CN104758928B - A kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use - Google Patents
A kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use Download PDFInfo
- Publication number
- CN104758928B CN104758928B CN201510030082.9A CN201510030082A CN104758928B CN 104758928 B CN104758928 B CN 104758928B CN 201510030082 A CN201510030082 A CN 201510030082A CN 104758928 B CN104758928 B CN 104758928B
- Authority
- CN
- China
- Prior art keywords
- virus
- sheep
- goatpox
- weak
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a kind of goatpox, sheep of virus bigeminy cell weak-toxic vaccine and its production and use.A kind of goatpox of the present invention, sheep of virus bigeminy cell weak-toxic vaccine, its active ingredient is goatpox, the weak poison of sheep infective pustule virus passage cause or its passage poison that inventor voluntarily separates and obtained after Attenuation culture, wherein goat capripoxvirus passage causes weak poison to be XY2010F43 plants of Goatpox Virus HN, and deposit number is CCTCC NO：V201503；Sheep infective pustule virus passage causes weak poison to be TS09F65 plants of Orf Virus HB, and deposit number is CCTCC NO：V201406.Protest test result shows that the goatpox, sheep of virus bigeminy cell weak-toxic vaccine prepared by the present invention are safe, and can produce effective immune protective efficiency.The present invention is for developing safe and efficient attenuated vaccine and being had important practical significance with low virulent strain large-scale production goatpox disclosed by the invention, sheep of virus bigeminy cell weak-toxic vaccine come prevention and control sheep pox and sore mouth virus.
Description
Technical field
The present invention relates to a kind of attenuated vaccine and its production and use, more particularly to a kind of goatpox, sore mouth virus two
Join cell weak-toxic vaccine and its production and use, the invention belongs to veterinary biological product field.
Background technology
Capripox virus (Capripoxvirus, CPV) be Poxviridae Chordopoxvirinae Capripoxvirus into
Member.Category member have goat capripoxvirus (Goatpox virus, GTPV), sheep pox virus (Sheep pox virus, SPV) and
Ox nodositas rash block disease is viral (Lumpy skin disease virus, LSDV).Sheep pox is a kind of as caused by capripox virus
Acute, contagious disease, one of 93 kinds of animal epidemics that must be reported are classified as by OIE (FAO/OIE),
China is classified as a class infectious disease.Its incidence of disease is up to more than 75%, and the death rate is up to more than 50%, and the lamb death rate is reachable
100%, while can also result in ewe miscarriage.Sheep pox is one of ancient disease, and Hansen reports Norway first within 1879
Goatpox.Later, the disease is reported in other regions of the world successively.Be distributed widely in Africa, the Middle East, the Indian subcontinent, Northern Europe,
Mediterranean various countries and Germany, Australia, the U.S. etc., the Countries in particularly Africa the north, the Middle East and Asia are popular more
Seriously.The surrounding countries bordered on China, such as India, Bangladesh, Ni Baier, Pakistan, Russia, many countries of Mongolia
There is this sick prevalence.The ground such as the Jiangsu of China, Guangdong, Guizhou, Shandong, Zhejiang, Guangxi, Gansu, Heilungkiang had in recent years
This disease is popular.The sick outburst and prevalence decline the productivity of domestic animal, and poultry living and Livestock Product Trade are stagnated, the hair to sheep husbandry
Exhibition causes great economic loss.
Sheep infective pustule (Contagious ecthyma, CE) also known as contagious ecthyma of sheep
(Contagious pustular dermatitis), is commonly called as " sore mouth virus (Orf) ", is by parapoxvirus member's sheep infective purulence
Blister virus (Orf virus, ORFV) infection causes a kind of acute, contact of goat, sheep and people and thermophilic epithelial people beast
Suffer from infectious disease altogether, with suffer from the lip of sheep, hoof, breast, vulva etc. skin and mucous membrane formation erythema, papule, running sore, ulcer and
Excipuliform thickness scab is characterized.The disease is classified as by OIE (OIE) need to declare class Animal diseases, and China is classified as one
Class animal epidemic.The disease is found in Europe earliest, at present, almost exists in all sheep raising countries and regions.In the case of nature
Main infringement sheep and goat, goat is more multiple.In addition, camel, ox, deer, musk-ox, cat, pup, monkey and having susceptible per capita
Property.This disease is often in the popular trend of mass-sending property, and 3~6 monthly age lamb most easy infections often cause colony to be fallen ill, especially raise close
The flock of sheep of collection.Fan Chunling etc. reports China and breaks out the disease at the sub- good fortune animal-breeding center in Beijing in 2006, and the disease propagates speed
Degree is fast, and the incidence of disease is up to 95.4%.Development and sheep recently as Mutton Sheep Industry are frequently flowed, and the disease happens occasionally, even
There is the sick prevalence in some large-scale sheep studs, huge economic loss is brought to sheep husbandry, seriously endanger Mutton Sheep Industry
Sound development.More seriously, this disease can by wound infection keeper, the infected shows as the back of the hand, refer between and
Forethiga bleb and ulceration.Such as 2005 Nian8Yue Fujian Province Yong'an Cities have 8 Yang Chang cultivation workmans because of infection sheep infective warts
Virus and fall ill.It can be seen that, sheep infective warts not only seriously endangers the sound development of Mutton Sheep Industry, and threatens the body of the mankind
Health, is a kind of to endanger more serious infectious diseases common to human beings and animals.
