CN101130075A - Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease - Google Patents
Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease Download PDFInfo
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Abstract
The invention discloses a foot-and-mouth disease O, A, Asia-1 tervalent inactivated vaccine, which comprises the following steps: setting the rate of O, A, Asia-1 foot-and-mouth disease virus antigen and adjuvant at 1: 1; blending 45% inactivated O virus antigen O/china/99 condensed into three times, 20% A virus antigen AF/72 and 35% Asia-1 virus disease antigen Asia-1/XJ/KLAMY/04; preparing toxic seed and vaccine; breeding virus; inactivating virus; detecting bacteria; condensing; dispensing vaccine; detecting the vaccine. The invention can prevent O, A, Asia-1 three serum foot-and-mouth disease with convenience and utility, which reduces immune labor, material and financial capability, therefore possessing widely applying prospect.
Description
Technical field
The present invention relates to the veterinary biological product technical field, is a kind of foot and mouth disease O, A, Asia-1 type tervalence inactivated vaccine specifically.
Background technology
Foot and mouth disease is a kind of acute, hot, the height contagiousness and the animal epidemic of long-distance communications fast that is caused by foot and mouth disease virus, infect and plant and other domestic and wild artiodactyl liking main poultry such as pig, cattle, sheep, susceptible animal is kind surplus in the of 70 nearly.Foot and mouth disease break out economic development, international trade, social stability and the international fame that will badly influence a country.Foot and mouth disease was broken out in China Taiwan in 1997, caused pig industry and related industry to be closed down, 700,000 people unemployment, and total economic loss reaches 8,000,000,000 dollars.Calendar year 2001, foot and mouth disease is at first broken out in Britain, causes direct losses to reach several hundred million pounds, but the indirect loss of foreign trade and related industry reaches 9,000,000,000 pounds, when 1.5 percentage points of annual rate of economic growth downward modulations.Blackwell (1984) once quoted an economic analysis report and claims: if foot and mouth disease takes place the very flourishing U.S. of animal husbandry, direct losses then and indirect loss will be respectively 4,000,000,000 and 40,000,000,000 dollars.In view of foot and mouth disease not only can cause tremendous economic loss and social influence, and can influence country reputation and image in the world, have the title of " political economy disease ", classified as first of category-A and the class zoonosis by FAO (Food and Agriculture Organization of the United Nation) (FAO), OIE (OIE) and the Chinese government, and list the great transnational animal epidemic whole world in and eliminate action plan, this has fully shown domestic and international degree of concern to foot and mouth disease.At present, foot and mouth disease popular total general layout in the world's is, developed country continues to enjoy no foot and mouth disease status, and developing country does not break away from the harm of foot and mouth disease yet.Seek advice from breadboard epidemic situation circular and detection statistics according to OIE and world's foot and mouth disease, the countries and regions of report foot and mouth disease morbidity have 82 (inferior) in the period of 2003～2005, and wherein Asia-accounts for 56.1% (46/82).The countries and regions of foot and mouth disease take place in, there are 10 to border on China, epidemic situation is very serious, long-term popular O, A and Asia-1 type foot and mouth disease, more than the 20000 kilometer long national boundary that be continuous, it is a lot of to import factor into, and institute is compromised very big.China's foot and mouth disease since liberation is popular constantly, in the past based on the O type, main infected cattle pig, along with the development of reform and opening-up, international exchange is frequent in recent years, artificial abortion's logistics is accelerated, though dropped into lot of manpower and material resources control and put out foot and mouth disease, domestic epidemic situation is constantly complicated, not only has O type foot and mouth disease popular, some areas also have Asia-1 type foot and mouth disease popular, seriously hinder China's animal husbandry to develop in a healthy way.
Vaccination is major measure, key link and the last line of defense that prevents that eqpidemic disease from breaking out and avoiding bringing about great losses.There is being the popular country of foot and mouth disease all will carry out planned Vaccination and Immunoprophylaxis every year; No foot and mouth disease country popular or that eliminated foot and mouth disease has also laid in the war preparedness vaccine of some in vaccine library; Be subjected to foot and mouth disease to threaten country except that carrying out strict import quarantine, regular vaccinoprophylaxis is also carried out in the border area.Hostis Picornaviridae, viral genome have the characteristic of picornavirus quasispecies, and pressure selects to cause virus very easily making a variation aspect virulence and the antigenicity.Determined 7 serotypes such as O, A, C, Asia-I, SATI, SATII, SATIII according to cross-protection test, do not had cross protection between type, the hereditary variation in the type between the different strains can cause antigenic difference.Foot and mouth disease is the pandemic infection disease, and popular serotype in various countries and strain exist for above-mentioned reasons than big-difference, and therefore, all numerous and confused development is suitable for the vaccine of own country, to prevent and to control the popular of this worldwide great animal epidemic.Once sum up attenuated vaccine, inactivated vaccine and new generation vaccine three major types of the research foot-and-mouth disease vaccine using and developing in the world.From the whole research and the applicable cases of foot-and-mouth disease vaccine, the malicious vaccine of living is because himself inherent defective, abandoned by many countries in the world.Though new generation vaccine is obtained some very promising results, in immune efficacy and the present economically ability of competing that still do not exist with conventional vaccine.Before new generation vaccine enters practical application, also need the immunologic mechanism and the molecular biological characteristic of this type of vaccine are done research more in depth.Conventional inactivated vaccine remains the main vaccine of our times foot and mouth disease immunity prevention and control.Aspect development of Inactivated Vaccine, representative in the world production of vaccine technology is: " the seedling strain that screening is excellent is with rolling bottle monolayer passage cell culture method or Maitland culture production seedling foot-and-mouth disease virus antigen; With acetyl group aziridine AEl or divinyl imines BEl virus is carried out deactivation; Utilize oily adjuvant emulsion preparation vaccine." China has succeeded in developing and the inactivated vaccine that is used for actual epidemic prevention has: cattle foot and mouth disease O type inactivated vaccine, Schweineseuche O-shaped inactivated vaccine, Schweineseuche O-shaped inactivated vaccine (II) they are Schweineseuche O-shaped efficient inactivation vaccine etc.
