CN101721694B - Hircine primary atypical pneumonia combined inactivated vaccine and preparation method thereof - Google Patents
Hircine primary atypical pneumonia combined inactivated vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a hircine primary atypical pneumonia combined inactivated vaccine and a preparation method thereof. The preparation method comprises the following steps of: a. a culture medium for vaccine preparation and a preparation method thereof: using an improved KM2 culture medium as the culture medium for vaccine preparation; b. a procedure for propagating mycoplasma; c. a procedure for inactivating the mycoplasma; and d. a vaccine preparation procedure: using the one-step emulsion process to prepare bi-phase oil emulsion vaccine; the liquid-phase composition: accounting for 45-50% of the total amount of the vaccine, and adding 1% Merthiolate solution which accounts for 1% of the total amount of antigens after mixing protonema mycoplasma hircine subspecies and ovine mycoplasma pneumoniae inactivated antigens in a ratio of 1:1; the oil-phase composition: accounting for 50-55% of the total amount of the vaccine and containing ISA206 oil adjuvant; and emulsifying into a water/oil/water (w/o/w) bi-phase oil emulsion inactivated vaccine with slight viscosity. The invention first combines the two dominant pathogens to prepare the combined vaccine, and can simultaneously prevent the two pathogens, thereby avoiding the defect that the single-pathogen preparation vaccine can be only used for one pathogen but not for the other, and being more applicable to the practical production in China.
Description
Technical field
The present invention relates to mycoplasma capri pneumovax field, is a kind of Hircine primary atypical pneumonia combined inactivated vaccine and preparation method thereof specifically.
Background technology
The mycoplasma capri pneumonia refers to a kind of contact respiratory infectious disease that betides goat that caused by mycoplasma, it faces to examine and is characterized as high heat, cough, breast and pleura generation serosity and fibrinous inflammation, get acute and chronic process, sickness rate is 22～30%, have up to 60～80%, mortality rate is 15～30%.Primary disease is common in each sheep raising countries and regions, the world, has a strong impact on the development that world's sheep husbandry is produced.In China, primary disease is for a long time popular, literature record can be traced back to nineteen twenty-two the earliest, especially along with developing rapidly of China's sheep husbandry in recent years, this sick epidemic regions increases, and more than the ten provinces and cities reports such as existing Gansu, Ningxia, Xinjiang, Qinghai, Sichuan, Guizhou, Guangxi, Hebei, Liaoning, Jiangsu, Inner Mongol, Jiangsu, Hunan, Henan are popular so far, and the state of an illness is complicated, harm increases the weight of, and annual sheep husbandry production to China has brought serious economic loss.
The anti-system of mycoplasma capri pneumonia faces larger difficulty, and main cause is that its Etiology factor is complicated.Except the popular advantage cause of disease Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma of China, this disease pathogen of having reported in the world at present also comprises mycoplasma capri goat pneumonia subspecies, mycoplasma capri goat subspecies and mycoplasma mycoides subsp macrocolony type.Between these cause of diseases without cross protection; and the popular advantage cause of disease in various countries is not quite similar; add that the mycoplasma genome is less; the same mycoplasma also easily morphs and causes antigenic difference, so countries in the world are generally all developed pointedly the inactivated vaccine that is suitable for this area according to the concrete popularity in this locality and carried out immunoprophylaxis.
Be popular in and lack cross protection between the former Mycoplasma mycoides subsp.capri of mycoplasma capri pneumonia disease of China and the pneumonia of sheep mycoplasma; and associated diseases is difficult to distinguish clinically; and often understand mixed infection; therefore use the very difficult adaptation of vaccine China production reality that single cause of disease is made, and China does not have also to prevent simultaneously the bivalent inactivated vaccine of these two kinds of cause of diseases at present.
Summary of the invention
The purpose of this invention is to provide a kind of bivalent inactivated vaccine that can prevent simultaneously Mycoplasma mycoides subsp.capri and these two kinds of cause of diseases of pneumonia of sheep mycoplasma and preparation method thereof, be used for the immunoprophylaxis of mycoplasma capri pneumonia.
The objective of the invention is to realize by following technical proposals.