In recent years, domestic and international sheep pox, sore mouth virus frequently break out, and sheep pox, sore mouth virus there is no effective treatment method, are immunized
Inoculation is the unique effective measure of prevention sore mouth virus.A kind of goatpox for being capable of anti-two disease of a pin, sore mouth virus are there is no at present
Bigeminy cell weak-toxic vaccine.Current sheep pox, the research and development of sore mouth virus vaccine and application are concentrated mainly on conventional vaccine, such as inactivated vaccine
And cell weak-toxic vaccine, and new generation vaccine, on such as cyst membrane subunit vaccine, nucleic acid vaccine and recombinant vaccine.But at present, sheep
Acne, the development of sore mouth virus new generation vaccine are also in experimental stage, and often preparation process is cumbersome, technical requirements are high for new generation vaccine,
There is very long stretch to walk applied to clinic.Although sheep pox, sore mouth virus inactivated vaccine domestic and foreign scholars have research, it is exempted from
Epidemic disease effect is limited, never real to be applied to clinic.At present, clinically widely used is still that sheep pox, sore mouth virus are weak
Malicious vaccine, because it has consumption few, can induce body to produce stronger cellular immunity and humoral immunity ability, and produce immune
The characteristics of time of protection is lasting.At home, although sore mouth virus attenuated vaccine is ground in Livestock Research and Veterinary Academy of Qinghai Prov. animal doctor
Studying carefully institute and Gansu Livestock and Veterinary Inst. has development, but its complex manufacturing, a backwardness, and it is all that freeze drying protectant is thermo-labile etc.
Many production technology bottleneck problems, be the vaccine can not volume production, the current vaccine, which is in, situation of the product without seedling.In addition, the weak poison
All it is the strain before and after the eighties in last century, popular poison domestic at present may morph, it is difficult to ensure that its immune validity.
There is production in Ji Jia vaccines producer to Attenuated Goat Pox Virus Vaccine at home, but the vaccine production process needs Testis Caprae seu Ovis primary cell,
Freeze drying protectant is thermo-labile to wait technological problemses, makes the production of vaccine, transport and preservation cost too high.Goatpox, sheep of virus
Bigeminy cell weak-toxic vaccine is studied, and still belongs to blank.Domestic and foreign scholars are existing it has proven convenient that sheep pox attenuated vaccine is in equal Yang Kou
Sore virus has partial intersection immunoprotection.Therefore, a kind of spectrotype of development is wide, immunogenicity is good, the simple mountain of seedling technique
Sheep pox, sheep of virus bigeminy cell weak-toxic vaccine, are more than to the domestic sheep pox of prevention, sore mouth virus outburst and prevalence with one-plus-one
Two realistic meaning.
The present invention from voluntarily separate acquisition ORFV Hubei Tongshan County isolated strain (HB09 plants of Orf virus) and
GTPV District, Xinyang Area, Henan Provinces city isolated strain (HN2010 plants of Goatpox virus) as candidate's strain of vaccine development, using ox/
Testis Caprae seu Ovis sertoli cell system and chick embryo fibroblast primary cell Attenuation, cultivate and meet " new biological product quality mark for animals
ORFV the and GTPV attenuated vaccine strains of standard ", match a certain proportion of heat-resisting lyophilized protecting agent and prepare lyophilized attenuated vaccine.Attack poison
Protection test result shows that prepared goatpox, sore mouth virus bigeminy cell weak-toxic vaccine can produce effective immunoprotection
Power.Therefore, the present invention using the popular strain of ORFV, GTPV develop safe and efficient attenuated vaccine for sheep pox, sore mouth virus it is anti-
Control and have important practical significance.
The content of the invention
For existing sheep pox, the deficiency of sore mouth virus attenuated vaccine, it is an object of the invention to provide a kind of goatpox, Yang Kou
Sore bigeminy cell weak-toxic vaccine and preparation method thereof, to realize with simple and practical preparation method production yield height, spectrotype
Extensively, stability is good, cost is low, safe and reliable vaccine.
The purpose of the present invention is achieved by the following technical solution：
To Hubei strain sore mouth positive material adapt to HB-TS09 plants of diseases of Orf Virus of separation with MDBK cell lines
Poison was passaged to for 17 generations, then the cell toxicant is inoculated with into new born bovine testis primary cell progress Attenuation, was carried out when being passaged to for 55 generation
The experiment of this animal safety finds that the cell toxicant has caused weak.Then proceed to be passaged to for 85 generations, virus (titre TCID is taken every five generations50
It is not less than 10-6.0/ 0.1mL) cell toxicant carry out the experiment of this animal safety, it was demonstrated that the strain is passaged to after 55 generations to sheep lamb
Sheep, adult sheep and kidlet and adult goat are safe.Wherein, in selection Orf Virus the 65th generations of HB-TS09, are thin
Born of the same parents' Attenuation poison is named as the cause of sheep infective pustule virus passage weak as sheep infective pustule virus attenuated vaccine strain
Malicious HB-TS09F65 plants of Orf Virus, Classification And Nomenclature is that sheep infective pustule virus passage causes weak poison, and in 2014 3
The moon delivers China typical culture collection center preservation, preserving number CCTCC NO on the 19th：V201406, preservation address is big in Wuhan
Learn.