Because the present some areas of China have O type and Asia-1 type foot and mouth disease popular in addition, the O of surrounding countries type, A type and the Asia-1 type foot and mouth disease of adjoining with China are popular without restraint, thereby have formed in domestic trouble and foreign invasion aspect the foot and mouth disease prevention and control, the situation of being attacked in the front and the rear.In view of China does not also prevent the inactivated vaccine of O, A, Asia-1 type three type foot and mouth disease at present simultaneously, and safe, efficient, fine polyvalent inactivation epidemic disease commonly used is in the world come the successful experience of effective many serotype of prevention and control foot and mouth disease, develop inactivated vaccine safe, efficient, that can prevent O, A, Asia-1 type three type foot and mouth disease simultaneously and be prevention foot and mouth disease urgent problem, significant.
Summary of the invention
The purpose of this invention is to provide a kind of efficient, safe and reliable tervalence inactivated vaccine that can prevent O, A, Asia-1 three type foot and mouth disease simultaneously, be used for the immunoprophylaxis of O, A, Asia-1 type foot and mouth disease.
The present invention is achieved through the following technical solutions its goal of the invention:
A kind of foot and mouth disease O, A, Asia-1 type tervalence inactivated vaccine, make by following proportioning by O type foot-and-mouth disease virus antigen, A type foot-and-mouth disease virus antigen, Asia-1 type foot-and-mouth disease virus antigen and adjuvant:
The ratio of vaccine preparation is antigen: adjuvant=1: 1;
Wherein antigen is prepared by following weight percentage:
45% deactivation also mixes through 3 times of spissated O type virus antigen O/china/99,20% A type virus antigen AF/72 and 35% Asia-1 type virus antigen Asia-1/XJ/KLAMY/04;
Adjuvant: adopt to cover to get very much 206 adjuvants---Montanide ISA 206 adjuvants;
Above-mentioned raw materials is prepared from according to following process and order:
A, seed culture of viruses
A.1, O type seed culture of viruses: cattle source O type foot and mouth disease virus O/china/99 strain cell toxicant;
A.2, A type seed culture of viruses: cattle source A type foot and mouth disease virus AF/72 strain cell toxicant;
A.3, Asia-1 type seed culture of viruses: cattle source Asia-1 type foot and mouth disease virus Asia-1/XJ/KLAMY/04 strain cell toxicant;
The preparation of b, seedling material
The seedling material is the BHK-21 cell monolayer, and the cultivation of cell is undertaken by general method for tissue culture;
The propagation of c, virus
Breed seedling O, A, Asia-1 type virus antigen with 15L rolling bottle BHK-21 cell respectively by concentrating culture method, the Rotary Machine rotating speed is 10～12r/h, gathers in the crops viral liquid, freezes to preserve under-15 ℃;
The program of d, inactivation of virus comprises:
D.1 filter; The O that will freeze, A, Asia-1 type seedling virus liquid melt respectively, cross the filtering cell debris through 60～80 order copper gauzes, use 7.5%NaHCO
3Transfer PH to 7.6～7.8, and add the penicillin of 200u/mL, the streptomycin of 200 μ g/mL;
D.2 deactivation; Adding BEI divinyl imines respectively in filtering O, A, Asia-1 type virus liquid makes its concentration reach 2mmol/L, in 37 ℃ of greenhouses or fermentation tank chuck water-bath circulation be warming up to 30 ℃, and at 30 ℃ of following lasting deactivation 26h, every therebetween 4h shake 1 time, the deactivation amortization period adds 50% hypo solution of filtration sterilization, making its whole content is 2%, puts 2～8 ℃ of freezers or fermentation tank brine ice and is incubated to be checked;
D.3 safety check; O, A, Asia-1 type inactivation of viruses liquid are carried out safety check respectively, and with every type inactivation of viruses liquid, nape portion subcutaneous injection 2～3 age in days neonatal rats, 0.2mL/ only establishes the contrast neonatal rat, observes 7d, and whole neonatal rats the foot and mouth disease symptom do not occur and morbidity is dead, and the side is that deactivation is thorough;
D.4 bacterium inspection;
O, A, Asia-1 type inactivation of viruses liquid are carried out steriling test by " Chinese veterinary drug allusion quotation " respectively;
E. concentrate O type, A type, Asia-1 type seedling virus liquid difference physics are concentrated 3 times;
F, vaccine preparation
Vaccine emulsifying, earlier oily adjuvant phase negative pressure of vacuum is fed in the mulser, in the whipping process, with the viral liquid of deactivation in proportion negative pressure of vacuum be fed to oily adjuvant mutually in, after circulation is stirred to antigen and adjuvant fully mixes, be emulsified into the slightly two-phase oil emulsion vaccine of viscosity by homogenizer;
G, vaccine test comprise:
