CN106492210A - Goats contagious pleuropneumonia inactivated vaccine and production method thereof - Google Patents
Goats contagious pleuropneumonia inactivated vaccine and production method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of goats contagious pleuropneumonia inactivated vaccine and preparation method thereof. the present invention adopts mycoplasma capri goat pneumonia subspecies C87001 strain, inoculation appropriate media, results culture, after concentrated, thimerosal deactivation, add mineral oil adjuvant mixing and emulsifying and make. for the goats contagious pleuropneumonia of preventing mycoplasma capri goat pneumonia subspecies to cause; The fluid nutrient medium production goats contagious pleuropneumonia inactivated vaccine that adopts the present invention to propose, there is obvious advantage compared with basic stitch inactivated vaccine, greatly reduce bio-safety risk in production of vaccine process, reduced production cost, and prepared vaccine is not prone to allergic reaction, and security is higher.
Description
Technical field
The present invention relates to a kind of goats contagious pleuropneumonia inactivated vaccine and production method thereof. belong to veterinary biologics field.
Background technology
The development of sheep husbandry and national economy and people of all nationalities' growth in the living standard relation are very close, particularly enter after 21 century, China's sheep husbandry is rapidly developed, achievement is remarkable, the proportion of sheep husbandry in national economy also improves year by year. according to FAO (Food and Agriculture Organization of the United Nation) (FAO) data, 2013, the sheep cultivation amount of CONTINENTAL AREA OF CHINA reaches 1.91 hundred million, goat 1.83 hundred million, add up to 3.74 hundred million .2014, the sheep cultivation amount of CONTINENTAL AREA OF CHINA is 2.02 hundred million, 1.88 hundred million of goats, although add up to 3.90 hundred million. China's sheep raising number of bits occupies first place in the world, but compared with the country of sheep husbandry prosperity, control and prevention of disease water flat weaker, this has restricted the development .2014 of China's sheep husbandry on certain procedures, National modern Mutton Sheep Industry technical system respectively to the Inner Mongol, national mutton sheep main producing region, Xinjiang, Ningxia, Gansu, (the district of Jin60Ge county of 14 provinces, autonomous regions and municipalities such as Qinghai, flag) in " national Mutton Sheep Industry investigation report in 2014 " that Mutton Sheep Industry current situation carries out forming after special investigations, point out: from investigation general status see, current have sheep Eaton agent pneumonia (infectiousness pleura to China's the most serious infectious disease of Mutton Sheep Industry harm pneumonia), streptococcosis, clostridial disease, sheep pox, sheep infective warts (sheep aphtha), lamb dysentery and sheep enterotoxemia. these are all sick Cause different degrees of death, the loss of sheep raising family is serious. is wherein the most noticeable is that serious sheep mycoplasma occurs in recent years pneumonia, raise intensive intensive sheep farm and very high through the incidence of disease of a sheep sheep Eaton agent pneumonia of long-distance transport, dead tight Weight.When Ningxia, Anhui, Yunnan and Sichuan are investigated, it is found that all of intensive sheep farm all has infectious pleuropneumonia in sheep, only It is the degree difference of harm.
Goats contagious pleuropneumonia (Contagious caprine pleurpneumonia, CCPP) is by goat (M.capricolum subsp.capripneumoniae, Mccp, a kind of goat for causing are distinctive for substance goat pneumonia subspecies Acute or chronic highly contagious disease, is characterized with assuming fibrinous pneumonia and pleuritis, sickness rate be 22%～30%, indivedual areas are up to 60%～80%, the death rate 15%～30%.The disease is on the dozens ofs such as Africa, Asia country and ground There is prevalence in area, brings more serious loss to goat aquaculture, is classified as B class infectious disease by OIE. China is starting in Gansu in nineteen forty-seven, then in the Inner Mongol, Sichuan, Shandong, Hubei, Hebei, Yunnan, Jiangxi, Jiangsu Deng Di lands Supervention is existing.In recent years, there is the report of CCPP in the area such as Hunan, Henan, Guizhou, Chongqing, Inner Mongol, cause to goat aquaculture Larger economic loss.
At present, China's prevention goats contagious pleuropneumonia, relies primarily on " the goat of China Veterinery Drug Inspection Office's research and development Contagious pleuropneumonia inactivated vaccine ", the vaccine are widely used nearly 60 years in China, and long-term clinical practice proves the vaccine There is fabulous clinical immune protection effectiveness .But former vaccine injects goat using strong poison pipe, collection lung tissue makes goat biography Metachromia pleuropneumonia Attenuated vaccine, the vaccine are not suitable for industrialized production, there is the danger for dissipating poison; In lung tissue, miscellaneous bacteria contains Amount is high, and anaphylaxiss easily occurs in prepared vaccine.