To Henan Strain goatpox pathological material of disease adapt to the Goatpox virus HN2010 of separation with Testis Caprae seu Ovis primary cell
Strain virus is passaged to 16 generations, GTPV poison valencys lgTCID50When >=3.5, the cell toxicant inoculated into chick embryo is carried out into fiber primary cell
Attenuation, carries out the experiment discovery of this animal safety, the cell toxicant has caused weak when being passaged to for 28 generation when being passaged to for 36 generation.Then
Continuation was passaged to for 56 generations in Testis Caprae seu Ovis primary cell, take respectively Attenuation malicious 21 generations, 25 generations, 30 generations, 35 generations, 40 generations, 45 generations,
The generation cell toxicant of 50 generation 7 (each generation cell virus TCID50Not less than 103.5/ 0.1mL) carry out the experiment of this animal safety, it was demonstrated that connect
The sheep group of each HN-XY2010 plants of attenuated IBDVs of generation Goatpox virus is planted within the observation period, each test group sheep, in mouth
There is red point in transmucosal, dies away within 3 days after inoculation.Compare sheep normal.Prove attenuated IBDVs Goatpox virusHN-
XY2010 plants safe and free of pathogenicity to sheep, goat, is the vaccine low virulent strain of one plant of safety.Select Goatpox virus
The generation attenuated IBDVs of HN-XY2010 strains the 43rd are named as the cause of goat capripoxvirus passage weak as capripox virus attenuated vaccine strain
2010F43 plants of vaccine strain Goatpox Virus HN-XY, Classification And Nomenclature is the strain of goat capripoxvirus passage attenuated vaccine
2010F43 plants of Goatpox Virus HN-XY, and deliver on January 14th, 2015 China typical culture collection center guarantor
Hide, preserving number CCTCC NO：V201503, preservation address is in Wuhan University.
A kind of goatpox of the present invention, sheep of virus bigeminy cell weak-toxic vaccine, it is characterised in that its active ingredient bag
Including goat capripoxvirus passage causes weak poison and the cause of sheep infective pustule virus passage weak, wherein, described goatpox
Virocyte Attenuation poison is HN-XY2010F43 plants of goatpox poison cell Attenuation poison Goatpox Virus, is deposited in
China typical culture collection center, the preservation time is on January 14th, 2015, and deposit number is CCTCC NO：V201503, institute
The sheep infective pustule virus passage stated causes weak poison to cause weak malicious Orf Virus for sheep infective pustule virus passage
HB-TS09F65 plants, China typical culture collection center is deposited in, the preservation time is on March 19th, 2014, and deposit number is
CCTCC NO：V201406.
In addition, goatpox, the method for sheep of virus bigeminy cell weak-toxic vaccine are prepared present invention also offers a kind of, its
It is characterised by comprising the following steps：
(1) preparation of viral antigen
Well-grown is selected to be covered with the new born bovine testis primary cell and newborn Testis Caprae seu Ovis primary cell of individual layer, nutrition of inclining
Liquid, is inoculated with described sheep infective pustule virus passage by the 1-10% of former nutrient solution volume and causes weak malicious Orf respectively
HB-TS09F65 plants of Virus and goat capripoxvirus passage cause weak malicious HN-XY2010F43 plants of Goatpox Virus'
Virus liquid, absorption adds DMEM maintaining liquids after 5-15 minutes, adjusts PH to 7.0~7.2, plus penicillin, streptomysin each 100 units/ml,
Cultivated in 37 DEG C, 5%CO2 concentration incubators；40~72h is cultivated, harvest virus when cytopathy reaches more than 75%,
Put -20 DEG C of refrigerator multigelations twice, then carry out steriling test, safety verification, mycoplasma inspection, exogenous virus inspection, reach
Arrive《People's Republic of China's veterinary biological product quality standard》Specified in indices, and virus titer is not less than 10- 3.5TCID50During/0.1ml, weak malicious antigen is caused as goatpox, sheep infective pustule virus passage, it is standby；
(2) preparation of vaccine and lyophilized
Indices prepared by step (1) all qualified goatpox, sheep infective pustule virus passage cause weak poison
Antigen is mixed with the ︰ 1 of volume ratio 1, then after being mixed with heat-resisting lyophilized protecting agent with the ︰ 1 of volume ratio 1 ratio, with 2mL/ bottles of sterile bars
It is packaged in chain bottle, is freezed in low-temperature freeze-drying machine, as goatpox, sheep of virus bigeminy cell weak-toxic under part
Vaccine, it is preserved at 2 DEG C -8 DEG C.
In method of the present invention, it is preferred that be inoculated with described sheep infective respectively by the 5% of former nutrient solution volume
Pustule virus passage causes weak poison and goat capripoxvirus passage causes weak malicious virus liquid, and absorption adds DMEM after 10 minutes
Maintaining liquid, adjusts PH to 7.0~7.2, plus penicillin, streptomysin each 100 units/ml, enters in 37 DEG C, 5%CO2 concentration incubators
Row culture.
In method of the present invention, it is preferred that contain 30~70g/L of trehalose in the heat-resisting lyophilized protecting agent,
10~30g/L of skimmed milk power, 10~30g/L of polyvinylpyrrolidone, 5~15g/L of gelatin, 0.5~4g/L of arginine, vitamin
1~5g/L of C, residual components are ultra-pure water.It is furthermore preferred that containing trehalose 50g/L, degreasing in the heat-resisting lyophilized protecting agent
Milk 20g/L, polyvinylpyrrolidone 20g/L, gelatin 10g/L, arginine 1.5g/L, vitamin C 2.5g/L, residual components are
Ultra-pure water.
The preparation method of described heat-resisting lyophilized protecting agent includes：(1) pair can autoclaved material：Trehalose, poly- second
Alkene pyrrolidone (PVP), skimmed milk power, gelatin are dissolved in ultra-pure water in respective ratio, are heated to after 50~60 DEG C of dissolvings, 116
DEG C sterilizing 30 minutes；(2) the material arginine of non-refractory, vitamin C are also dissolved in ultra-pure water in respective ratio, use aperture
It is degerming for 0.22 μm of membrane filtration：Each group respectively mixes (1), (2) two components, obtains described heat-resisting lyophilized protecting agent.