G.1, physical behavior inspection; This vaccine is the mineral oil emulsion type, and outward appearance is white in color or rose pink slightly viscous liquid.Get a cleaning suction pipe, draw a little vaccine and drip, be the cloud diffusion in cleaning cold water surface; Draw vaccine 10mL, add in the centrifuge tube, with 3000r/min centrifugal 15 minutes, water was separated out and should be no more than 0.5mL; (outlet internal diameter 1.2mm) draws 25 ± 1 ℃ vaccine 1mL with the 1mL suction pipe, makes its vertical 0.4mL that flows out naturally, and required time should be less than or equal to 8s;
G.2, steriling test;
G.3, safety verification
G.3.1 use 2 of the Cavia porcelluss of body weight 350～450g, every subcutaneous injection vaccine 2mL; With 5 of the mices of body weight 18～22g, every subcutaneous injection vaccine 0.5mL observed 7 continuously, dead or significantly local response or the general reaction that causes because of vaccinate all should not occur;
G.3.2 use 3 of the healthy susceptible cattle at least 6 monthly ages, 20 points of every dorsal surface of tongue intradermal injection, every some 0.1mL vaccine, day by day observe 4d at least, afterwards, every cattle intramuscular injection vaccine 9mL continues to observe 6d day by day the toxic reaction that the foot and mouth disease symptom all do not occur or significantly cause because of vaccinate;
G.4, efficacy test: 45 of the healthy susceptible cattle at least 6 monthly ages of usefulness are divided into 3 groups, 15 every group.Vaccine to be checked is divided into 1 part, 1/3 part, 3 dosage groups of 1/9 part, each dosage group behind the inoculation 21d, at random is divided into O type, A type, Asia-1 type three rose poison group with each dosage group immune cattle respectively at 15 cattle of musculi colli injection, together with each 2 of contrast cattle, carry out counteracting toxic substances respectively; O type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease O type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50A type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease A type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50Asia-1 type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease Asia-1 type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50Observe 10d continuously; Blister or ulcer appear in few 3 hoof of contrast Adeps Bovis seu Bubali, and immune cattle only blister or ulcer occur at lingual surface, and other position is judged to protection when not having pathological changes, is judged to when typical foot and mouth disease blister or ulcer appear in arbitrary position except that lingual surface and does not protect; According to the protection number of immune cattle, calculate the PD of tested vaccine by the Karber method
50Every part vaccine should reach 3PD at least respectively to the immune efficacy of O type, A type, Asia-1 type foot and mouth disease
50
China is animal husbandry big country, and the animal husbandry output value has accounted for more than 1/3 of the general production value of agriculture.According to statistics, to the main domestic animal amount of livestock on hand in the whole nation in the end of the year 2004,4.82 hundred million of pigs, 1.38 hundred million of cattle, 3.66 hundred million of sheep increase by 3.4%, 2.3%, 7.6% respectively than the last year.Country has brought up to the strategic position of transform agricultural production and increase farmers' income, solve " rural economy, rural development and rural demography " and building a New Socialist Countryside developing animal husbandry.Foot and mouth disease is one of most important worldwide animal epidemic, is called as " the No.1 killer " of animal husbandry, and the annual economic loss that causes because of this disease reaches tens billion of units.In addition, also relate to food safety and public health.The popular history of China's foot and mouth disease for a long time, the epidemic situation more complicated.At present, except that popular O type foot and mouth disease, some areas also popular Asia-1 type foot and mouth disease.Seriously restrict health, the sustainable development of animal husbandry, greatly affecting the international trade of animals and animal product.And many countries of bordering on China, long-term popular O, A and Asia-l type foot and mouth disease, epidemic situation are very serious.In order to tackle China's foot and mouth disease accident, also refuse the strategic technological reserve of neighbouring country's foot and mouth disease outside the gateway of a country in order to provide, we use for reference the successful experience that the popular country of many serotype foot and mouth disease in the world adopts multivalent inactivated vaccine prevention and control foot and mouth disease mostly, under the subsidy of the chief of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences fund project and Ministry of Agriculture's animal epidemics monitoring project, foot and mouth disease O, A, Asia-I type tervalence inactivated vaccine have been succeeded in developing.