Content of the invention
Present invention aim at preparing the goat contagious pleura lung produced using solution culture fermentation culture process
Scorching inactivated vaccine, for preventing goats contagious pleuropneumonia.
Technical solution of the present invention
1. a kind of goats contagious pleuropneumonia inactivated vaccine, it is characterised in that the inactivated vaccine contains using EZH liquid
The mycoplasma capricolum subsp. pneumonia antigen of goats of body culture medium culturing.
2. goats contagious pleuropneumonia inactivated vaccine of the present invention, it is characterised in that the mycoplasma capri Lung in Goats
Scorching subspecies antigen production bacterial strain is C87001 strains.
3. goats contagious pleuropneumonia inactivated vaccine of the present invention, it is characterised in that the fluid medium is EZH
Fluid medium, is made up of following component (W/W):Lactoalbumin hydrolysate 3.5%, yeast powder 1.5%, glucose 1.5%, PPLO meat
Powder 10.5%, 1% phenol red 0.1%, balance of water for injection；PH value is adjusted to 7.8 with the sodium hydroxide solution of sterilizing 1mol/L,
Through 0.22 μm of filtration sterilization.
4. the preparation method of goats contagious pleuropneumonia inactivated vaccine of the present invention, it is characterised in that use the goat
The strain of mycoplasma goat pneumonia subspecies C87001 is inoculated with appropriate media as production of vaccine bacterial strain, harvests culture warp after culture
Concentration, inactivation, plus mineral oil adjuvant mixing and emulsifying make goats contagious pleuropneumonia inactivated vaccine.
The specific embodiment of the invention
1. the preparation of goats contagious pleuropneumonia inactivated vaccine
(1) production and inspection strain mycoplasma capri goat pneumonia subspecies C87001 strain production basic bacteria, goatThe strain of mycoplasma goat pneumonia subspecies C87002 is vaccine test strain, (shouldBacterium original Classification And Nomenclature is thread mycoplasma goat pneumonia subspecies (Mycoplasma mycoides suhsp.capri Edwardand Freundt) (asks for an interview China Veterinery Drug Inspection Office to and being prepared and provided by China Veterinery Drug Inspection Office, veterinary microorganism culture presevation administrative center of China writes, China beast Doctor's strain catalogue (second edition), Scientia Agricultura Sinica technology publishing house, 2002, p89) .).
(2) first order seed breeding and identification take C87001 strains freeze-drying lactobacillus 1, EZH liquid of the inoculation containing 15% horse serum
Culture medium, puts 37 DEG C and cultivates 3～5, when Medium's PH Value drops to 7.0 or so, as seedling first order seed.
(3) secondary seed breeding and identification take first order seed, and EZH fluid medium of the inoculation containing 15% horse serum puts 37
DEG C culture 3～5 days, when Medium's PH Value drops to 7.0 or so, as seedling secondary seed.
(4) antigen for vaccine is prepared and takes qualified secondary seed, contains 15% horse serum by 2% inoculation of culture medium total amountEZH fluid mediums, 37 DEG C culture, when culture pH value drops to 7.0 or so, terminate culture.Sampling is purely examinedTest and viable bacteria titer determination. viable bacteria titre answers >=1 × 109.0CCU/ml.
(5) antigen concentration is concentrated by ultrafiltration system using 20,000 molecular cut offs (MW) and concentrates 15 times (V/V).
(6) 0.01% addition thimerosal of concentrated antigen total amount is pressed in inactivation, is fully mixed, little in 15～25 DEG C of effects 10
When, period stirs for several times.After inactivation terminates, 2～8 DEG C of preservations should be less than 30, and sampling carries out the inspection of semifinished product.
(7) vaccine formulation imports oily adjuvant in oil phase tank, matches somebody with somebody autoclaving 30 minutes with least 121, is cooled to room temperature
Standby.According to inactivation before viable bacteria titer determination result, with PBS (0.01mol/L, pH value 7.2) by concentration after antigen diluent extremely
Contain 1m10 per 1ml9.0CCU thalline.Oil phase is added in emulsion tank, is stirred with 80～100r/min, while pressing 1:The ratio of 1 (V/V)
Example, is slowly added to water phase, stirs 5～10min, with mulser with 5600r/min, 10～20min of circulating emulsion after adding.Emulsifying
Sampling is tested afterwards, qualified rear subpackage.