Compared to prior art, the advantage of the invention is that：
1st, weak poison is caused to prepare vaccine from the passage of popular strain, it is ensured that the immune protective efficiency of vaccine；
2nd, using the heat-resisting lyophilized protecting agent for GTPV and ORFV viruses of this laboratory development, poxvirus work is solved
Vaccine is only capable of the Pinch technology of the storage and transport under the conditions of -15 DEG C, makes vaccine storage, transport more convenient.
The present invention is by for clinically for sheep pox, the prevention and control of sore mouth and sheep pox, sheep of virus in flock of sheep
Remove and provide important material base and technical support, have broad application prospects.
Embodiment
The invention will now be further described with reference to specific embodiments, and advantages of the present invention and feature will be with specific implementations
The description of example is apparent.But these embodiments are only exemplary, and few the scope of the present invention constitutes any limitation.Ability
Field technique personnel should be understood that without departing from the spirit and scope of the present invention can be to technical scheme and details
Form and form are modified or replaced, but these modifications and replacement are each fallen within the scope of protection of the invention.
The separation identification of the sheep infective pustule virus of embodiment 1 (HB-TS09 plants of Orf Virus), kind poison cause weak, seed
Criticize and set up and heat-resisting lyophilized protecting agent development
Sheep infective pustule virus seedling strain (HB-TS09 plants of Orf Virus) separation is identified, causes weak, seed lot to set up
With heat-resisting lyophilized protecting agent development, in the application for a patent for invention of the present inventor：《A kind of sheep infective pustule virus cell weak-toxic epidemic disease
Seedling and its preparation method and application》(application number：CN201410160855.0, publication number：) and patent of invention CN104017776A
Application《Sheep infective pustule virus cell weak-toxic vaccine heat-resisting lyophilized protecting agent and its preparation method and application》(application number：
CN201410101828.6, publication number：CN103893776A described in).Inventor is successfully separated out sore mouth virus Hubei poison
Strain (HB-TS09 plants of Orf Virus), and by its Attenuation, establish seedling strong malicious seed lot and weak malicious seed lot.Its
In, the generation passages of selection Orf Virus HB-TS09 the 65th cause weak poison as sheep infective pustule virus attenuated vaccine strain,
It is named as sheep infective pustule virus passage and causes weak malicious HB-TS09F65 plants of Orf Virus, Classification And Nomenclature is sheep infective
Pustule virus passage causes weak poison, and delivers China typical culture collection center preservation, preserving number on March 19th, 2014
CCTCC NO：V201406, preservation address is in Wuhan University.
In addition, inventor designs six kinds of heat-resisting lyophilized protecting agent prescriptions for sheep of virus, therefrom filter out a kind and be directed to
The preferable heat-resisting lyophilized protecting agent of sheep of virus protecting effect.
It is formulated and is：Trehalose 50g/L, defatted milk 20g/L, polyvinylpyrrolidone 20g/L, gelatin 10g/L, arginine
1.5g/L, vitamin C 2.5g/L, residual components are ultra-pure water.
Preparation method includes：(1) pair can autoclaved material：Trehalose, polyvinylpyrrolidone (PVP), defatted milk
Powder, gelatin are dissolved in ultra-pure water in respective ratio, are heated to after 50~60 DEG C of dissolvings, and 116 DEG C sterilize 30 minutes；(2) will be intolerant to
Material arginine, the vitamin C of high temperature are also dissolved in ultra-pure water in respective ratio, are that 0.22 μm of membrane filtration is degerming with aperture：
Each group respectively mixes (1), (2) two components, obtains described heat-resisting lyophilized protecting agent.
The separation and cause of the goat capripoxvirus of embodiment 2 (Goatpox virus HN-XY 2010) are weak
Separation, the identification of 2.1 goat capripoxviruses (Goatpox virus HN-XY 2010)
Inventor replicates to the positive pathological material of disease of goatpox that picks up from District, Xinyang Area, Henan Province for 2010, virus purification, case, most
Strain virus strain is obtained eventually, and the Strain is identified through viral physicochemical property, for the specific PCR of P 32 gene of goat capripoxvirus
Detection and sequencing prove that the Strain is that goat capripoxvirus virus (is named as HN-XY10 plants of Gotepox Virus, referred to as
GTPV/HN/2010 plants).
The cause of 2.2 goat capripoxviruses (HN-XY2010 plants of Goatpox virus) seedling strain is weak and seed culture of viruses seed lot is built
It is vertical
16 generations, GTPV poison valencys lgTCID are passaged to GTPV/HN/2010 strain virus with Testis Caprae seu Ovis primary cell50>=3.5,
From the SPF chicken embryos of the age in days of 9 ages in days~11, aseptic condition is rejected after the head, four limbs, internal organ of chicken embryo, by remaining tissue, after shredding
Chick embryo fibroblast primary cell is prepared into mixed enzyme digestion method.The cell toxicant is inoculated with each of well-grown asepsis pollution-free
Item index reaches the chick embryo fibroblast primary cell of " new biological product quality standard for animals ", carries out Attenuation, and set up cause
Weak seed culture of viruses seed lot.36 generations (good per generation cell toxicant cytopathogenic effect (CPE)) are passaged to, malicious valency is 103.5/ 0.1mL with
On, return this animal carry out this animal safety experiment discovery, the cell toxicant be passaged to 28 generations caused it is weak.Will be thin with chick embryo fibroblast
Born of the same parents cause weak goat capripoxvirus (HN-XY2010 plants of F36 of Goatpox virus) inoculation Testis Caprae seu Ovis primary cell to carry out passage training
Support to 56 generations, set up seed culture of viruses seed lot.