This vaccine system is with the cell adapted poison of cattle source O type hoof-and-mouth disease strain, cattle source A type hoof-and-mouth disease strain and cattle source Asia-1 type hoof-and-mouth disease strain, inoculation BHK-21 cell, the harvesting culture, after (BEI) deactivation of divinyl imines and physics concentrate, the adjuvant mixing and emulsifying that refuels is made, be used for the immunoprophylaxis of O type, A type and Asia-1 type foot and mouth disease, to foot and mouth disease susceptible animal safety.Once inoculate effective duration of immunity 6 months; 2～8 ℃ of following vaccine storage lives 12 months; Immunizing dose is every cattle 3mL.By general international standard PD
50Method is carried out efficacy test, 4 batches of vaccines (040920,041213,050112 and 050401) immune cattle, and the immune efficacy of anti-foot and mouth disease O type homology strong virus attack is respectively 6.50,4.65,3.74 and 5.80 PD
50/ head part, average 5.17 PD
50/ head part; The immune efficacy of anti-A type homology strong virus attack is respectively 7.22,5.19,8.10,7.22 PD
50/ head part, average 6.93 PD
50/ head part; The immune efficacy of anti-Asia-1 type homology strong virus attack is respectively 7.22,8.10,8.10,8.10 PD
50/ head part, average 7.88 PD
50/ head part, the then equal 2/2 4 hoof morbidity of contrast cattle.Show that this vaccine has surpassed the immune efficacy of O, A, Asia-1 type foot and mouth disease and " is not less than 3 PD
50/ head part " domestic or international standard.
Compare with having the foot and mouth disease inactivated vaccine both at home and abroad now, this vaccine has following characteristics:
1) this vaccine seedling seed culture of viruses is to determine through advanced person's hoof-and-mouth disease poison strain storehouse technology screening, not only immunogenicity is good, spectrotype is wide, heritability is stable, laboratory animal and seedling material are had adaptability, and is the popular representative strain of foot and mouth disease in recent years.As the seedling strain, epidemic prevention has more specific aim to reality with it.
2) BHK-21 cell concentration culture method propagative viruses antigen, (BEI) deactivation of divinyl imines and two-phase oil adjuvant emulsion technology are adopted in the vaccine manufacturing.In addition, with foot-and-mouth disease virus antigen concentrate, preservation, dilution technology be applied to the vaccine manufacturing, improve and optimized production of vaccine technology.For guaranteeing product quality, the management of standard seed culture of viruses, set up seedling and counteracting toxic substances with seed culture of viruses and seedling cell seed lot, worked out the vaccine quality standard that integrates with international standard.
3) safety of this vaccine reaches international like product advanced level; The average immune efficacy of A type and Asia-1 type foot and mouth disease is respectively 6.93 and 7.88PD
50/ head part has surpassed standard (6 PD of the emergent vaccine of strategy in the world
50/ head part), be 5.17PD to the average immune efficacy of O type FMD
50/ head part has surpassed the standard (3PD of conventional in the world prevention with Seedling
50/ head part).Therefore, be foot and mouth disease O, A, Asia-1 type tervalence inactivated vaccine efficiently, have broad application prospects.
4) once inoculate the foot and mouth disease that to prevent O, A, three serotypes of Asia-1 type simultaneously.Therefore, have now, can reduce the human and material resources and the financial resources that are used to prevent epidemic with vaccine be beyond one's reach convenience and practicality.
The specific embodiment
Embodiment 1
A kind of foot and mouth disease O, A, Asia-1 type tervalence inactivated vaccine, by deactivation and through 3 times of spissated O type foot-and-mouth disease virus antigen O/China/99 account for the vaccine total amount 22.5%, A type foot-and-mouth disease virus antigen AF/72 account for the vaccine total amount 10%, Aisa-1 type foot-and-mouth disease virus antigen Asia-1/XJ/KLAMY/04 account for the vaccine total amount 17.5% with Montanide ISA 206 adjuvants of producing by French SEPPIC company, form through autoclaving, account for the 50% formulated jointly of vaccine total amount, realize by following process and order:
1 seed culture of viruses
1.1O type seed culture of viruses: O/china/99 is isolating O type foot and mouth disease cattle source tongue skin poison in 1999, identifies, preserves and supply by country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences foot and mouth disease reference laboratory.After 1: 10000 times of dilution of this seed culture of viruses, inoculate the cattle of no foot and mouth disease O type serum antibody, typical foot and mouth disease blister pathological changes appears in 4 hoof in 48h, to the median infective dose ID of cattle
50/ 0.1mL is 10
-9.00After 1: 10 times of dilution of cow tongue skin seed culture of viruses, tangible paralysis symptom and final dead appears in inoculation 2～3 age in days neonatal rats in 24h, and blind passage to the is during 3 generations, and the morbidity death time is 18～20h, to the median lethal dose(LD 50) LD of neonatal rat
50/ 0.2mL is 10
-8.50Adapt to the BHK-21 cell with this seed culture of viruses, in the 5th generation,, malicious incubation time was 8h, the median infective dose TCID of pair cell
50/ mL is 10
-8.60Through nucleotide sequence analysis, this seed culture of viruses belongs to " PanAsia " pedigree on genetic classification, with the VP1 gene nucleotide series homology of the Pan Asia type strain of delivering in the world more than 95%; Connecting on the PK15 cell passed for 60 generations after, the nucleotide sequence of main immunogene with go down to posterity before compare with originality to be 99.22%, to illustrate that the heritability of this seed culture of viruses is stable; Analyze by the cell neutralization test, other O type foot and mouth disease isolated strain antigen of this seed culture of viruses and China is in close relations, is 0.66～1.13 to the r value of other O type virus, and average 0.98.