The above fluid medium of the invention is EZH fluid mediums, is made up of following component (W/W):The newborn egg of hydrolysis
White 3.5%, yeast powder 1.5%, glucose 1.5%, PPLO gravy powders 10.5%, 1% phenol red 0.1%, balance of water for injection；
PH value is adjusted to 7.8 with the sodium hydroxide solution of sterilizing 1mol/L, through 0.22 μm of filtration sterilization.
2. the inspection of goats contagious pleuropneumonia inactivated vaccine
(1) character
Appearance milky white or faint yellow Emulsion.
Dosage form water-in-oil type (W/O).A cleaning suction pipe is taken, a small amount of vaccine is drawn and is dripped in cold water, should not expand in oil droplet shape
Dissipate.
Stability is drawn vaccine 10ml and is added in centrifuge tube, is centrifuged 15min with 3000r/min, and the water that ttom of pipe is separated out is corresponding
Must not exceed 0.5ml.
Viscosity is by existing《Chinese veterinary pharmacopoeia》Annex is measured, and should meet regulation.
(2) loading quantity inspection is pressed《Chinese veterinary pharmacopoeia》(Chinese veterinary pharmacopoeia committee, Republic of China Veterinary Pharmacopoeia, two
1 year version three, Chinese agriculture publishing house, 2011, hereinafter referred to as《Chinese veterinary pharmacopoeia》) annex checked, should meet regulation.
(3) steriling test is pressed《Chinese veterinary pharmacopoeia》Annex is tested, and answers asepsis growth.
(4) safety verification 1.5～2.0kg of body weight healthy rabbits 5, each intramuscular injection vaccine 1.0ml；With 1 monthly age
The susceptible goat of health 2, each branch intramuscular injection vaccine 4.0ml are observed 10.Work should be all good for.
(5) efficacy test body weight is at least 20kg, the healthy susceptible goat 4 of 1～3 years old, and each cervical region is subcutaneously or intramuscularly
Vaccinate 2.0ml.Together with control goat 3 after 21 days, each trachea injection mycoplasma capri goat pneumonia subspecies are malicious by force
C87002 strain lung tissue Emulsion 4.0ml, observe 30.Control goat all should fall ill, and immune goat should at least protect 3.
(6) the antiseptic mercurials determination of residual amount is pressed《Chinese veterinary pharmacopoeia》Annex is measured, and should meet regulation.
The present invention relates to biomaterial resource information
Mycoplasma capri goat pneumonia subspecies (M.capricolum subsp.capripneumoniae, Mccp) C87001
Strain production basic bacteria, the strain of mycoplasma capri goat pneumonia subspecies C87002 are vaccine test strain, and such bacterium is former to classify
It is named as thread mycoplasma goat pneumonia subspecies (Mycoplasma mycoides suhsp.capri Edward and
Freundt) C87001 strains and C87002 strains, are prepared by China Veterinery Drug Inspection Office and are provided and (ask for an interview Chinese veterinary medicament
Supervision institute, Chinese veterinary microorganism culture presevation administrative center write, Chinese veterinary's strain catalogue (second edition), Chinese agriculture section
Technology publishing house, 2002, p89).
The positive effect of the present invention
The present invention relates to a kind of goats contagious pleuropneumonia inactivated vaccine and preparation method thereof.The present invention is using goat
Substance goat pneumonia subspecies C87001 strain, is inoculated with appropriate media, harvests culture, after the inactivation of concentrated, thimerosal, plus mineral
Oily adjuvant mixing and emulsifying is made, for the goats contagious pleuropneumonia for preventing mycoplasma capri goat pneumonia subspecies to cause.This
Invention produces goats contagious pleuropneumonia inactivated vaccine using solution culture fermentation culture process, compared with basic stitch inactivated vaccine
There is obvious advantage, that is, greatly reduce the bio-safety risk in vaccine production process, reduce production cost, and made
Standby vaccine is less prone to anaphylaxiss, and safety is higher.
Embodiment
Following examples are not construed as limiting to claimed technical scheme for further illustrating the present invention.
Embodiment 1
Vaccine is prepared and is checked
According to the production technology for determining, 3 batches of goats contagious pleuropneumonia inactivated vaccines are prepared for, now result is reported such as
Under.
1. material
(1) strain production strain：Goats contagious pleuropneumonia mycoplasma C87001 strain, F8 generations, viable bacteria titre are 1
×109.0CCU/ml；Inspection strain：Goats contagious pleuropneumonia C87002 strain tissue lyophilizing poison, code name 87002, generation：
52 generations, specification：1.0g/, identified by China Veterinery Drug Inspection Office, take care of and supplied.
(2) culture medium EZH fluid medium and steriling test culture medium, by China Veterinery Drug Inspection Office's culture medium
Group is provided.
The EZH fluid mediums, are made up of following component (W/W):Lactoalbumin hydrolysate 3.5%, yeast powder 1.5%, Portugal
Grape sugar 1.5%, PPLO gravy powders 10.5%, 1% phenol red 0.1%, balance of water for injection；Sodium hydroxide with sterilizing 1mol/L
Solution adjusts pH value to 7.8, through 0.22 μm of filtration sterilization.
(3) oily adjuvant Montanide ISA 50V adjuvants, French SEPPIC companies production.
(4) goat is used in laboratory animal test, and in 1 years old age, health, without dermatosiss, parasitic infection, through goat contagious breast
The serologic tests such as film mycoplasma pneumoniae F38 strains, mycoplasma ovine pneumoniae Y98 strains and brucellosiss are feminine gender, from
Sheep raising factory of Gansu Province.Rabbit, 1.5～2.0kg of body weight, health, from Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.
Laboratory animal field.
(5) filter (Polygard CR, 0.22 μm)：Retort (Foochow Fu Erte companies)；Ultrafiltration concentration equipment
(Millipore Products, Pellicon2 Cassette filters, using 20,000 molecular cut offs of Biomax, improve polyether sulfone materials,
0.5m2Membrane area membrane stack)；Emulsifying device (Germany produces 2000/4 type mulsers of IKA).
2. antigen is prepared and inspection
(1) goats contagious pleuropneumonia mycoplasma C87001 strain production seed is connect in the ratio of culture medium total amount 2%
Plant in the horse serum EZH fluid mediums containing 15%, cultivate in 37 DEG C, treat that culture pH value drops to 7.0 or so, harvest culture
Thing, harvests bacterium solution.3 batches of bacterium solutions are continuously prepared using the method, while sampling carries out purely inspection, viable bacteria titer determination.
(2) antigen detection
1) purely inspection is pressed《Chinese veterinary pharmacopoeia》Annex method is checked, purely.
2) with the EZH fluid mediums containing 15% horse serum, to carry out 10 times of series dilute by testing sample for viable bacteria titer determination
Release to 10-12, while set not plus bacterium solution containing 15% horse serum EZH fluid mediums as negative control.Put 37 DEG C to cultivate 7,
The highest dilution of culture medium color change is the titre of testing sample.With the average titer of 3 parts of samples, as bacterium solution titre
(CCU/ml).Assay is shown in Table 1.
The pure inspection of table 1 laboratory, 3 batches of product culture bacterium solutions and viable bacteria titre assay
Note："-" represents that pure assay is pure.
As it can be seen from table 13 batches of products of laboratory are pure, front bacterium solution viable bacteria titre is concentrated up to 7 × 109.0～1 ×
1010.0CCU/mll.
(3) concentration of seedling antigen
Qualified antigen culture is checked by 3 batches, respectively with 15 times of 20,000 molecular weight inner pressed ultrafiltration membrane stack ultrafiltration concentration,
Amount of antigen for stock solution amount 1/15 after concentration.
(4) inactivation of bacterium solution
Thimerosal is added concentration bacterium solution in 0.01% ratio, is fully mixed, respectively in the inactivation 10 hours of 20 DEG C of room temperature, phase
Between stir 5 times.After inactivation, 2～8 DEG C of preservations, and aseptic sampling carries out the inspection of semifinished product.
(5) inspection of semifinished product
1) every batch of inactivation antigen of steriling test is by existing《Chinese veterinary pharmacopoeia》Annex method is tested, without antibacterial and
Fungus growth.
2) inactivation inspection takes EZH fluid medium 2 of the inactivation antigen inoculation containing 15% horse serum, and every 0.2ml puts
37 DEG C are cultivated 5, are grown without mycoplasma.Assay is shown in Table 2.
The inspection of semifinished product result of 23 batches of products of table
Note："-" is indicated without thin, bacterium fungus growth.
From table 2 it can be seen that the antigens inactive of 3 batches of products of laboratory is complete.
(6) prepared by vaccine
1) according to viable bacteria titer determination result before inactivation, with PBS, (0.01mol/L, after 7.2) pH value will concentrate antigen diluent
Antigen diluent and adjust concentration, make every 1ml contain 1m109.0CCU thalline.
2) oil phase prepares SEPPIC Montanide ISA 50V2 oil adjuvant through 121 DEG C of sterilizing 30min, standby.
3) oil phase is pressed in vaccine emulsifying：Water is mutually 1:1 ratio is added in emulsion tank, is fully stirred with the rotating speed of 90r/min
Mix, then the cutter by rotating speed for 5600r/min is sheared, and makes vaccine be emulsified into Water-In-Oil dosage form.
4), after subpackage vaccine is sufficiently mixed, aseptically quantitative separating, seals bottleneck, 2～8 DEG C of preservations.
(7) product inspection
1) character detection
Outward appearance：3 batches of vaccines of trial-production are milky Emulsion.
Dosage form：Every batch of vaccine sampling is drawn a small amount of vaccine and is dripped in cold water, in water dust shape not respectively with a cleaning suction pipe
Diffusion.
Viscosity：By existing《Chinese veterinary pharmacopoeia》Annex method is detected that viscosity is respectively less than 200cP.
2) stability inspection：Every batch of vaccine is drawn 10ml and is added in centrifuge tube, is centrifuged 15 minutes with 3000 revs/min, pipe
The water that bottom separates out is mutually not less than 0.5ml.
3) loading quantity inspection：By existing《Chinese veterinary pharmacopoeia》Annex method detected, all should be not less than 100ml/ bottles.
4) steriling test：Every batch of product randomly selects 10 bottles, by existing《Chinese veterinary pharmacopoeia》Method is tested, without thin
Bacterium, mycete etc. grow.
5) the antiseptic mercurials determination of residual amount：By existing《Chinese veterinary pharmacopoeia》Carry out, meet veterinary biologicses inspection
General provision.
6) safety verification
With 1.5～2.0kg of body weight healthy rabbits 5, intramuscular injection 1.0ml/ is only；Healthy susceptible goat (mountain with 1 years old age
Sheep mycoplasma goat pneumonia subspecies antibody I HA potency is not higher than 1: 4) 2, and branch intramuscular injection 4.0ml/ is only.Observe 10,
All strong work.
7) efficacy test
More than body weight 20kg, the healthy susceptible goat of 1 years old 4, each cervical region subcutaneously or intramuscularly injects 2.0ml.After 21 days,
Goat 3 is compareed together with condition identical, each trachea injects the strong poison C87002 strain lung tissues of mycoplasma capri goat pneumonia subspecies
Agent 4.0ml (containing 15 minimum morbidity amounts), observes 30.Control goat 3/3 falls ill, and equal 4 of immune goat is all protected.
Embodiment 2
Report with comparable product comparative study
In order to compare goats contagious pleuropneumonia inactivated vaccine with comparable product in safety, effect and immune generation phase
Etc. aspect difference, take laboratory trial-production 3 batches of goats contagious pleuropneumonia inactivated vaccine products and domestic comparable product by 1
Part/only inoculation 1 years old age goat, compare the safety of two kinds of vaccinations and counteracting toxic substances protective rate etc. after 21 days.
1. test material
(1.) 3 batches of goats contagious pleuropneumonia inactivated vaccines of experimental vaccine：Lot number is 201001001,201001002
With 201001003.
(2) control vaccine goats contagious pleuropneumonia inactivated vaccine, lot number is 200901, by Harbin Pharmaceutical Group's biology epidemic disease
Seedling company limited produces；Mycoplasma capri goat pneumonia subspecies C87002 organize lyophilizing poison
(3) the strong poison C87002 strains of mycoplasma capri goat pneumonia subspecies, code name 87002, generation：52 generations, specification：1.0g/
, identified by China Veterinery Drug Inspection Office, take care of and supplied.
(4) laboratory animal 1) to test and use goat, in 1 years old age, purchased from certain plant, 20～40kg of body weight, health, without skin
Disease, parasitic infection, through mycoplasma capri goat pneumonia subspecies F38 strain, mycoplasma ovine pneumoniae Y-98 strains and brucella
The serologic tests such as disease are feminine gender.2) Cavia porcelluss, 350～450g of body weight, health；Rabbit, 1.5～2.0kg of body weight, health, Beijing
Gold is herded YANG excess and tests animal cultivation Co., Ltd, Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd..
2. safety verification
(1) safety to goat
(lot number is 201001001,201001002 to 3 batches of goats contagious pleuropneumonia inactivated vaccines that laboratory is manufactured experimently
With 201001003) respectively with 2.0ml/ subcutaneous vaccination 1 years old age goat, while taking Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.
Comparable product is observed 10 by a 1 part/subcutaneous vaccination 1 years old age goat, if occur locally and systemically reacting.
(2) safety to non-target animals (Cavia porcelluss and rabbit)
(lot number is 201001001,201001002 to 3 batches of goats contagious pleuropneumonia inactivated vaccines that laboratory is manufactured experimently
With the comparable product difference intramuscular injection Cavia porcelluss and rabbit each 2 for 201001003) producing with Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.
Only, 2.0ml/ only, is observed 10, sees whether occur locally and systemically reacting.
The comparable product of 3 batches of laboratory products and Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.'s production is to testing goat, Cavia porcelluss
With rabbit injection daily ought have tenderness, secondary disappear in the future, locally and systemically react without other, table 3.
3 laboratory products of table and comparable product injection Cavia porcelluss and the local response observation result in rabbit 7 days
Note："/" is represented and is not vaccinated.
3. immune generation phase test
(lot number is 201001001,201001002 to 3 batches of goats contagious pleuropneumonia inactivated vaccines that laboratory is manufactured experimently
With 201001003) respectively with 2.0ml cervical regions subcutaneous vaccination 1 years old age goat, while taking Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.'s life
The comparable product of product presses 1 part/cervical region subcutaneous vaccination 1 years old age goat, the 14th day and counteracting toxic substances on the 21st after inoculation.It is shown in Table 4.
4. this animal protest test
After immunity 21 days, immune sheep with compare sheep while with mycoplasma capri goat pneumonia subspecies by force poison C87002 strain lung groups
Knit Emulsion trachea inoculation 4.0ml (containing 15 minimum morbidity amounts), clinical observation 30 days.
The immune generation phase measurement result of the vaccine of 4 present invention of table and domestic comparable product
From table 4, it can be seen that the after the comparable product of 3 batches of products of inoculation and Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.'s production
14, all goat counteracting toxic substances protections reached counteracting toxic substances protection on the more than 3/4, the 21st and reach 4/4；Counteracting toxic substances matched group goat 3/3 falls ill.
The production of 3 batches of products of laboratory is using EZH fluid medium culture mycoplasma capri goat pneumonia subspecies
C87001 strain strains, harvest 15 times of bacterium solution concentration, thimerosal inactivation and import 50V oil adjuvants and press 1:After 1 (v/v) mixing and emulsifying
Subpackage is made.Compared by carrying out the aspects such as two kinds of products safeties, effect and immune generation phase, as a result shown：Laboratory
The comparable product that product and Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. produce is suitable in effect and in terms of the immune generation phase.
Embodiment 3
Minimum immune dosage determines
In order to check immune effect of the goats contagious pleuropneumonia inactivated vaccine various dose to target animals, laboratory is taken
3 batches of goats contagious pleuropneumonia inactivated vaccines of trial-production are inoculated with 1 years old age goat by various dose, carry out counteracting toxic substances protection after 21 days
Test, determines the minimum immune dosage of the product.Now result is reported as follows.
1. material
(1) 3 batches of goats contagious pleuropneumonia inactivated vaccines of vaccine, lot number are 201001001,201001002 and
201001003 batches, prepared by this laboratory.
(2) goats contagious pleuropneumonia C87002 organizes lyophilizing poison mycoplasma capri goat pneumonia subspecies malicious by force
C87002 strains, code name 87002, generation：52 generations, specification：1.0g/, identified by China Veterinery Drug Inspection Office, take care of and supplied
Should.
(3) laboratory animal Gansu one cultivates factory goat, 1 years old age, health, free from infection, dermatosiss and parasite sense inside the province
Dye, examines through serology such as mycoplasma capri goat pneumonia subspecies F38 strain, mycoplasma ovine pneumoniae Y98 strains and brucellosiss
Test and be feminine gender.
2. counteracting toxic substances protection
3 batches of products of laboratory (lot number be 201001001,201001002 and 201001003 batches) respectively by 0.5,1.0,
1.5th, the immunizing dose of 2.0ml is injected to goat, carries out within 21 days after injection counteracting toxic substances, and different immunizing dose counteracting toxic substances protective rates determine knot
Fruit is shown in Table 5,6.
Counteracting toxic substances protective rate after the difference immunizing dose inoculation of 5 vaccine of table
6 counteracting toxic substances matched group goat counteracting toxic substances protective rate of table
From table 3 and table 4 as can be seen that 3 batches of products are protected by counteracting toxic substances on the 21st after 1.0,1.5,2.0ml immunizing doses inoculation goat
Shield rate reaches 3/4 more than (75%)；Counteracting toxic substances matched group goat 3/3 (100%) falls ill.
3. clinicing symptom observation result
After counteracting toxic substances, the clinical symptoms of daily observation immune group and matched group goat, Continuous Observation 30 days.201001001 batches
Immune group 2, No. 4, No. 7 goats occur coughing, subtract food, body temperature and raise, the 201001002 batches of immune group 18, No. 19 No. 24,
No. 27 goats go out now cough, subtract food, body temperature rising；There is cough, subtract food, body temperature 201001003 batches of immune group in No. 34 goats
Raise, remaining goat clinical manifestation of immune group is normal；3 sheep of matched group occur coughing, subtract food, the high symptom of body temperature liter, its
The death in 24 days after counteracting toxic substances of middle C1 goats.
4. cut open inspection result
30 days after counteracting toxic substances, all test goats are dissected, as a result 201001001 batches of 3 mountains for clinical symptoms occur
Sheep finds that there are different degrees of characteristic pathological changes in pulmonary after dissecting；After 201001002 batches of 4 goats for clinical symptoms occur are dissected
It was found that there are different degrees of characteristic pathological changes in pulmonary；2010011003 batches of No. 34 goats for clinical symptoms occur dissect rear left lung diaphragm
2/3 liver of leaf becomes, and section is in marble-like.Remaining immune goat pulmo is normal；Matched group C1 is dead to dissect rear left apex pulmonis
2/3 liver of leaf becomes, and section is in marble-like；Remaining 2 control goats expire to dissect and different degrees of pulmonary's marble sample occur
Characteristic pathological changes.
This result of the test shows, can not produce reliable protected effect using 0.5ml immunizing doses；Using 1.0,1.5 with
Without significant difference, after 3 batches of vaccines are inoculated with 21 for 1 time, counteracting toxic substances can play a protective role 2.0ml immunizing doses to goat, it was demonstrated that this
Vaccine is inoculated with 1 years old age goat with 1.0ml, and after 21 days, counteracting toxic substances can be protected, and be this vaccine minimum immune dosage.Therefore, for guaranteeing
Immune effect, immunizing dose suggestion are 2.0ml/.
Embodiment 4
The duration of immunity of vaccine is determined
The counteracting toxic substances protective rate of this test different time after the product of determination experiment room 3 batches is to the inoculation of 1 monthly age goat, comments
Determine immune efficacy of the vaccine to target animals.
1. material is with embodiment 3.
The using dosage of 1 part of vaccine is：2.0ml/ only, is subcutaneously or intramuscularly injected using cervical region.
2. counteracting toxic substances protective rate
3 batches of products of laboratory (lot number is 201001001,201001002 and 201001003 batches), 1 part immune programme for children pair
Goat is injected, and the 6th, 9,12 and 15 months counteracting toxic substances after injection, different time points counteracting toxic substances protective rate measurement result is shown in Table 7,8.
Counteracting toxic substances protective rate (1) after the inoculation of 7 Different immunizing schedule of table
Counteracting toxic substances protective rate (2) after the inoculation of 8 Different immunizing schedule of table
From table 3,4 result of table can be seen that 3 batches of products be once inoculated with and twice be inoculated with 1 part immune goat after 6,9,12,
Counteracting toxic substances protective rate reaches 3/4 more than (75%) within 15 months；Each time point counteracting toxic substances matched group goat 3/3 falls ill, and through cut open inspection, pulmonary has
Typical goats contagious pleuropneumonia pathological changes.Prove the goat using two kinds of immune programme for children inoculations, the protective rate of counteracting toxic substances in 15 months
Reach《Chinese veterinary pharmacopoeia》Regulation.
3. clinicing symptom observation result
After counteracting toxic substances, the clinical symptoms of daily observation immune group and matched group goat, Continuous Observation 30 days.Goat counteracting toxic substances are protected
Test group clinical manifestation is normal；201001003 batches of 1 skill of handling needles respectively have 1 goat to occur respectively after immunity after 9,12 month counteracting toxic substances
Cough, subtract food, body temperature rising, 12 months counteracting toxic substances have 1 goat cough occur, subtract food, body after 201001002 batches of 1 skill of handling needles immunity
Temperature rise, after 201001001 batches of 2 skill of handling needles immunity, 15 months counteracting toxic substances have 1 goat cough occur, subtract food, body temperature rising, remaining mountain
Sheep clinical manifestation is normal, and matched group goat occurs coughing, subtracts food, the high symptom of body temperature liter.
4. cut open inspection result
30 days after counteracting toxic substances, to occurred cough, subtract the elevated goat of food, body temperature and counteracting toxic substances matched group goat solves
Cut open, as a result, different degrees of marble-like pathological changes occurs in pulmonary, clinical symptoms goat pulmo do not occur normal.1 skill of handling needles mountain
Sheep immune vaccine after 6,9,12,15 months protest test morbidity result of determination be shown in Table 6, table 8,12,2 pin of table 10 and table respectively
Protest test morbidity result of determination after method goat immune vaccine is shown in Table 9, table 10, table 11 respectively.