The pure property of 2.3 seeds culture of viruses is examined
Attenuation malicious 21 generations, 25 generations, 30 generations, 35 generations, 40 generations, 45 generations, the generation cell toxicant of 50 generation 7 are taken respectively, by existing
《Chinese veterinary pharmacopoeia》, 2010 editions, annex progress steriling tests of page 42.Each generation cell toxicant is without colony growth；By existing《Chinese beast
Pharmacopeia》, 2010 editions, annex progress mycoplasma inspections of page 49.Each generation cell toxicant grows without mycoplasma；By existing《Chinese veterinary drug
Allusion quotation》, 2010 editions, annex progress exogenous virus inspections of page 40.Each generation cell toxicant pollutes without exogenous virus；Prove that each generation is caused weak
Cell toxicant is pure, pollution-free.
2.4 sheep pox attenuated IBDVs safety examinations
Attenuation malicious 21 generations, 25 generations, 30 generations, 35 generations, 40 generations, 45 generations, generation cell toxicant (each generation of 50 generation 7 are taken respectively
Cell virus TCID50Not less than 103.5/ 0.1mL), per generation cell toxicant, it is not less than 10000 with goatpox, sheep of virus
TCID50 immunizing doses, (sheep mucous membrane of mouth and root of the tail intradermal vaccination) is inoculated with the silk floss of or so 1 age healthy sheep and 3 monthly ages or so
Each 4 of lamb sheep (from the healthy sheep for meeting experimental animal standard for being not suffering from sheep pox, also not injecting sheep pox vaccine)；With
10000 TCID50 dosage of inoculation (sheep mucous membrane of mouth and root of the tail intradermal vaccination) is inoculated with or so 1 age healthy goat and 3 respectively
The kidlet at or so monthly age is (from the healthy mountain for meeting experimental animal standard for being not suffering from sheep pox, also not injecting sheep pox vaccine
Sheep) each 4,8 groups are inoculated with altogether.It is random select each one of sheep, goat with 1mL dose inoculations physiological saline (sheep mucous membrane of mouth and
Root of the tail intradermal vaccination) compare.The daily body temperature and oral mucosa for determining inoculation sheep and control sheep after oral mucosa streak inoculation
Lesion situation, observe 15 days, carry out attenuated IBDVs safety evaluatio.
As a result：
The sheep group of each HN-XY2010 plants of attenuated IBDVs of generation Goatpox virus is inoculated with the observation period, each experiment
Only, there is red point in oral mucosa, died away within 3 days after inoculation in group sheep.Compare sheep normal.Prove attenuated IBDVs
HN-XY2010 plants of Goatpox virus are safe and free of pathogenicity to sheep, goat, are the vaccine low virulent strains of one plant of safety.
The generation attenuated IBDVs of Goatpox virus HN-XY2010 strains the 43rd are selected as capripox virus attenuated vaccine strain, life
Entitled capripox virus passage causes weak malicious Goatpox Virus HN-XY2010F43, and on January 14th, 2015 delivers
State's Type Tissue Collection preservation, preserving number CCTCC NO：V201503, preservation address is in Wuhan University.
The goatpox of embodiment 3, the trial-production of sheep of virus bigeminy cell weak-toxic vaccine laboratory
The preparation of the weak malicious antigen of 3.1 goatpoxes, sheep infective pustule virus
Select well-grown to be covered with newborn Testis Caprae seu Ovis primary cell (LT) and the new born bovine testis primary cell (BT) of individual layer, incline
Nutrient solution is removed, being inoculated with goat capripoxvirus passage respectively by the 5% of former nutrient solution volume causes weak malicious Goatpox Virus HN-
XY2010F43(CCTCC NO：) and sheep infective pustule virus passage causes weak malicious Orf Virus HB- V201503
TS09F65 virus liquid (CCTCC NO：V201406), DMEM maintaining liquids are added after adsorbing 10 minutes, PH to 7.0, plus mould is adjusted
Element, each 100 units of streptomysin/ml, are cultivated in 37 DEG C, 5%CO2 concentration incubators.Cultivate 48h, cytopathy (CPE)
Harvested when reaching 80%, put -20 DEG C of refrigerator multigelations twice, then carried out steriling test, safety verification, mycoplasma and examine conjunction
Lattice reach《People's Republic of China's veterinary biological product quality standard》Specified in indices, Reed-Muench methods determine
Goatpox Virus HN-XY2010F43 virus titer (TCID50/ 0.1ml) it is 104.0/ 0.1ml and Orf Virus HB-
TS09F65 virus titer (TCID50/ 0.1ml) it is 106.0/0.1ml.It is each to prepare goatpox, sheep infective pustule virus cell
Attenuation viral disease venom 3000ml is used as the viral antigen for preparing vaccine.
The preparation of 3.2 vaccines and lyophilized
The indices that first 2.1 are prepared all qualified goatpox, sheep infective pustule virus cell weak-toxic antigen, with body
After ratio of the product than 1 ︰ 1 is mixed, then with heat-resisting lyophilized protecting agent (trehalose 50g/L, defatted milk 20g/L, polyvinylpyrrolidone
20g/L, gelatin 10g/L, arginine 1.5g/L, vitamin C 2.5g/L, residual components are ultra-pure water, are obtained through aseptic process
Protective agent.) mixed with 1 ︰ 1 ratio, after stirring, to be packaged under 2mL/ bottles of aseptic conditions in chain bottle, in cryogenic freezing
Freezed in drying machine.As finished product attenuated vaccine, by it, temperature is preserved at 2 DEG C -8 DEG C.Weak malicious freeze dried vaccine is prepared altogether
Three batches every batch 1700 bottles, lot number is：20140511、20140520、20140528.