1.2A type seed culture of viruses: AF/72 is isolating A type foot and mouth disease cattle source tongue skin poison in 1972, identifies, preserves and supply by country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences foot and mouth disease reference laboratory.After 1: 10000 times of dilution of this seed culture of viruses, inoculate the cattle of no foot and mouth disease A type serum antibody, typical foot and mouth disease blister pathological changes appears in 4 hoof in 48h, to the median infective dose ID of cattle
50/ 0.1mL is 10
-8.00With cow tongue skin seed culture of viruses dilution back inoculation in 1: 10 2～3 age in days neonatal rats, tangible paralysis symptom and final dead appears in 48h, and blind passage to the is during 3 generations, and the morbidity death time is 21～23h, to the median lethal dose(LD 50) LD of neonatal rat
50/ 0.2mL is 10
-7.67Adapt to the BHK-21 cell with this seed culture of viruses, in the 8th generation,, malicious incubation time was 12h, the median infective dose TCID of pair cell
50/ mL is 10
-7.67Through nucleotide sequence analysis, the homology of this seed culture of viruses and surrounding countries and China's cattle source 50～sixties of 20th century A type foot and mouth disease isolated strain is more than 91%, on genetic classification symbolic animal of the birth year with gene hypotype; Analyze by the cell neutralization test, this seed culture of viruses and China other A type foot and mouth disease isolated strain antigen r value to other A type virus in close relations is 0.78～1.00, average 0.88.
1.3Asia-1 type seed culture of viruses: Asia-1/XJ/KLAMY/04 is isolating Asia-1 type foot and mouth disease cattle source tongue skin poison in 2004, identifies, preserves and supply by country of Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences foot and mouth disease reference laboratory.After 1: 10000 times of dilution of this seed culture of viruses, inoculate the cattle of no foot and mouth disease Asia-1 type serum antibody, typical foot and mouth disease blister pathological changes appears in 4 hoof in 48h, to the median infective dose ID of cattle
50/ 0.1mL is 10
-7.50After 1: 10 times of dilution of cow tongue skin seed culture of viruses, tangible paralysis symptom and final dead appears in inoculation 2～3 age in days neonatal rats in 48h, and blind passage to the is during 3 generations, and the morbidity death time is 25～30h, to the median lethal dose(LD 50) LD of neonatal rat
50/ 0.2mL is 10
-8.00Adapt to the BHK-21 cell with this seed culture of viruses, in the 5th generation,, malicious incubation time was 8h, the median infective dose TCID of pair cell
50/ mL is 10
-8.33Through nucleotide sequence analysis, this seed culture of viruses is 97.8%～99.1% with the popular malicious homology of states such as the Tajikistan that adjoins with China, India, Pakistan, belongs to height homology poison; Analyze by the cell neutralization test, other Asia-1 type foot and mouth disease isolated strain antigen of this seed culture of viruses and China is in close relations, is 0.82～1.00 to the r value of other Asia-1 type virus, and average 0.89.
The program of 2 virus of proliferation
2.1 the preparation of seedling material: the seedling material is the BHK-21 cell monolayer, and the cell seed is provided by China Veterinery Drug Inspection Office.The used calf serum of cell preparation should not have foot and mouth disease virus antibody on inspection, and the every batch of serum all should filtration sterilization, steriling test.The cultivation of nutritional solution preparation and BHK-21 cell is undertaken by general method for tissue culture.