A kind of Hircine primary atypical pneumonia combined inactivated vaccine, contain Mycoplasma mycoides subsp.capri (Latin name: Mycoplasma mycoides subsp.capri strain M87-1) and the pneumonia of sheep mycoplasma (Latin name: Mycoplasma ovipneumnoiae strain MoGH3-3) inactivation antigen and ISA206 oil adjuvant, described Hircine primary atypical pneumonia combined inactivated vaccine prepares according to following step and method:
The a seedling is with culture medium and preparation method thereof: the seedling culture medium is improvement KM2 culture medium;
A.1 medium component: minimum key element culture medium MEM accounts for 50% of total amount, 1.7% lactoalbumin hydrolysate Hank ' s buffer accounts for 35% of total amount, 25% yeast extract accounts for 2% of total amount, healthy horse serum accounts for 10% of total amount, 1% thaliium acetate solution accounts for 1% of total amount, 20,000 IU penicillins account for 1%, 0.4% phenol red 1% of the total amount that accounts for;
A.2 culture medium preparation method: with 1.7% lactoalbumin hydrolysate Hank ' s buffer, 25% yeast extract, 1% thaliium acetate solution and 0.4% phenol red through 10 pounds of 20 minutes autoclavings, the minimum key element culture medium MEM, healthy horse serum and the penicillin that add Entkeimung after the cooling, adjust pH value to 7.4～7.8 with the 1mol/L sodium hydroxide solution, aseptic subpackaged, 2～8 ℃ save backup;
The program of b propagation mycoplasma
B.1 the propagation of mycoplasma: Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma are inoculated respectively improvement KM2 culture medium, cultivated through 3～5 days, when the culture pH value drops to 6.8～7.0, carry out continuous amplification culture 3～5 times by 5% of culture medium total amount, when the culture pH value drops to 6.8～7.0, results mycoplasma bacterium liquid is measured the growth titre and is carried out ultrafiltration and concentration;
B.2 mycoplasma is concentrated: with ultrafiltration membrane system mycoplasma bacterium liquid is concentrated, make Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma bacterial concentration all reach 6.0～10.0 * 10
8Ccu/ml;
C. the program of mycoplasma deactivation
C.1 deactivation: the formalin of adding respectively concentration 40% for concentrated Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma bacterium liquid, make the concentration of formaldehyde in the bacterium liquid reach 0.2%, 37 ℃ of lower each lasting deactivations 10 hours, shake was 1 time in per 2～4 hours during this time, deactivation stops, and carries out the deactivation check;
C.2 deactivation check: the mycoplasma bacterium liquid of deactivation is sampled, and 2 of each inoculation improvement KM2 culture medium are carried out 10 times of serial dilutions to 10
-5, put 37 ℃ and cultivated 10, it is complete for deactivation all to give birth to the elder without mycoplasma;
D. vaccine preparation procedure
Adopt a step emulsion process, make the two-phase oil emulsion vaccine;
Liquid phase ingredient: after 1: 1 by volume equal proportion of Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma inactivation antigen is mixed, press 1% of antigen total amount and add 1% thimerosal solution; Liquid phase ingredient accounts for 45～50% of vaccine total amount;
Oil-phase component: ISA206 oil adjuvant, oil-phase component accounts for 50～55% of vaccine total amount;
Emulsification method: first 206 oily adjuvants are fed in the homogenizer, again with the liquid phase ingredient charging, circulation stirring by homogenizer, is emulsified into slightly W/O/W type (w/o/w) the two-phase oil-emulsion inactivated vaccine of viscosity to fully mixing under 36～38MPa pressure.
The check of e vaccine
E.1 physical behavior check: this vaccine is mineral oil two-phase emulsion-type, and outward appearance should be creamy white or rose pink slightly viscous liquid.Centrifugal 15 minutes of 3000rpm should be not stratified.Be the 1ml suction pipe of 1.2mm with the outlet internal diameter, draw 25 ℃ vaccine 1ml, make its vertical natural flow out, record flows out the 0.4ml required time, should be within 8 seconds.
E.2 steriling test: undertaken by 15 pages of People's Republic of China's veterinary drug allusion quotation (version in 2005) appendix, should be without bacterial growth.
E.3 safety verification: use 5 of healthy susceptible goats (looking into without Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma serum antibody) about 12 monthly ages available from Pest-or disease-free area, each cervical region subcutaneous injection vaccine 6ml, observed 30, should be without subtracting the untoward reaction such as food, depressed, the body temperature reaction of spirit, injection site inflammation, and all strong work.
