 2018
No. 3 China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1 are common
The preservation of microorganism center, deposit number are CGMCC No.15244；In vaccine the content of inactivation antigen be CCU not less than 1.5 ×
108/ head part.
2. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the vaccine susceptible piglet 5 of 2~3 week old health, each flesh
Meat vaccinates 4.0ml, observes 14, the locally or systemically adverse reaction caused by vaccine, and all strong work should not occurs.
3. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the efficacy test result of vaccine Immunization method with it is right
It is compared according to group, the pneumonia lesion slip of immune group test pig is not less than 75%.
4. a kind of mycoplasma hyopneumoniae inactivated vaccine, it is characterised in that the vaccine preserves 27 months at 2~8 DEG C.
5. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine of the present invention, it is characterised in that preparation method is such as
Under：
(1) production seed is prepared：Freeze-drying lactobacillus is broken seal, strain is inoculated in fluid nutrient medium by 10% inoculum concentration, in
37~37.5 DEG C cultivate 3~7 days, and Medium's PH Value be 6.8 when, obtain level-one production seed；Two level production kind is taken with legal system
Son；
(2) bacterium solution culture：It is packed by the 60% of fermenter volume and improves GoodwinShi fluid nutrient mediums, 121 DEG C of sterilizing 20min,
When culture medium temperature is cooled to 37 DEG C, 20% healthy Swine serum and 250 units/ml penicillin is added, stirs evenly, by culture
10% inoculation two level of base total amount produces seed, and 100r/min stir cultures 3~7 days under the conditions of 37 DEG C wait for that culture medium color becomes
It is yellow, harvest bacterium solution when being down to 6.5 or so by 7.5 in slight muddy, pH value, the viable bacteria titre in bacterium solution should be not less than 1 ×
108CCU/ml；
(3) it inactivates and blocks：The 0.2mol/L BEI solution of sterilizing is added in fermentation tank, makes the final concentration of 5% (V/ of inactivator
V), stir evenly, then fermentation tank bacterium solution is squeezed into and is heated up in 37 DEG C of inactivation tank in advance, 37 DEG C of inactivations are stirred 24 hours；
After inactivation stops, sampling carries out inactivation inspection, while final concentration of 1.5%~2.0% thio sulphur being added in inactivated bacterial liquid
Acid sodium solution, 22~25 DEG C are stirred continuously 2 hours, neutralize remaining BEI；
(4) it concentrates：Harvest bacterium solution is concentrated into the 1/10 of original volume through 200Ku molecular weight ultrafiltration membrane packets；
(5) match seedling：1 part of Montanide GEL 01RP water-soluble adjuvants ratio is added to carry out with seedling, stirring at low speed in 9 parts of bacterium solutions,
1% thimerosal solution of preceding addition is stirred terminating, its final concentration is made to be no more than 0.01%.
6. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine of the present invention, which is characterized in that described in step (1)
Culture medium is improvement GoodwinShi A26 fluid nutrient mediums.
7. a kind of preparation method of mycoplasma hyopneumoniae inactivated vaccine of the present invention, it is characterised in that the improvement
The group of GoodwinShi A26 fluid nutrient mediums becomes：PPLO powder 20.0g, 25% yeast leachate 100.0ml, glucose
5.0g, L- cysteine hydrochloride 0.1g, Sodium Pyruvate 2g, MEM 20g, ampicillin 0.25g, phenol red 0.02g, water for injection
Add to 1000ml.
8. a kind of efficacy test of mycoplasma hyopneumoniae inactivated vaccine of the present invention, it is characterised in that following method optionally its
One：
(1) IHA methods：With the susceptible rabbit 5 of 1.5~2.0kg health, each leg muscle vaccinates 0.5ml, by identical after 14 days
Dosage and approach carry out two and exempt from；Other 5 are not inoculated with as a contrast, and two exempt from 30 days afterwards, together with control rabbit blood sampling, are examined with IHA methods
Rabbit anteserum mycoplasma hyopneumoniae antibody titer is surveyed, control group Serum Antibodies of Rabbits answers all feminine genders, and immunizing rabbit should at least 4
Serum Antibodies of Rabbits potency is not less than 1:16；
(2) Immunization method：With the susceptible piglet 5 of 2~3 week old health, each musculi colli vaccinates 2.0ml, phase is pressed after 3 weeks
With mode and dosage two are carried out to exempt from, two exempt from after 35 days, together with control pig 5, each intratracheal injection mycoplasma hyopneumoniae poison ZY by force
The suspension 5.0ml of lung tissue containing viral disease is observed 28, and mycoplasma hyopneumoniae infection classical symptom, off-test should occur in control pig
When cut open and kill total Test pig, score the lung of each test pig disease lesion of breathing, and compare by Goodwin55 minute marks point-score
Group is compared, and the pneumonia lesion slip of immune group test pig reaches 79.8%~83.8%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109468248A (en) * | 2018-12-14 | 2019-03-15 | 南京天邦生物科技有限公司 | A kind of porcine mycoplasmal pneumonia antigen high-efficient culture method |
| CN110438029A (en) * | 2019-06-25 | 2019-11-12 | 成都天邦生物制品有限公司 | A kind of novel ablation method of mycoplasma hyopneumoniae |
| CN114836341A (en) * | 2022-04-15 | 2022-08-02 | 辽宁益康生物股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN115181709A (en) * | 2022-08-31 | 2022-10-14 | 天津瑞普生物技术股份有限公司 | Mycoplasma hyorhinis culture medium |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109468248A (en) * | 2018-12-14 | 2019-03-15 | 南京天邦生物科技有限公司 | A kind of porcine mycoplasmal pneumonia antigen high-efficient culture method |
| CN110438029A (en) * | 2019-06-25 | 2019-11-12 | 成都天邦生物制品有限公司 | A kind of novel ablation method of mycoplasma hyopneumoniae |
| CN114836341A (en) * | 2022-04-15 | 2022-08-02 | 辽宁益康生物股份有限公司 | Mycoplasma hyopneumoniae culture medium and preparation method thereof |
| CN115181709A (en) * | 2022-08-31 | 2022-10-14 | 天津瑞普生物技术股份有限公司 | Mycoplasma hyorhinis culture medium |
| CN115181709B (en) * | 2022-08-31 | 2023-07-11 | 天津瑞普生物技术股份有限公司 | Mycoplasma hyopneumoniae culture medium |
| 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 |
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Inventor after: Song Yanli Inventor after: Wang Baohui Inventor after: Xu Huan Inventor after: Lu Zhongwen Inventor after: Guo Limin Inventor before: Song Yanli Inventor before: Zhu Ping Inventor before: Wang Baohui Inventor before: Xu Huan Inventor before: Lu Zhongwen Inventor before: Han E Inventor before: Guo Limin Inventor before: Si Xiangling |
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Application publication date: 20180814 |