2.2 the propagation of virus: breed seedling O, A, Asia-1 type virus antigen with 15L rolling bottle BHK-21 cell respectively by concentrating culture method (Xue Jingshan, [21] ZL patent No. 93114885.5), but the Rotary Machine rotating speed is 10～12 commentaries on classics/h.Microscopy rolling bottle cell CPE can gather in the crops viral liquid when reaching 90% in incubation, freezes to preserve under-15 ℃.Sampling is measured, TCID
50All be not less than 10
-7.0
The program of 3 inactivation of virus
3.1 filter: the O that will freeze, A, Asia-1 type seedling virus liquid melt respectively, cross the filtering cell debris through 60～80 order copper gauzes, transfer PH to 7.6～7.8 with 7.5%NaHCO3, and add the penicillin of 200u/mL, the streptomycin of 200 μ g/mL.
3.2 deactivation: in filtering O, A, Asia-1 type virus liquid, add BEI respectively and make its concentration reach 2mmol/L, in 37 ℃ of greenhouses or fermentation tank chuck water-bath circulation be warming up to 30 ℃, and at 30 ℃ of following lasting deactivation 26h, every therebetween 4h shake 1 time, the deactivation amortization period adds 50% hypo solution of filtration sterilization, and making its whole content is 2%.Sampling, and it is to be checked to put the insulation of 2～8 ℃ of freezers or fermentation tank brine ice.
3.3 safety check: O, A, Asia-1 type inactivation of viruses liquid are carried out safety check respectively.With every type inactivation of viruses liquid, 4 of nape portion subcutaneous injection 2～3 age in days neonatal rats, 0.2mL/ only establishes 1 of contrast neonatal rat, observes 7d, and whole neonatal rats the foot and mouth disease symptom all do not occur and morbidity is dead, and the side is that deactivation is thorough.
3.4 bacterium inspection: O, A, Asia-1 type inactivation of viruses liquid are carried out steriling test by " Chinese veterinary drug allusion quotation " respectively, answer asepsis growth.
4 concentrate: with O type, A type, Asia-1 type seedling virus liquid, physics concentrates 3 times respectively.
5 vaccine preparation procedures: the vaccine configuration proportion is V (antigen): V (adjuvant)=1: 1; Vaccine emulsifying is Montanide ISA 206 (French SEPPIC company product) with oily adjuvant; Vaccine emulsifying antigen is that deactivation is thorough, the bacterium inspection is qualified and through spissated O, A, Asia-1 type mixing foot-and-mouth disease virus antigen, three kinds of antigenic ratios are " O: A: Asia-1 type=45%: 20%: 35% ".During emulsifying, earlier oily adjuvant phase negative pressure of vacuum is fed in the mulser, in the whipping process, with the viral liquid of deactivation in proportion slowly negative pressure of vacuum be fed to oily adjuvant mutually in.After feeding intake, after circulation is stirred to antigen and adjuvant fully mixes,, be emulsified into the slightly two-phase oil emulsion vaccine of viscosity by homogenizer.
6 vaccine tests
6.1 physical behavior inspection: this vaccine is the mineral oil emulsion type, and outward appearance is white in color or rose pink slightly viscous liquid.Get a cleaning suction pipe, draw a little vaccine and drip, should be the cloud diffusion in cleaning cold water surface; Draw vaccine 10mL, add in the centrifuge tube, with 3000r/min centrifugal 15 minutes, water was separated out and should be no more than 0.5mL; (outlet internal diameter 1.2mm) draws about 25 ℃ vaccine 1mL with the 1mL suction pipe, makes its vertical 0.4mL that flows out naturally, and required time should be less than or equal to 8s.
6.2 steriling test: carry out steriling test by " Chinese veterinary drug allusion quotation ", answer asepsis growth.
6.3 safety verification: 1) Cavia porcellus of usefulness body weight 350～450g is 2, every subcutaneous injection vaccine 2mL; With 5 of the mices of body weight 1 8～22g, every subcutaneous injection vaccine 0.5mL.Observe 7d continuously, dead or significantly local response or the general reaction that causes because of vaccinate all should not occur.2) the healthy susceptible cattle (foot and mouth disease cell NAT is less than or equal to 1: 8 or the neonatal rat NAT is less than or equal to 1: 4) at least 6 monthly ages of usefulness is 3,20 points of every dorsal surface of tongue intradermal injection, and every some 0.1mL vaccine is observed 4d at least day by day.Afterwards, every cattle intramuscular injection vaccine 9mL continues to observe day by day 6.The toxic reaction that the foot and mouth disease symptom all should not occur or significantly cause because of vaccinate.