The goatpox of embodiment 4, sore mouth virus bigeminy cell weak-toxic vaccine product inspection
4.1 physical behavior
Product is slightly yellow Sponge Porosity agglomerate, is easily departed from bottle wall, plus dissolved rapidly after dilution.
4.2 steriling test
From preparing preserve 20140511,20140520,20,140,528 3 batches of goatpox, sore mouth virus bigeminy cell weak-toxic epidemic diseases
Every batch of seedling takes three bottles at random in seedling, by existing《Chinese veterinary pharmacopoeia》, 2010 editions, annex progress of page 42, vaccine finished product is given birth to without bacterium
It is long.
4.3 mycoplasmas are examined
From preparing preserve 20140511,20140520,20,140,528 3 batches of goatpox, sore mouth virus bigeminy cell weak-toxic epidemic diseases
Every batch of seedling takes three bottles at random in seedling, by existing《Chinese veterinary pharmacopoeia》, 2010 editions, annex progress mycoplasma inspections of page 49.Each generation
Cell toxicant grows without mycoplasma；
4.4 exogenous viruses are examined
From preparing preserve 20140511,20140520,20,140,528 3 batches of goatpox, sore mouth virus bigeminy cell weak-toxics
Every batch of seedling takes three bottles at random in vaccine, by existing《Chinese veterinary pharmacopoeia》, 2010 editions, annex progress exogenous virus inspections of page 40.Respectively
Generation cell toxicant pollutes without exogenous virus；Prove that each generation causes weak cell toxicant pure, pollution-free.
4.5 lyophilized goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine residual moisture are determined
From preparing preserve 20141011,20141020,20,141,028 3 batches of goatpox, sore mouth virus bigeminy cell weak-toxic epidemic diseases
Every batch of seedling takes three bottles at random in seedling, by existing《Chinese veterinary pharmacopoeia》, 2010 editions, annex page 38 is measured, and three batches of vaccines are accorded with
Close regulation.
4.6 lyophilized goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine vacuum are determined
From preparing preserve 20140511,20140520,20,140,528 3 batches of goatpox, sore mouth virus bigeminy cell weak-toxic epidemic diseases
Every batch of seedling takes three bottles at random in seedling, by existing《Chinese veterinary pharmacopoeia》, 2010 editions, annex page 48 is measured, and three batches of vaccines are accorded with
Close regulation.
The goatpox of embodiment 5, sheep of virus bigeminy cell weak-toxic vaccine safety test
All indexs prepared by embodiment 3 all reach three batches of goatpoxes, the sheep of " new biological product quality standard for animals "
Aphtha bigeminy cell weak-toxic vaccine, is sampled immediately, with goatpox, not less than 10000 TCID50 immunizing doses of sheep of virus,
(sheep mucous membrane of mouth and root of the tail intradermal vaccination) is inoculated with the sheep lamb of or so 1 age healthy sheep and 3 monthly ages or so (from being not suffering from
Sheep pox, the healthy sheep for meeting experimental animal standard for also not injecting sheep pox vaccine) each 4；With connecing for 10000 TCID50
Plant the kid that dosage (sheep mucous membrane of mouth and root of the tail intradermal vaccination) is inoculated with or so 1 age healthy goat and 3 monthly ages or so respectively
Each 4 of sheep (from the healthy goat for meeting experimental animal standard for being not suffering from sheep pox, also not injecting sheep pox vaccine), is inoculated with 8 altogether
Group.It is random to select each one of sheep, goat and done pair with 1mL dose inoculations physiological saline (sheep mucous membrane of mouth and root of the tail intradermal vaccination)
According to.The daily body temperature and the lesion situation of oral mucosa for determining inoculation sheep and control sheep, observation 15 after oral mucosa streak inoculation
Day, carry out attenuated IBDVs safety evaluatio.
Result of the test finds that the sheep group of each batch vaccine is within the observation period, and each test group sheep only, goes out in oral mucosa
Existing red point, dies away for 3 days after inoculation.Compare sheep normal.Prove goatpox, sheep of virus bigeminy cell weak-toxic vaccine pair
Sheep and lamb, goat and lamb are safe and free of pathogenicity.Prove goatpox, sheep of virus bigeminy cell weak-toxic vaccine pair
Sheep is only safe.
The goatpox of embodiment 6, sheep of virus bigeminy cell weak-toxic vaccine potency test
6.1 from preparing preserve 20140511,20140520,20,140,528 3 batches of goatpox, sore mouth virus bigeminy cells it is weak
Every batch of seedling takes three bottles at random in malicious vaccine, dilutes and mixes by commercial weight, and median infective dose is determined with bovine testicle primary cell
(TCID50), it is desirable to sheep infective pustule virus TCID50It is not less than 106/ 0.1mL, goat capripoxvirus TCID50It is not less than 103.5/
0.1mL。
6.2 every batch of vaccine immunity adult sheep and lamb, adult goat and each 5 of lamb, adult sheep inoculation 0.4ml (mouths
Transmucosal streak inoculation 0.2ml/ and root of the tail intradermal vaccination 0.2ml/ are only), lamb halves.Select sheep, goat each one at random
Only compareed with 0.4mL dose inoculations physiological saline (sheep mucous membrane of mouth and root of the tail intradermal vaccination).It is immunized after concentrating 21, together with
Condition identical compares sheep, and random equivalent is divided into goatpox group and sore mouth virus group, the goatpox group strong poison the 16th of goatpox cell
Generation (Goatpox virus HN-XY 2010F16) cell is malicious by force；Sore mouth virus group the 17th generation (Orf of the strong poison of sore mouth virus cell
Virus HB-TS09F17), with 100 ID50 dosage, carry out challenge test.Observation 10~15 days, as a result such as the institute of table 1 and 2
Show.Potency test proves weak using goatpox low virulent strain (2010 plants of F43 of Goatpox virus HN-XY) and sore mouth virus cell
Bigeminy cell weak-toxic vaccine prepared by strain (HB-TS09F65 plants of Orf Virus) is good to sheep and goat immune protective efficiency,
The infection of sheep pox and sheep of virus can be prevented with clinical immunization.