E.4 efficacy test: use 10 of healthy susceptible goats (looking into without Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma serum antibody) about 12 monthly ages available from Pest-or disease-free area, per 51 components are immune I group and II group, each cervical region subcutaneous injection vaccine 3ml, in addition with 10 of basic identical with the immune sheep goats of condition, per 51 components are organized for contrast I group and II.Inoculate rear 30 days, each trachea inoculation pneumonia of sheep mycoplasma culture 10ml (5 * 10 of immune I group and contrast I group goat
8Ccu/ml), observed 30, the contrast sheep all falls ill, and immune sheep is protected 4 at least, or 4 morbidities of contrast sheep, and immune sheep all protection is qualified.Each trachea inoculation Mycoplasma mycoides subsp.capri culture 10ml (5 * 10 of immunity II group and contrast II group goat
8Ccu/ml), observed 30, the contrast sheep all falls ill, and immune sheep is protected 4 at least, or 4 morbidities of contrast sheep, and immune sheep all protection is qualified.
The invention has the advantages that: (1) has used efficient mycoplasma culture medium, and Mycoplasma mycoides subsp.capri and the pneumonia of sheep mycoplasma growth titre in improvement KM2 culture medium all can reach 1 * 10
8Ccu/ml; (2) first these two kinds of advantage cause of diseases are united the preparation bigeminy vaccine, can prevent simultaneously two kinds of cause of diseases, avoided preparing the shortcoming that vaccine is attended to one thing and lose sight of another with single cause of disease, be more suitable for China and produce actual;
(3) this vaccine is applicable to the goat at different cultivars, various ages to the equal safety of the goat at different physiological situations and age.No matter support only size, each cervical region subcutaneous injection vaccine 3ml, the immune protective rate of the mycoplasma capri pneumonia that Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma are caused all can reach more than 4/5.Effective duration of immunity that vaccine is once inoculated is 10 months.2～8 ℃ of lower storage lives are 12 months.
The specific embodiment
Embodiment 1,
1 preparation culture medium
11～12 March in 2005, preparation improvement KM2 culture medium 42000ml.With 1.7% lactoalbumin hydrolysate Hank ' s buffer 14700ml, 25% yeast extract 840ml, 1% thaliium acetate solution 420ml and 0.4% phenol red 420ml, mixing is by 10 pounds of 20 minutes autoclavings, add minimum key element culture medium MEM21000ml, healthy horse serum 4200ml, 20,000 IU penicillin 420ml with 0.22 μ m microporous filter membrane Entkeimung after the cooling, being 7.8,2 ℃ with 1mol/L sodium hydroxide solution adjustment pH value to pH value saves backup.
2 propagation mycoplasmas
2.1 the propagation of mycoplasma: on March 12nd, 2005 is with Mycoplasma mycoides subsp.capri M87-1 strain, inoculation improvement KM2 culture medium 5ml, cultivated through 3 days, the culture pH value drops to 7.0, carry out continuous amplification culture 3 times by 5% of culture medium total amount, the culture pH value drops at 7.0 o'clock, results Mycoplasma mycoides subsp.capri M87-1 bacterium liquid 18000ml, and measuring its growth titre is 6.7 * 10
7Ccu/ml.
On March 12nd, 2005, inoculation improvement KM2 culture medium 10ml cultivated through 3 days with pneumonia of sheep mycoplasma MoGH3-3 strain, and the culture pH value drops to 7.0.Carry out continuous amplification culture 3 times by 5% of culture medium total amount, the culture pH value drops at 7.0 o'clock, results pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid 18000ml, and measuring its growth titre is 1 * 10
8Ccu/ml.
2.2 mycoplasma is concentrated: with ultrafiltration membrane system (PALL Centrasette 5) Mycoplasma mycoides subsp.capri M87-1 bacterium liquid is carried out 10 times and concentrate, making bacterial concentration is 6.7 * 10
8Ccu/ml; Pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid is carried out 10 times concentrate, making bacterial concentration is 10.0 * 10
8Ccu/ml.
3 deactivation mycoplasmas
3.1 deactivation: make its concentration reach 0.2% (40% formalin) to concentrated Mycoplasma mycoides subsp.capri M87-1 and pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid adding formalin (40%) respectively, 37 ℃ of lower each lasting deactivations 10 hours, during per 2 hours shakes 1 time.