6.4 efficacy test: 45 of the healthy susceptible cattle at least 6 monthly ages of usefulness, foot and mouth disease cell NAT is less than or equal to 1: 8 or the neonatal rat NAT is less than or equal to 1: 4, is divided into 3 groups, 15 every group.Vaccine to be checked is divided into 1 part, 1/3 part, 3 dosage groups of 1/9 part, and each dosage group is respectively at 15 cattle of musculi colli injection.Behind the inoculation 21d, each dosage group immune cattle is divided into O type, A type, three counteracting toxic substances groups of Asia-1 type at random,, carries out counteracting toxic substances respectively together with each 2 of contrast cattle.O type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease O type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50A type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease A type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50Asia-1 type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease Asia-1 type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50Observe 10d continuously.Blister or ulcer appear in contrast Niu Junying at least 3 hoof.Immune cattle only blister or ulcer occur at lingual surface, and other position is judged to protection when not having pathological changes, is judged to when typical foot and mouth disease blister or ulcer appear in arbitrary position except that lingual surface and does not protect.According to the protection number of immune cattle, calculate the PD of tested vaccine by the Karber method
50, every part vaccine reaches 3PD at least respectively to the immune efficacy of O type, A type, Asia-1 type foot and mouth disease
50
Claims (1)
1. a foot and mouth disease O, A, Asia-1 type tervalence inactivated vaccine, make by following proportioning by O type foot-and-mouth disease virus antigen, A type foot-and-mouth disease virus antigen, Asia-1 type foot-and-mouth disease virus antigen and adjuvant:
The ratio of vaccine preparation is antigen: adjuvant=1: 1;
Wherein antigen is prepared by following weight percentage:
45% deactivation also mixes through 3 times of spissated O type virus antigen O/china/99,20% A type virus antigen AF/72 and 35% Asia-1 type virus antigen Asia-1/XJ/KLAMY/04;
Adjuvant: adopt to cover to get very much 206 adjuvants---Montanide ISA 206 adjuvants;
Above-mentioned raw materials is prepared from according to following process and order:
A, seed culture of viruses
A.1, O type seed culture of viruses: cattle source O type foot and mouth disease virus O/china/99 strain cell toxicant;
A.2, A type seed culture of viruses: cattle source A type foot and mouth disease virus AF/72 strain cell toxicant;
A.3, Asia-1 type seed culture of viruses: cattle source Asia-1 type foot and mouth disease virus Asia-1/XJ/KLAMY/04 strain cell toxicant;
The preparation of b, seedling material
The seedling material is the BHK-21 cell monolayer, and the cultivation of cell is undertaken by general method for tissue culture;
The propagation of c, virus
Breed seedling O, A, Asia-1 type virus antigen with 15L rolling bottle BHK-21 cell respectively by concentrating culture method, the Rotary Machine rotating speed is 10～12r/h, gathers in the crops viral liquid, freezes to preserve under-15 ℃;
The program of d, inactivation of virus comprises:
D.1 filter; To freeze 0, A, the seedling of Asia-1 type virus liquid melts respectively, crosses the filtering cell debris through 60～80 order copper gauzes, uses 7.5%NaHCO
3Transfer PH to 7.6～7.8, and add the penicillin of 200u/mL, the streptomycin of 200 μ g/mL;
D.2 deactivation; Adding BEI divinyl imines respectively in filtering O, A, Asia-1 type virus liquid makes its concentration reach 2mmol/L, in 37 ℃ of greenhouses or fermentation tank chuck water-bath circulation be warming up to 30 ℃, and at 30 ℃ of following lasting deactivation 26h, every therebetween 4h shake 1 time, the deactivation amortization period adds 50% hypo solution of filtration sterilization, making its whole content is 2%, puts 2～8 ℃ of freezers or fermentation tank brine ice and is incubated to be checked;
D.3 safety check; O, A, Asia-1 type inactivation of viruses liquid are carried out safety check respectively, and with every type inactivation of viruses liquid, nape portion subcutaneous injection 2～3 age in days neonatal rats, 0.2mL/ only establishes the contrast neonatal rat, observes 7d, and whole neonatal rats the foot and mouth disease symptom do not occur and morbidity is dead, and the side is that deactivation is thorough;
D.4 bacterium inspection;
O, A, Asia-1 type inactivation of viruses liquid are carried out steriling test by " Chinese veterinary drug allusion quotation " respectively;
E. concentrate O type, A type, Asia-1 type seedling virus liquid difference physics are concentrated 3 times;
F, vaccine preparation
Vaccine emulsifying, earlier oily adjuvant phase negative pressure of vacuum is fed in the mulser, in the whipping process, with the viral liquid of deactivation in proportion negative pressure of vacuum be fed to oily adjuvant mutually in, after circulation is stirred to antigen and adjuvant fully mixes, be emulsified into the slightly two-phase oil emulsion vaccine of viscosity by homogenizer;
G, vaccine test comprise:
G.1, physical behavior inspection; This vaccine is the mineral oil emulsion type, and outward appearance is white in color or rose pink slightly viscous liquid.Get a cleaning suction pipe, draw a little vaccine and drip, be the cloud diffusion in cleaning cold water surface; Draw vaccine 10mL, add in the centrifuge tube, with 3000r/min centrifugal 15 minutes, water was separated out and should be no more than 0.5mL; (outlet internal diameter 1.2mm) draws 25 ± 1 ℃ vaccine 1mL with the 1mL suction pipe, makes its vertical 0.4mL that flows out naturally, and required time should be less than or equal to 8s;
G.2, steriling test;
G.3, safety verification
G.3.1 use 2 of the Cavia porcelluss of body weight 350～450g, every subcutaneous injection vaccine 2mL; With 5 of the mices of body weight 18～22g, every subcutaneous injection vaccine 0.5mL observed 7 continuously, dead or significantly local response or the general reaction that causes because of vaccinate all should not occur;
G.3.2 use 3 of the healthy susceptible cattle at least 6 monthly ages, 20 points of every dorsal surface of tongue intradermal injection, every some 0.1mL vaccine, day by day observe 4d at least, afterwards, every cattle intramuscular injection vaccine 9mL continues to observe 6d day by day the toxic reaction that the foot and mouth disease symptom all do not occur or significantly cause because of vaccinate;
G.4, efficacy test: 45 of the healthy susceptible cattle at least 6 monthly ages of usefulness are divided into 3 groups, 15 every group.Vaccine to be checked is divided into 1 part, 1/3 part, 3 dosage groups of 1/9 part, each dosage group behind the inoculation 21d, at random is divided into O type, A type, Asia-1 type three counteracting toxic substances groups with each dosage group immune cattle respectively at 15 cattle of musculi colli injection, together with each 2 of contrast cattle, carry out counteracting toxic substances respectively; O type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease O type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50A type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease A type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50Asia-1 type counteracting toxic substances group, 2 strong poison of intradermal injection cattle foot and mouth disease Asia-1 type virus are divided in every cow tongue upper surface both sides, every some 0.1mL, 0.2mL contains 10 altogether
4ID
50Observe 10d continuously; Blister or ulcer appear in few 3 hoof of contrast Adeps Bovis seu Bubali, and immune cattle only blister or ulcer occur at lingual surface, and other position is judged to protection when not having pathological changes, is judged to when typical foot and mouth disease blister or ulcer appear in arbitrary position except that lingual surface and does not protect; According to the protection number of immune cattle, calculate the PD of tested vaccine by the Karber method
50, every part vaccine should reach 3PD at least respectively to the immune efficacy of 0 type, A type, Asia-1 type foot and mouth disease
50
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100185436A CN101130075A (en) | 2007-07-23 | 2007-07-23 | Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100185436A CN101130075A (en) | 2007-07-23 | 2007-07-23 | Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease |
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| Publication Number | Publication Date |
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| CN101130075A true CN101130075A (en) | 2008-02-27 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100185436A Pending CN101130075A (en) | 2007-07-23 | 2007-07-23 | Tervalence inactivated vaccine for type O, A, Asia-1 hoof-and-mouth disease |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864434B (en) * | 2009-12-21 | 2012-08-15 | 中国农业科学院兰州兽医研究所 | Sheep aphthovirus Asial type multi-epitope recombinant vaccine and preparation method thereof |
| CN102988970A (en) * | 2011-09-19 | 2013-03-27 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease purification vaccine, preparation method and applications thereof |
| CN103382480A (en) * | 2013-07-31 | 2013-11-06 | 中国农业科学院兰州兽医研究所 | Asia 1 type foot and mouth disease virus antigen and preparation and application thereof |
| CN106986925A (en) * | 2017-05-08 | 2017-07-28 | 中牧实业股份有限公司 | Ox aftosa is O-shaped, A type divalence synthetic peptide vaccines and its preparation method and application |
| CN108079288A (en) * | 2018-02-09 | 2018-05-29 | 天康生物股份有限公司 | Vaccine, the emulsifying process of vaccine and application |
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2007
- 2007-07-23 CN CNA2007100185436A patent/CN101130075A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864434B (en) * | 2009-12-21 | 2012-08-15 | 中国农业科学院兰州兽医研究所 | Sheep aphthovirus Asial type multi-epitope recombinant vaccine and preparation method thereof |
| CN102988970A (en) * | 2011-09-19 | 2013-03-27 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease purification vaccine, preparation method and applications thereof |
| CN102988970B (en) * | 2011-09-19 | 2014-06-18 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease purification vaccine, preparation method and applications thereof |
| CN103382480A (en) * | 2013-07-31 | 2013-11-06 | 中国农业科学院兰州兽医研究所 | Asia 1 type foot and mouth disease virus antigen and preparation and application thereof |
| CN103382480B (en) * | 2013-07-31 | 2015-06-24 | 中国农业科学院兰州兽医研究所 | Asia 1 type foot and mouth disease virus antigen and preparation and application thereof |
| CN106986925A (en) * | 2017-05-08 | 2017-07-28 | 中牧实业股份有限公司 | Ox aftosa is O-shaped, A type divalence synthetic peptide vaccines and its preparation method and application |
| CN106986925B (en) * | 2017-05-08 | 2020-08-25 | 中牧实业股份有限公司 | O-type and A-type bivalent synthetic peptide vaccine for bovine foot and mouth disease and preparation method and application thereof |
| CN108079288A (en) * | 2018-02-09 | 2018-05-29 | 天康生物股份有限公司 | Vaccine, the emulsifying process of vaccine and application |
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