9 vaccine immunity of table, 6 months goat protest test morbidity result of determination
10 vaccine immunity of table, 12 months goat protest test morbidity result of determination
12 vaccine immunity of table, 15 months goat protest test morbidity result of determination
Embodiment 5
Vaccine stability (storage life) is tested
Result of the test is shown in Table table 12-16.
It is residual that 12 3 batches of vaccines of table preserve different time character inspection, steriling test, loading amount and antiseptic mercurials at 2-8 DEG C
Allowance measurement result
13 3 batches of vaccines of table preserve different time safety verification result at 2～8 DEG C
2～8 DEG C of 14 laboratory products of table preserve 0,3 and 6 months counteracting toxic substances protective rates
2～8 DEG C of 15 laboratory products of table preserve 9,12 and 18 months counteracting toxic substances protective rates
2～8 DEG C of 16 vaccine of table preserves 21,24 and 30 months counteracting toxic substances protective rates
From table 14,15 and 16 as can be seen that 3 batches of products are preserved and pressed within 30 months 21 days after 2.0ml immunizing doses inoculation goat
Though counteracting toxic substances protective rate reaches 3/4 more than (75%), protective rate has substantially reduction compared with preserving 21 months；Counteracting toxic substances matched group
Goat 3/3 (100%) falls ill, and has typical goats contagious pleuropneumonia pathological changes through cut open inspection pulmonary.
Claims (4)
1. a kind of goats contagious pleuropneumonia inactivated vaccine, it is characterised in that the inactivated vaccine contains to be trained using EZH liquid
The mycoplasma capricolum subsp. pneumonia antigen of goats of foster base culture.
2. goats contagious pleuropneumonia inactivated vaccine as claimed in claim 1, it is characterised in that the mycoplasma capri goat
Pneumonia subspecies antigen production bacterial strain is C87001 strains.
3. goats contagious pleuropneumonia inactivated vaccine as claimed in claim 1, it is characterised in that the fluid medium is EZH
Fluid medium, is made up of following component (W/W):Lactoalbumin hydrolysate 3.5%, yeast powder 1.5%, glucose 1.5%, PPLO meat
Powder 10.5%, 1% phenol red 0.1%, balance of water for injection；PH value is adjusted to 7.8 with the sodium hydroxide solution of sterilizing 1mol/L,
Through 0.22 μm of filtration sterilization.
4. the preparation method of goats contagious pleuropneumonia inactivated vaccine as claimed in claim 1, it is characterised in that use the mountain
The strain of sheep mycoplasma goat pneumonia subspecies C87001 is inoculated with appropriate media as production of vaccine bacterial strain, harvests culture after culture
Concentrated, inactivation, plus mineral oil adjuvant mixing and emulsifying makes goats contagious pleuropneumonia inactivated vaccine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109453371A (en) * | 2018-11-23 | 2019-03-12 | 中国兽医药品监察所 | A kind of sheep pox, goats contagious pleuropneumonia bivalent inactivated vaccine and its production method |
| CN113481178A (en) * | 2021-07-07 | 2021-10-08 | 苏州米迪生物技术有限公司 | Goat infectious pleuropneumonia genetic engineering subunit vaccine and preparation method and application thereof |
| CN115044513A (en) * | 2022-06-30 | 2022-09-13 | 山西农业大学 | Mycoplasma filiformis subspecies capri SXTG01 and application thereof, mycoplasma filiformis subspecies capri vaccine and preparation method thereof |
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| CN101721694A (en) * | 2009-11-07 | 2010-06-09 | 中国农业科学院兰州兽医研究所 | Hircine primary atypical pneumonia combined inactivated vaccine and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109453371A (en) * | 2018-11-23 | 2019-03-12 | 中国兽医药品监察所 | A kind of sheep pox, goats contagious pleuropneumonia bivalent inactivated vaccine and its production method |
| CN113481178A (en) * | 2021-07-07 | 2021-10-08 | 苏州米迪生物技术有限公司 | Goat infectious pleuropneumonia genetic engineering subunit vaccine and preparation method and application thereof |
| CN115044513A (en) * | 2022-06-30 | 2022-09-13 | 山西农业大学 | Mycoplasma filiformis subspecies capri SXTG01 and application thereof, mycoplasma filiformis subspecies capri vaccine and preparation method thereof |
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