The goatpox of table 1, sheep of virus bigeminy cell weak-toxic vaccine adult sheep efficacy test
The goatpox of table 2, sheep of virus bigeminy cell weak-toxic vaccine lamb efficacy test
Claims (6)
1. a kind of goatpox, sheep of virus bigeminy cell weak-toxic vaccine, it is characterised in that active ingredient includes goat capripoxvirus
Passage causes weak poison and sheep infective pustule virus passage to cause weak poison, wherein, described goat capripoxvirus cell is passed
In generation, causes weak poison to cause weak malicious HN-XY2010F43 plants of Goatpox Virus for goat capripoxvirus passage, is deposited in Chinese Typical Representative
Culture collection, the preservation time is on January 14th, 2015, and deposit number is CCTCC NO：V201503, described sheep passes
Metachromia pustule virus passage causes weak poison to cause weak malicious Orf Virus HB- for sheep infective pustule virus passage
TS09F65 plants, China typical culture collection center is deposited in, the preservation time is on March 19th, 2014, and deposit number is
CCTCC NO：V201406.
2. the goatpox, sheep of virus bigeminy cell weak-toxic vaccine described in claim 1 prevent and treat sheep pox and Yang Kou in preparation
Application in sore medicine.
3. a kind of method of goatpox prepared described in claim 1, sheep of virus bigeminy cell weak-toxic vaccine, its feature exists
In comprising the following steps：
(1) preparation of viral antigen
Well-grown is selected to be covered with the new born bovine testis primary cell and newborn Testis Caprae seu Ovis primary cell of individual layer, incline nutrient solution, presses
The 1-10% of former nutrient solution volume be inoculated with respectively sheep infective pustule virus passage described in claim 1 cause weak poison and
Goat capripoxvirus passage causes weak malicious virus liquid, and absorption adds DMEM maintaining liquids after 5-15 minutes, adjusts PH to 7.0~7.2, plus
Penicillin, streptomysin each 100 units/ml, are cultivated in 37 DEG C, 5%CO2 concentration incubators；Cultivate 40~72h, cell
Harvest virus, puts -20 DEG C of refrigerator multigelations twice when lesion reaches more than 75%, then carry out steriling test, safety verification,
Mycoplasma is examined, exogenous virus is examined, and is reached《People's Republic of China's veterinary biological product quality standard》Specified in it is every
Index, and virus titer is not less than 10-3.5TCID50During/0.1ml, caused as goatpox, sheep infective pustule virus passage
Weak malicious antigen, it is standby；
(2) preparation of vaccine and lyophilized
Indices prepared by step (1) all qualified goatpox, sheep infective pustule virus passage cause weak malicious antigen
Mixed with the ︰ 1 of volume ratio 1, then after being mixed with heat-resisting lyophilized protecting agent with the ︰ 1 of volume ratio 1 ratio, with 2mL/ bottles of aseptic conditions
It is packaged in chain bottle, is freezed in low-temperature freeze-drying machine, as goatpox, sheep of virus bigeminy cell weak-toxic epidemic disease
Seedling, it is preserved at 2 DEG C -8 DEG C.
4. method as claimed in claim 3, it is characterised in that be inoculated with claim 1 institute respectively by the 5% of former nutrient solution volume
The sheep infective pustule virus passage stated causes weak poison and goat capripoxvirus passage causes weak malicious virus liquid, absorption 10
Add DMEM maintaining liquids after minute, PH to 7.0~7.2, plus penicillin, streptomysin each 100 units/ml are adjusted, in 37 DEG C, 5%CO2It is dense
Cultivated in degree incubator.
5. method as claimed in claim 3, it is characterised in that contain 30~70g/ of trehalose in the heat-resisting lyophilized protecting agent
L, 10~30g/L of skimmed milk power, 10~30g/L of polyvinylpyrrolidone, 5~15g/L of gelatin, 0.5~4g/L of arginine, dimension life
Plain 1~5g/L of C, residual components are ultra-pure water.