3.2 deactivation check: inactivation time arrives, with the Mycoplasma mycoides subsp.capri M87-1 of deactivation and the pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid 0.2ml that respectively samples, and 2 of each inoculation improvement KM2 culture medium, every 0.1ml carries out 10 times of serial dilutions to 10
-5, put 37 ℃ and cultivated 10, all grow without mycoplasma.
The preparation of 4 vaccines
Adopt a step emulsion process, make the two-phase oil emulsion vaccine.
Liquid phase ingredient: Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma inactivation antigen mix altogether 3600ml in 1: 1 ratio, press 1% of antigen total amount and add 1% thimerosal solution 36ml, and 3636ml accounts for 50% of total amount altogether.
Oil-phase component: ISA206 oil adjuvant 3636ml accounts for 50% of total amount.
Emulsification method: first ISA 206 oily adjuvants are fed in the homogenizer, again with the liquid phase ingredient charging, circulation stirring is to fully mixing, under 36MPa pressure, pass through homogenizer, be emulsified into slightly W/O/W type (w/o/w) the two-phase oil-emulsion inactivated vaccine of viscosity, lot number 050502, in batches 6500ml.
5 vaccine tests
5.1 the physical behavior check: this vaccine is mineral oil two-phase emulsion-type, and outward appearance is creamy white.Centrifugal 15 minutes of 3000rpm is not stratified.Be the 1ml suction pipe of 1.2mm with the outlet internal diameter, draw 25 ℃ vaccine 1ml, make its vertical natural flow out, flowing out the 0.4ml required time is 3 seconds.
5.2 steriling test: undertaken by 15 pages of People's Republic of China's veterinary drug allusion quotation (version in 2005) appendix, all without bacterial growth.
5.3 safety verification: use 5 of healthy susceptible goats (looking into without Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma serum antibody) about 12 monthly ages available from Pest-or disease-free area, each cervical region subcutaneous injection vaccine 6ml, observed 30, without subtracting the untoward reaction such as food, depressed, the body temperature reaction of spirit, injection site inflammation, and all be good for alive.
5.4 efficacy test: use 10 of healthy susceptible goats (looking into without Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma serum antibody) about 12 monthly ages available from Pest-or disease-free area, per 51 components are immune I group and II group, each cervical region subcutaneous injection vaccine 3ml, in addition with 10 of basic identical with the immune sheep goats of condition, per 51 components are organized for contrast I group and II.Inoculate rear 30 days, and each trachea inoculation pneumonia of sheep mycoplasma MoGH3-3 culture 10ml of immune I group and contrast I group goat (5 * 10^8ccu/ml), observed 30.The result contrasts all (5/5) morbidities of sheep, immune sheep 4/5 protection.Each trachea inoculation Mycoplasma mycoides subsp.capri M87-1 culture 10ml (5 * 10 of immunity II group and contrast II group goat
8Ccu/ml), observed 30.The result contrasts sheep 4/5 morbidity, and immune sheep is (5/5) protection all.
Embodiment 2,
1 preparation culture medium
1～2 April in 2005, preparation improvement KM2 culture medium 42000ml.With 1.7% lactoalbumin hydrolysate Hank ' s buffer 14700ml, 25% yeast extract 840ml, 1% thaliium acetate solution 420ml, 0.4% phenol red 420ml mixes by 10 pounds of 20 minutes autoclavings, adds minimum key element culture medium (MEM) 21000ml with 0.22 μ m microporous filter membrane Entkeimung after the cooling, healthy horse serum 4200ml, 20,000 IU penicillin 420ml are 7.4,8 ℃ with 1mol/L sodium hydroxide solution adjustment pH value to pH value and save backup.
2 propagation mycoplasmas
2.1 the propagation of mycoplasma: on April 10th, 2005, inoculation improvement KM2 culture medium 10ml cultivated through 5 days with Mycoplasma mycoides subsp.capri M87-1 strain, and the culture pH value drops to 6.8.Carry out continuous amplification culture 3 times by 5% of culture medium total amount, the culture pH value drops at 6.8 o'clock, results Mycoplasma mycoides subsp.capri M87-1 bacterium liquid 20000ml, and measuring its growth titre is 1 * 10
8Ccu/ml.
On April 10th, 2005, inoculation improvement KM2 culture medium 10ml cultivated through 5 days with pneumonia of sheep mycoplasma MoGH3-3 strain, and the culture pH value drops to 6.8.Carry out continuous amplification culture 3 times by 5% of culture medium total amount, the culture pH value drops at 6.8 o'clock, results pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid 20000ml, and measuring its growth titre is 1 * 10
8Ccu/ml.
2.2 mycoplasma is concentrated: with ultrafiltration membrane system (PALL Centrasette 5) Mycoplasma mycoides subsp.capri M87-1 bacterium liquid is carried out 6 times and concentrate, making bacterial concentration is 6.0 * 10
8Ccu/ml; Pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid is carried out 6 times concentrate, making bacterial concentration is 6.0 * 10
8Ccu/ml.
3 deactivation mycoplasmas
3.1 deactivation: make its concentration reach 0.2% (40% formalin) to concentrated Mycoplasma mycoides subsp.capri M87-1 and pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid adding formalin (40%) respectively, 37 ℃ of lower each lasting deactivations 10 hours, during per 4 hours shakes 1 time.
3.2 the deactivation check: inactivation time arrives, with the Mycoplasma mycoides subsp.capri M87-1 of deactivation and the pneumonia of sheep mycoplasma MoGH3-3 bacterium liquid 0.2ml that respectively samples, 2 of each inoculation improvement KM2 culture medium, every 0.1ml, carry out 10 times of serial dilutions to 10^-5, put 37 ℃ and cultivated 10, all grow without mycoplasma.
The preparation of 4 vaccines
Adopt a step emulsion process, make the two-phase oil emulsion vaccine.
Liquid phase ingredient: Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma inactivation antigen mix altogether 4000ml by 1: 1 equal proportion, press 1% of antigen total amount and add 1% thimerosal solution 40ml, altogether 4040ml (45%).
Oil-phase component: ISA206 oil adjuvant 4940ml accounts for 55% of total amount.
Emulsification method: first 206 oily adjuvants are fed in the homogenizer, again with the liquid phase ingredient charging, circulation stirring is to fully mixing, under 38MPa pressure, pass through homogenizer, be emulsified into slightly W/O/W type (w/o/w) the two-phase oil-emulsion inactivated vaccine of viscosity, lot number 050603, in batches 8000ml.
5 vaccine tests
5.1 the physical behavior check: this vaccine outward appearance is slightly pink milky Emulsion.3000rpm did not have layering in centrifugal 15 minutes.Be the 1ml suction pipe of 1.2mm with the outlet internal diameter, draw 25 ℃ vaccine 1ml, make its vertical natural flow out, flowing out the 0.4ml required time is 4 seconds.
5.2 steriling test: undertaken by 15 pages of People's Republic of China's veterinary drug allusion quotation (version in 2005) appendix, all without bacterial growth.
5.3 safety verification: use 5 of healthy susceptible goats (looking into without Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma serum antibody) about 12 monthly ages available from Pest-or disease-free area, each cervical region subcutaneous injection vaccine 6ml, observed 30, without subtracting the untoward reaction such as food, depressed, the body temperature reaction of spirit, injection site inflammation, and all be good for alive.
5.4 efficacy test: use about 12 monthly ages available from Pest-or disease-free area 10 of healthy susceptible goats to look into without Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma serum antibody, per 51 components are immune I group and II group, each cervical region subcutaneous injection vaccine 3ml, in addition with 10 of basic identical with the immune sheep goats of condition, per 51 components are organized for contrast I group and II.Inoculate rear 30 days, each trachea inoculation pneumonia of sheep mycoplasma MoGH3-3 culture 10ml (5 * 10 of immune I group and contrast I group goat
8Ccu/ml), observed 30.The result contrasts sheep 4/5 morbidity, immune sheep 5/5 protection.Each trachea inoculation Mycoplasma mycoides subsp.capri M87-1 culture 10ml (5 * 10 of immunity II group and contrast II group goat
8Ccu/ml), observed 30.The result contrasts sheep 5/5 morbidity, immune sheep 4/5 protection.
Claims (1)
1. Hircine primary atypical pneumonia combined inactivated vaccine, it is characterized in that containing Mycoplasma mycoides subsp.capri, Latin name: Mycoplasma mycoides subsp.capri strain and pneumonia of sheep mycoplasma, Latin name: Mycoplasma ovipneumnoiae strain inactivation antigen and ISA206 oil adjuvant, adopt following methods and step to be prepared from:
The a seedling is with culture medium and preparation method thereof: the seedling culture medium is improvement KM2 culture medium;
A.1 medium component: minimum key element culture medium MEM accounts for 50% of total amount, 1.7% lactoalbumin hydrolysate Hank ' s buffer accounts for 35% of total amount, 25% yeast extract accounts for 2% of total amount, healthy horse serum accounts for 10% of total amount, 1% thaliium acetate solution accounts for 1% of total amount, 20,000 IU penicillins account for 1%, 0.4% phenol red 1% of the total amount that accounts for;
A.2 culture medium preparation method: with 1.7% lactoalbumin hydrolysate Hank ' s buffer, 25% yeast extract, 1% thaliium acetate solution and 0.4% phenol red through 10 pounds of 20 minutes autoclavings, the minimum key element culture medium MEM, healthy horse serum and the penicillin that add Entkeimung after the cooling, adjust pH value to 7.4～7.8 with the 1mol/L sodium hydroxide solution, aseptic subpackaged, 2～8 ℃ save backup;
The program of b propagation mycoplasma
B.1 the propagation of mycoplasma: Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma are inoculated respectively improvement KM2 culture medium, cultivated through 3～5 days, when the culture pH value drops to 6.8～7.0, carry out continuous amplification culture 3～5 times by 5% of culture medium total amount, when the culture pH value drops to 6.8～7.0, results mycoplasma bacterium liquid is measured the growth titre and is carried out ultrafiltration and concentration;
B.2 mycoplasma is concentrated: with ultrafiltration membrane system mycoplasma bacterium liquid is concentrated, make Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma bacterial concentration all reach 6.0～10.0 * 10
8Ccu/ml;
C. the program of mycoplasma deactivation
C.1 deactivation: the formalin of adding respectively concentration 40% for concentrated Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma bacterium liquid, make the concentration of formaldehyde in the bacterium liquid reach 0.2%, 37 ℃ of lower each lasting deactivations 10 hours, shake was 1 time in per 2～4 hours during this time, deactivation stops, and carries out the deactivation check;
C.2 deactivation check: the mycoplasma bacterium liquid of deactivation is sampled, and 2 of each inoculation improvement KM2 culture medium are carried out 10 times of serial dilutions to 10
-5, put 37 ℃ and cultivated 10, it is complete for deactivation all to give birth to the elder without mycoplasma;
D. vaccine preparation procedure
Adopt a step emulsion process, make the two-phase oil emulsion vaccine;
Liquid phase ingredient: after 1: 1 by volume equal proportion of Mycoplasma mycoides subsp.capri and pneumonia of sheep mycoplasma inactivation antigen is mixed, press 1% of antigen total amount and add 1% thimerosal solution; Liquid phase ingredient accounts for 45～50% of vaccine total amount;
Oil-phase component: ISA206 oil adjuvant, oil-phase component accounts for 50～55% of vaccine total amount;
Emulsification method: first 206 oily adjuvants are fed in the homogenizer, again with the liquid phase ingredient charging, circulation stirring by homogenizer, is emulsified into slightly W/O/W type (w/o/w) the two-phase oil-emulsion inactivated vaccine of viscosity to fully mixing under 36～38MPa pressure.
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| CN103110583A (en) * | 2013-03-05 | 2013-05-22 | 西南民族大学 | Preparation technology of mycoplasma ovipneumoniae inactivated vaccine |
| CN105325505A (en) * | 2014-08-07 | 2016-02-17 | 王秀红 | Total nutrient convenient lunch bag for field travel survival people and preparation method for same |
| CN105859845B (en) * | 2016-04-14 | 2019-03-12 | 石河子大学 | Mycoplasma ovine pneumoniae multi-epitope fusion antigen MO-meAg5 and its preparation method and application |
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| CN111789941B (en) * | 2019-04-09 | 2022-04-19 | 内蒙古华希生物科技有限公司 | Bivalent inactivated vaccine for mycoplasma pneumonia and chlamydia psittaci disease of goats and preparation method thereof |
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