6. method as claimed in claim 3, it is characterised in that contain trehalose 50g/L in the heat-resisting lyophilized protecting agent, takes off
Fat milk 20g/L, polyvinylpyrrolidone 20g/L, gelatin 10g/L, arginine 1.5g/L, vitamin C 2.5g/L, residual components
For ultra-pure water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510030082.9A CN104758928B (en) | 2015-01-21 | 2015-01-21 | A kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510030082.9A CN104758928B (en) | 2015-01-21 | 2015-01-21 | A kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104758928A CN104758928A (en) | 2015-07-08 |
| CN104758928B true CN104758928B (en) | 2017-09-22 |
Family
ID=53641239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510030082.9A Active CN104758928B (en) | 2015-01-21 | 2015-01-21 | A kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104758928B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106215186B (en) * | 2016-08-02 | 2019-10-22 | 甘肃省畜牧兽医研究所 | The preparation method of sore mouth virus live-vaccine heat-proof protective agent and its freeze-dried powder and the freeze-dried powder |
| CN106075429B (en) * | 2016-08-16 | 2020-05-26 | 中国农业科学院兰州兽医研究所 | Goat pox, sheep pox and aphtha triple cell attenuated vaccine and preparation method and application thereof |
| CN106139141B (en) * | 2016-08-16 | 2020-05-22 | 中国农业科学院兰州兽医研究所 | Sheep pox and orf bivalent cell attenuated vaccine and preparation method and application thereof |
| CN112126628B (en) * | 2020-07-20 | 2022-09-13 | 天康制药(苏州)有限公司 | Goat pox virus propagation method, goat pox live vaccine, preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2325437C1 (en) * | 2006-09-22 | 2008-05-27 | ГНУ ВНИИ ветеринарной вирусологии и микробиологии | Attenuated strain of goatpox virus |
| CN102302774A (en) * | 2008-10-08 | 2012-01-04 | 中国农业科学院哈尔滨兽医研究所 | Peste des petits ruminants recombinant goat pox virus live vector vaccines |
-
2015
- 2015-01-21 CN CN201510030082.9A patent/CN104758928B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2325437C1 (en) * | 2006-09-22 | 2008-05-27 | ГНУ ВНИИ ветеринарной вирусологии и микробиологии | Attenuated strain of goatpox virus |
| CN102302774A (en) * | 2008-10-08 | 2012-01-04 | 中国农业科学院哈尔滨兽医研究所 | Peste des petits ruminants recombinant goat pox virus live vector vaccines |
Non-Patent Citations (4)
| Title |
|---|
| Animal poxvirus vaccines: a comprehensive review;Veerakyathappa et al;《Expert Rev. Vaccines》;20121231;第11卷(第11期);第1355-1374页具体1360右栏最后一段、1361页、1362页第2段、1363页第2段 * |
| Comparative efficacy of live replicating sheeppox vaccine strains in Ovines;R. Yogisharadhya et al;《Biologicals》;20111231;第39卷;第417-423页 * |
| 活毒苗防治山羊传染性脓疱病的临床应用;王芬等;《Ｊｏｕｒｎａｌ ｏｆ Ａｎｉｍａｌ Ｓｃｉｅｎｃｅ ａｎｄ Ｖｅｔｅｒｉｎａｒｙ Ｍｅｄｉｃｉｎｅ》;20121231;第31卷(第3期);第84-85页 * |
| 羊传染性脓疱皮炎细胞弱毒苗的研究;薛双虎等;《甘肃畜牧兽医》;19891231(第5期);第1-3页具体第1页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104758928A (en) | 2015-07-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103627678B (en) | A kind of porcine pseudorabies virus variant PRV-ZJ01 and application | |
| CN102220287B (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN104758928B (en) | A kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use | |
| CN101514334A (en) | Chicken infectivity bronchitis virus attenuated vaccine strain and application thereof | |
| CN103849632B (en) | S1 gene of low virulent strain of infectious bronchitis and low virulent strain and application thereof | |
| CN104017776B (en) | A kind of sheep infective pustule virus cell weak-toxic vaccine and its preparation method and application | |
| CN106075429B (en) | Goat pox, sheep pox and aphtha triple cell attenuated vaccine and preparation method and application thereof | |
| CN103497934B (en) | Avian infectious bronchitis virus vaccine strain (HF2 strain) and application thereof | |
| CN104800842B (en) | A kind of goatpox, sheep pox divalence cell weak-toxic vaccine and its preparation method and application | |
| CN104258386B (en) | A kind of mink viral enteritis inactivated vaccine-canine distemper live vaccine combination | |
| CN108066758B (en) | Raccoon dog canine distemper-parvovirus enteritis bigeminal live vaccine and preparation method and application thereof | |
| CN104056265B (en) | Porcine circovirus 2 type, Porcine reproductive and respiratory syndrome bigeminy vaccine and preparation method thereof | |
| CN106190988B (en) | Inactivated vaccine of feline calicivirus CH-JL5 strain | |
| CN101745106A (en) | Porcine parvnvirus living vaccine and preparation method thereof | |
| CN103784951B (en) | Prevent and treat antigen composition of respiratory disease of scabies secondary infection of pig and its preparation method and application | |
| CN102727877A (en) | Method for preparing highly pathogenic porcine reproductive and respiratory syndrome live vaccine (JXA1-R strain) by utilizing bioreactor and application thereof | |
| CN110302374B (en) | Canine vaccine and preparation method and application thereof | |
| CN102228685A (en) | Preparation method of iridovirus cell inactivated vaccine for Chinese giant salamanders, and application method thereof | |
| CN106139141B (en) | Sheep pox and orf bivalent cell attenuated vaccine and preparation method and application thereof | |
| CN104388396B (en) | Porcine pseudorabies poison strain and inactivated vaccine prepared therefrom and application | |
| CN104069489B (en) | Newcastle disease and infectious bursa of Fabricius bivalent inactivated vaccine and preparation method thereof | |
| CN101380470B (en) | Pig parvovirus live vaccine | |
| CN109010814A (en) | The production method of haemophilus parasuis and mycoplasma hyopneumoniae bivalent inactivated vaccine | |
| CN105582535A (en) | Preparation method of CSF (Classical Swine Fever) and PR (Pseudorabies) bivalent live vaccine and product of CSF and PR bivalent live vaccine | |
| CN111073863B (en) | Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |