CN104758928A - Goatpox virus-orf virus combined cell attenuated vaccine and its preparation method and use - Google Patents
Goatpox virus-orf virus combined cell attenuated vaccine and its preparation method and use Download PDFInfo
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Abstract
The invention discloses a goatpox virus-orf virus combined cell attenuated vaccine and its preparation method and use. The goatpox virus-orf virus combined cell attenuated vaccine contains an active component which comprises goatpox and contagious pustular dermatitis virus cell passage-attenuated viruses or passaged viruses separated by the inventor and subjected to passage attenuation culture, wherein the goatpox virus cell passage-attenuated virus is a Goatpox Virus HN-XY2010F43 strain and has a preservation number of CCTCC NO: V201503 and the contagious pustular dermatitis virus cell passage-attenuated virus is an Orf Virus HB-TS09F65 strain and has a preservation number of CCTCC NO: V201406. A toxin counteracting protection test result shows that the goatpox virus-orf virus combined cell attenuated vaccine is safe and can produce an effective immunization protection function. The goatpox virus-orf virus combined cell attenuated vaccine and its preparation method have important practical meanings for development of safe and efficient attenuated vaccines and large-scale production of the goatpox virus-orf virus combined cell attenuated vaccine for preventing and controlling goatpox and orf from the disclosed attenuated strain.
Description
Technical field
The present invention relates to a kind of attenuated vaccine and its production and use, particularly a kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and its production and use, the invention belongs to veterinary biological product field.
Background technology
Capripox virus (Capripoxvirus, CPV) is Poxviridae Chordopoxvirinae Capripoxvirus member.This genus member has the sick virus (Lumpy skin disease virus, LSDV) of goat capripoxvirus (Goatpox virus, GTPV), sheep pox virus (Sheep pox virus, SPV) and cattle nodositas rash block.Sheep pox is that the one caused by capripox virus is acute, contagious disease, is classified as one of 93 kinds of animal epidemics that must report by OIE (FAO/OIE), and China is classified as a class infectious disease.Its sickness rate can reach more than 75%, and mortality rate can reach more than 50%, and lamb mortality rate can reach 100%, and ewe also can be caused to miscarry simultaneously.Sheep pox is one of ancient disease, Hansen reported first in 1879 goatpox of Norway.Afterwards, this disease was 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., particularly the Countries in northern, Middle East, Africa and Asia is popular comparatively serious.The surrounding countries of bordering on China, all there be the popular of primary disease in countries as many in India, Bangladesh, Ni Baier, Pakistan, Russia, Mongolia etc.The ground such as Jiangsu, Guangdong, Guizhou, Shandong, Zhejiang, Guangxi, Gansu, Heilungkiang of China all had primary disease popular in recent years.The outburst of this disease the and popular productivity of domestic animal is declined, live poultry and Livestock Product Trade are stagnated, and cause great economic loss to the development of sheep husbandry.
Sheep infective pustule (Contagious ecthyma, CE) contagious ecthyma of sheep (Contagious pustular dermatitis) is had another name called, be commonly called as " sore mouth virus (Orf) ", by parapoxvirus member sheep infective pustule virus (Orf virus, ORFV) infect cause goat, sheep and people a kind of acute, contact and addicted to epithelial Arbo infectious disease, to form the thick crust of erythema, pimple, abscess, ulcer and excipuliform for feature at place's skin such as lip, hoof, breast, pudendum and mucosa of suffering from sheep.This disease is classified as by OIE (OIE) need declare class Animal diseases, and China is classified as a class animal epidemic.This disease is found in Europe the earliest, at present, almost all exists in all sheep raising countries and regions.Mainly encroach on sheep and goat in nature situation, goat is comparatively multiple.In addition, camel, cattle, deer, musk-ox, cat, pup, monkey and have susceptibility per capita.Primary disease is often in the popular trend of mass-sending property, and the most easy infection of 3 ~ 6 monthly age lamb, often causes colony to fall ill, especially raise intensive flock of sheep.Fan Chunling etc. report China and break out this disease at good fortune animal cultivation center, Asia, Beijing in 2006, and this sick spread speed is fast, and sickness rate is up to 95.4%.In recent years along with the development of Mutton Sheep Industry and sheep are frequently flowed, this disease happens occasionally, and even has the popular of this disease at some large-scale sheep studs, has carried out huge economic loss, the sound development of serious harm Mutton Sheep Industry to sheep raising industrial belt.More seriously, this disease can pass through wound infection keeper, the infected shows as the back of the hand, refer between and forethiga herpes and ulceration.Such as Fujian Province's in August, 2005 Yongan municipalization 8 Yang Chang cultivate workman and fall ill because infecting sheep infective pustule virus.Visible, the sound development of sheep infective pustule not only serious harm Mutton Sheep Industry, and threaten the mankind healthy is the comparatively serious infectious diseases common to human beings and animals of a kind of harm.
In recent years, domestic and international sheep pox, sore mouth virus frequently break out, and sheep pox, sore mouth virus there is no effective Therapeutic Method, and immunity inoculation is the unique effective measure of prevention sore mouth virus.There is no at present and a kind ofly can prevent goatpox, the sore mouth virus bigeminy cell weak-toxic vaccine of two diseases by a pin.Research and development and the application of current sheep pox, sore mouth virus vaccine mainly concentrate on conventional vaccine, as inactivated vaccine and cell weak-toxic vaccine, and new generation vaccine, as on cyst membrane subunit vaccine, nucleic acid vaccine and recombiant vaccine.But at present, the development of sheep pox, sore mouth virus new generation vaccine is also under test, and new generation vaccine often preparation process is loaded down with trivial details, technical requirement is high, be applied to clinically to also have very long stretch to walk.Although sheep pox, sore mouth virus inactivated vaccine Chinese scholars all have research, its immune effect is limited, is never really applied to clinical.At present, widely usedly clinically remain sheep pox, sore mouth virus attenuated vaccine because it to have consumption few, body can be induced to produce stronger cellular immunization and humoral immunization ability, and the feature that the time producing immune protective efficiency is lasting.At home; although sore mouth virus attenuated vaccine all has development at Livestock Research and Veterinary Academy of Qinghai Prov.'s veterinary institute and Gansu Livestock and Veterinary Inst.; but its complex manufacturing, backwardness; many production technology bottleneck problems such as freeze drying protectant is thermo-labile; be that this vaccine cannot volume production, this vaccine is in the situation of product without Seedling at present.In addition, this weak poison is all the strain before and after the eighties in last century, and current domestic popular poison may morph, and is difficult to the effectiveness ensureing its immunity.Attenuated Goat Pox Virus Vaccine at home Ji Jia vaccine producer all has production, but this vaccine production process needs Testis Caprae seu Ovis primary cell, and freeze drying protectant is thermo-labile waits technological problems, makes this production of vaccine, transport and retain costs too high.Goatpox, sheep of virus bigeminy cell weak-toxic vaccine are studied, and still belong to blank.Chinese scholars now confirms, and sheep pox attenuated vaccine has partial intersection immunoprotection to being in equal sheep of virus.Therefore, develop that a kind of spectrotype is wide, immunogenicity good, the simple goatpox of seedling technique, sheep of virus bigeminy cell weak-toxic vaccine, to the domestic sheep pox of prevention, sore mouth virus outburst and the popular realistic meaning that there is one-plus-one and be greater than two.
The present invention selects Tongshan County, the ORFV Hubei isolated strain (Orf virus HB09 strain) that is separated acquisition voluntarily and GTPV District, Xinyang Area, Henan Province city isolated strain (Goatpox virus HN2010 strain) as candidate's strain of vaccine development, utilize cattle/Testis Caprae seu Ovis sustenticular cell system and chick embryo fibroblast primary cell Attenuation, cultivate ORFV and the GTPV attenuated vaccine strain meeting " new biological product quality standard for animals ", a certain proportion of heat-resisting lyophilized protecting agent of proportioning prepares lyophilizing attenuated vaccine.Goatpox prepared by the display of protest test result, sore mouth virus bigeminy cell weak-toxic vaccine can produce effective immune protective efficiency.Therefore, the present invention utilizes ORFV, GTPV epidemic isolates to develop safety, efficient attenuated vaccine has important practical significance for the control of sheep pox, sore mouth virus.
Summary of the invention
For the deficiency of existing sheep pox, sore mouth virus attenuated vaccine, the object of the present invention is to provide a kind of goatpox, sore mouth virus bigeminy cell weak-toxic vaccine and preparation method thereof, to realize producing by simple and practical preparation method the vaccine that output is high, spectrotype wide, good stability, cost are low, safe and reliable.
The object of the invention is to be achieved through the following technical solutions:
By MDBK cell line, be passaged to the Orf Virus HB-TS09 strain virus that Hubei strain sore mouth positive material carries out adapting to be separated 17 generations, again this cell toxicant inoculation new born bovine testis primary cell is carried out Attenuation, carry out the test of this animal safety when being passaged to for 55 generation to find, this cell toxicant has caused weak.Then continue to be passaged to for 85 generations, get virus (titre TCID every five generations
50be not less than 10
-6.0/ 0.1mL) cell toxicant carry out the test of this animal safety, confirm that this strain was passaged to after 55 generations all safe to sheep lamb, grow up sheep and kidlet and adult goat.Wherein, Orf Virus HB-TS09 the 65th generation passage is selected to cause weak poison as sheep infective pustule virus attenuated vaccine strain, called after sheep infective pustule virus passage causes weak malicious Orf Virus HB-TS09F65 strain, Classification And Nomenclature is that sheep infective pustule virus passage causes weak poison, and deliver China typical culture collection center preservation on March 19th, 2014, preserving number CCTCC NO:V201406, preservation address is in Wuhan University.
With Testis Caprae seu Ovis primary cell, be passaged to the Goatpox virus HN2010 strain virus that Henan Strain goatpox pathological material of disease carries out adapting to be separated 16 generations, GTPV poison valency lgTCID
50when>=3.5, become fiber primary cell to carry out Attenuation this cell toxicant inoculated into chick embryo, carry out the test of this animal safety when being passaged to for 36 generation and find, this cell toxicant has caused weak when being passaged to for 28 generation.Then continue to be passaged to for 56 generations at Testis Caprae seu Ovis primary cell, get Attenuation 21 generations of poison, 25 generations, 30 generations, 35 generations, 40 generations, 45 generations, 50 generation 7 generation cell toxicant (each generation cell virus TCID respectively
50be not less than 10
3.5/ 0.1mL) carry out the test of this animal safety, confirming that a sheep group of each generation Goatpox virus HN-XY2010 strain attenuated IBDVs of inoculation is within the observation period, only, all there is red point at oral mucosa, within 3 days, dies away after inoculation in each test group sheep.Contrast sheep is normal.Proving that attenuated IBDVs Goatpox virusHN-XY2010 strain is all safe to sheep, goat, no pathogenicity, is the vaccine low virulent strain of a strain safety.Select Goatpox virus HN-XY2010 strain the 43rd generation attenuated IBDVs as capripox virus attenuated vaccine strain, called after goat capripoxvirus passage attenuated vaccine strain Goatpox Virus HN-XY 2010F43 strain, Classification And Nomenclature is goat capripoxvirus passage attenuated vaccine strain Goatpox Virus HN-XY 2010F43 strain, and deliver China typical culture collection center preservation on January 14th, 2015, 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 characterized in that its effective ingredient comprises goat capripoxvirus passage and causes weak poison and sheep infective pustule virus passage causes weak, wherein, it is goatpox poison cell Attenuation poison Goatpox Virus HN-XY2010F43 strain that described goat capripoxvirus passage causes weak poison, be deposited in China typical culture collection center, the preservation time is on January 14th, 2015, deposit number is CCTCC NO:V201503, it is that sheep infective pustule virus passage causes weak malicious Orf Virus HB-TS09F65 strain that described sheep infective pustule virus passage causes weak poison, be deposited in China typical culture collection center, the preservation time is on March 19th, 2014, deposit number is CCTCC NO:V201406.
In addition, present invention also offers a kind of method preparing goatpox, sheep of virus bigeminy cell weak-toxic vaccine, it is characterized in that comprising the following steps:
(1) preparation of virus antigen
Well-grown is selected to be covered with the new born bovine testis primary cell of monolayer and newborn Testis Caprae seu Ovis primary cell, incline nutritional solution, inoculate described sheep infective pustule virus passage respectively by the 1-10% of former nutritional solution volume and cause the virus liquid that weak malicious Orf Virus HB-TS09F65 strain and goat capripoxvirus passage cause weak malicious Goatpox Virus HN-XY2010F43 strain, adsorb and add DMEM maintenance medium after 5-15 minute, adjust PH to 7.0 ~ 7.2, add penicillin, streptomycin each 100 units/ml, in 37 DEG C, cultivate in 5%CO2 concentration incubator; Cultivate 40 ~ 72h, results virus when cytopathy reaches more than 75%, put-20 DEG C of refrigerator multigelations twice, then steriling test, safety verification, mycoplasma inspection, exogenous virus inspection is carried out, reach the indices of regulation in " People's Republic of China's veterinary biological product quality standard ", and virus titer is not less than 10
-3.5tCID
50during/0.1ml, cause weak malicious antigen as goatpox, sheep infective pustule virus passage, for subsequent use;
(2) preparation of vaccine and lyophilizing
The goatpox that indices step (1) prepared is all qualified, sheep infective pustule virus passage cause weak malicious antigen and mix with volume ratio 1 ︰ 1; after mixing with the ratio of volume ratio 1 ︰ 1 with heat-resisting lyophilized protecting agent again; to be packaged in chain bottle under 2mL/ bottle aseptic condition; lyophilizing is carried out in low-temperature freeze-drying machine; be goatpox, sheep of virus bigeminy cell weak-toxic vaccine, it is preserved at 2 DEG C-8 DEG C.
In method of the present invention, preferably, inoculate described sheep infective pustule virus passage respectively by 5% of former nutritional solution volume and cause the virus liquid that weak poison and goat capripoxvirus passage cause weak poison, adsorb and add DMEM maintenance medium after 10 minutes, adjust PH to 7.0 ~ 7.2, add penicillin, streptomycin each 100 units/ml, in 37 DEG C, cultivate in 5%CO2 concentration incubator.
In method of the present invention, preferably, trehalose 30 ~ 70g/L is contained in described heat-resisting lyophilized protecting agent; defatted milk powder 10 ~ 30g/L, polyvinylpyrrolidone 10 ~ 30g/L, gelatin 5 ~ 15g/L; arginine 0.5 ~ 4g/L, vitamin C 1 ~ 5g/L, residual components is ultra-pure water.Preferred, containing trehalose 50g/L, defatted milk 20g/L, polyvinylpyrrolidone 20g/L, gelatin 10g/L, arginine 1.5g/L, vitamin C 2.5g/L in described heat-resisting lyophilized protecting agent, residual components is ultra-pure water.
The preparation method of described heat-resisting lyophilized protecting agent comprises: (1) is to can autoclaved material: trehalose, polyvinylpyrrolidone (PVP), defatted milk powder, gelatin are dissolved in ultra-pure water in respective ratio, be heated to 50 ~ 60 DEG C dissolve after, 116 DEG C of sterilizings 30 minutes; (2) the material arginine of non-refractory, vitamin C are also dissolved in ultra-pure water in respective ratio; be that 0.22 μm of membrane filtration is degerming with aperture: (1), (2) two components mix by each group respectively, obtain described heat-resisting lyophilized protecting agent.
Compared to prior art, the invention has the advantages that:
1, select the passage of epidemic isolates to cause weak poison to prepare vaccine, ensure that the immune protective efficiency of vaccine;
2, utilize the heat-resisting lyophilized protecting agent for GTPV and ORFV virus of this laboratory development, solve poxvirus live vaccine only can under-15 DEG C of conditions the Pinch technology of storage and transport, this vaccine is stored, transport more convenient.
The present invention, by for providing important material base and technical support for the prevention and control of sheep pox, sore mouth and sheep pox, the removing of sheep of virus in flock of sheep clinically, has broad application prospects.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear with the description of specific embodiment.But these embodiments are only exemplary, and few scope of the present invention forms any restriction.It will be understood by those skilled in the art that and not depart under spirit and scope of the invention and can technical scheme of the present invention and detail view and form modified or be replaced, but these amendments and replacing all fall in the scope of protection of the invention.
The isolation identification of embodiment 1 sheep infective pustule virus (Orf Virus HB-TS09 strain), plant poison and cause weak, seed lot and set up and heat-resisting lyophilized protecting agent development
Sheep infective pustule virus seedling strain (Orf Virus HB-TS09 strain) isolation identification, cause weak, seed lot is set up and heat-resisting lyophilized protecting agent development, application for a patent for invention the present inventor: " a kind of sheep infective pustule virus cell weak-toxic vaccine and its preparation method and application " (application number: CN201410160855.0, publication number: CN104017776A) and application for a patent for invention " 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) in describe.Inventor successfully isolates sore mouth virus Hubei strain (Orf Virus HB-TS09 strain), and by its Attenuation, establishes the strong malicious seed lot of seedling and weak malicious seed lot.Wherein, Orf Virus HB-TS09 the 65th generation passage is selected to cause weak poison as sheep infective pustule virus attenuated vaccine strain, called after sheep infective pustule virus passage causes weak malicious Orf Virus HB-TS09F65 strain, Classification And Nomenclature is that sheep infective pustule virus passage causes weak poison, and deliver China typical culture collection center preservation on March 19th, 2014, preserving number 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 filters out a kind for the good heat-resisting lyophilized protecting agent of sheep of virus protected effect.
Formula is: trehalose 50g/L, defatted milk 20g/L, polyvinylpyrrolidone 20g/L, gelatin 10g/L, arginine 1.5g/L, vitamin C 2.5g/L, and residual components is ultra-pure water.
Preparation method comprises: (1) is to can autoclaved material: trehalose, polyvinylpyrrolidone (PVP), defatted milk powder, gelatin are dissolved in ultra-pure water in respective ratio, be heated to 50 ~ 60 DEG C dissolve after, 116 DEG C of sterilizings 30 minutes; (2) the material arginine of non-refractory, vitamin C are also dissolved in ultra-pure water in respective ratio; be that 0.22 μm of membrane filtration is degerming with aperture: (1), (2) two components mix by each group respectively, obtain described heat-resisting lyophilized protecting agent.
The separation of embodiment 2 goat capripoxvirus (Goatpox virus HN-XY 2010) and cause weak
The separation of 2.1 goat capripoxviruses (Goatpox virus HN-XY 2010), qualification
Inventor is to the positive pathological material of disease of the goatpox picking up from District, Xinyang Area, Henan Province for 2010, virus purification, case copies, finally obtain a strain virus strain, this Strain is identified through viral physicochemical property, detect for the specific PCR of P 32 gene of goat capripoxvirus and order-checking proves that this Strain is goat capripoxvirus virus (called after Gotepox Virus HN-XY10 strain, be called for short GTPV/HN/2010 strain).
Weak and the seed culture of viruses seed lot of causing of 2.2 goat capripoxviruses (Goatpox virus HN-XY2010 strain) seedling strain is set up
With Testis Caprae seu Ovis primary cell, be passaged to GTPV/HN/2010 strain virus 16 generations, GTPV poison valency lgTCID
50>=3.5, select the SPF Embryo Gallus domesticus of 9 age in days ~ 11 ages in days, aseptic condition by remaining tissue, shreds rear mixed enzyme digestion method and is prepared into chick embryo fibroblast primary cell after rejecting the head of Embryo Gallus domesticus, extremity, internal organs.The indices of this cell toxicant inoculation well-grown asepsis pollution-free is reached the chick embryo fibroblast primary cell of " new biological product quality standard for animals ", carry out Attenuation, and foundation causes weak seed culture of viruses seed lot.Be passaged to 36 generations (every generation cell toxicant cytopathogenic effect (CPE) is good), malicious valency is all 10
3.5/ more than 0.1mL, return this animal and carry out the test of this animal safety and find, this cell toxicant was passaged to for 28 generations and has caused weak.Inoculate Testis Caprae seu Ovis primary cell carry out Secondary Culture to 56 generation by causing weak goat capripoxvirus (Goatpox virus HN-XY2010 strain F36) by chick embryo fibroblast, set up seed culture of viruses seed lot.
The pure property inspection of 2.3 seed culture of viruses
Get respectively Attenuation poison 21 generations, 25 generations, 30 generations, 35 generations, 40 generations, 45 generations, 50 generation 7 generation cell toxicant, by existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 42 pages carries out steriling test.Each generation cell toxicant is without colony growth; By existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 49 pages carries out mycoplasma inspection.Each generation cell toxicant grows without mycoplasma; By existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 40 pages carries out exogenous virus inspection.Each generation cell toxicant pollutes without exogenous virus; Prove that each generation causes weak cell toxicant pure, pollution-free.
2.4 sheep pox attenuated IBDVs safety examinations
Get Attenuation 21 generations of poison, 25 generations, 30 generations, 35 generations, 40 generations, 45 generations, 50 generation 7 generation cell toxicant (each generation cell virus TCID respectively
50be not less than 10
3.5/ 0.1mL), every generation cell toxicant, 10000 TCID50 immunizing doses are not less than, healthy sheep and each 4 of the sheep lamb about 3 monthly ages (never suffered from sheep pox, also do not inject the healthy sheep meeting laboratory animal standard of sheep pox vaccine) about (sheep oral mucosa and root of the tail intradermal vaccination) inoculated for 1 age with goatpox, sheep of virus; About inoculating for 1 age respectively with the dosage of inoculation of 10000 TCID50 (sheep oral mucosa and root of the tail intradermal vaccination), healthy goat and each 4 of the kidlet about 3 monthly ages (never suffered from sheep pox, also do not inject the healthy goat meeting laboratory animal standard of sheep pox vaccine), inoculate 8 groups altogether.Random choose sheep, each one of goat contrast with 1mL dose inoculation normal saline (sheep oral mucosa and root of the tail intradermal vaccination).After oral mucosa streak inoculation, every day measures inoculation sheep and contrasts the body temperature of sheep and the pathological changes situation of oral mucosa, observes 15, carries out attenuated IBDVs safety evaluatio.
Result:
Inoculating a sheep group of each generation Goatpox virus HN-XY2010 strain attenuated IBDVs within the observation period, only, all there is red point at oral mucosa, within 3 days, dies away after inoculation in each test group sheep.Contrast sheep is normal.Proving that attenuated IBDVs Goatpox virus HN-XY2010 strain is all safe to sheep, goat, no pathogenicity, is the vaccine low virulent strain of a strain safety.
Select Goatpox virus HN-XY2010 strain the 43rd generation attenuated IBDVs as capripox virus attenuated vaccine strain, called after capripox virus passage causes weak malicious Goatpox Virus HN-XY2010F43, and deliver China typical culture collection center preservation on January 14th, 2015, preserving number CCTCC NO:V201503, preservation address is in Wuhan University.
Embodiment 3 goatpox, sheep of virus bigeminy cell weak-toxic vaccine laboratory are manufactured experimently
The preparation of the weak malicious antigen of 3.1 goatpoxes, sheep infective pustule virus
Well-grown is selected to be covered with newborn Testis Caprae seu Ovis primary cell (LT) and the new born bovine testis primary cell (BT) of monolayer, incline nutritional solution, inoculate goat capripoxvirus passage respectively by 5% of former nutritional solution volume and cause the virus liquid (CCTCC NO:V201406) that weak malicious Goatpox Virus HN-XY2010F43 (CCTCC NO:V201503) and sheep infective pustule virus passage cause weak malicious Orf Virus HB-TS09F65, adsorb and add DMEM maintenance medium after 10 minutes, adjust PH to 7.0, add penicillin, streptomycin each 100 units/ml, in 37 DEG C, cultivate in 5%CO2 concentration incubator.Cultivate 48h, gather in the crops when cytopathy (CPE) reaches 80%, put-20 DEG C of refrigerator multigelations twice, then carry out steriling test, safety verification, mycoplasma be up to the standards and reach indices of regulation in " People's Republic of China's veterinary biological product quality standard ", Reed-Muench method measures the virus titer (TCID of Goatpox Virus HN-XY2010F43
50/ 0.1ml) be 10
4.0virus titer (the TCID of/0.1ml and Orf Virus HB-TS09F65
50/ 0.1ml) be 10
6.0/ 0.1ml.Each preparation goatpox, sheep infective pustule virus passage cause weak viral disease venom 3000ml as the virus antigen preparing vaccine.
The preparation of 3.2 vaccines and lyophilizing
First by all qualified for 2.1 indices prepared goatpox, sheep infective pustule virus cell weak-toxic antigen; after the mixing of the ratio of volume ratio 1 ︰ 1; again 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 is ultra-pure water, through the protective agent that aseptic process obtains.) mix with the ratio of 1 ︰ 1, after stirring, to be packaged in chain bottle under 2mL/ bottle aseptic condition, in low-temperature freeze-drying machine, carry out lyophilizing.Be finished product attenuated vaccine, its temperature at 2 DEG C-8 DEG C is preserved.Prepare weak malicious freeze dried vaccine three batches altogether and often criticize 1700 bottles, lot number is: 20140511,20140520,20140528.
Embodiment 4 goatpox, the product inspection of sore mouth virus bigeminy cell weak-toxic vaccine
4.1 physical behavior
Product is that micro-yellow spongy loosens agglomerate, easily and bottle wall depart from, dissolve rapidly after adding diluent.
4.2 steriling test
From 20140511,20140520,20,140,528 3 batches of goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine of preparation preservation, often criticize Seedling get three bottles at random, by existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 42 pages carries out, and vaccine finished product is without bacterial growth.
4.3 mycoplasma inspections
From 20140511,20140520,20,140,528 3 batches of goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine of preparation preservation, often criticize Seedling get three bottles at random, by existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 49 pages carries out mycoplasma inspection.Each generation cell toxicant grows without mycoplasma;
4.4 exogenous virus inspections
From 20140511,20140520,20,140,528 3 batches of goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine of preparation preservation, often criticize Seedling get three bottles at random, by existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 40 pages carries out exogenous virus inspection.Each generation cell toxicant pollutes without exogenous virus; Prove that each generation causes weak cell toxicant pure, pollution-free.
4.5 lyophilizing goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine residual moisture measure
From 20141011,20141020,20,141,028 3 batches of goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine of preparation preservation, often criticize Seedling get three bottles at random, by existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 38 pages measures, and three batches of vaccines all conform with the regulations.
4.6 lyophilizing goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine vacuum measure
From 20140511,20140520,20,140,528 3 batches of goatpoxes, sore mouth virus bigeminy cell weak-toxic vaccine of preparation preservation, often criticize Seedling get three bottles at random, by existing " Chinese veterinary pharmacopoeia ", 2010 editions, annex 48 pages measures, and three batches of vaccines all conform with the regulations.
Embodiment 5 goatpox, the safety test of sheep of virus bigeminy cell weak-toxic vaccine
All indexs embodiment 3 prepared all reach three batches of goatpoxes, the sore mouth virus bigeminy cell weak-toxic vaccine of " new biological product quality standard for animals ", sample immediately, 10000 TCID50 immunizing doses are not less than, healthy sheep and each 4 of the sheep lamb about 3 monthly ages (never suffered from sheep pox, also do not inject the healthy sheep meeting laboratory animal standard of sheep pox vaccine) about (sheep oral mucosa and root of the tail intradermal vaccination) inoculated for 1 age with goatpox, sheep of virus; About inoculating for 1 age respectively with the dosage of inoculation of 10000 TCID50 (sheep oral mucosa and root of the tail intradermal vaccination), healthy goat and each 4 of the kidlet about 3 monthly ages (never suffered from sheep pox, also do not inject the healthy goat meeting laboratory animal standard of sheep pox vaccine), inoculate 8 groups altogether.Random choose sheep, each one of goat contrast with 1mL dose inoculation normal saline (sheep oral mucosa and root of the tail intradermal vaccination).After oral mucosa streak inoculation, every day measures inoculation sheep and contrasts the body temperature of sheep and the pathological changes situation of oral mucosa, observes 15, carries out attenuated IBDVs safety evaluatio.
Result of the test finds, a sheep group of each batch of vaccine is within the observation period, and each test group sheep only, all occurs red point at oral mucosa, within 3 days, dies away after inoculation.Contrast sheep is normal.Prove goatpox, sheep of virus bigeminy cell weak-toxic vaccine is to sheep and lamb, goat and lamb are all safe, no pathogenicity.Prove that goatpox, sheep of virus bigeminy cell weak-toxic vaccine are to a sheep safety.
Embodiment 6 goatpox, sheep of virus bigeminy cell weak-toxic vaccine potency test
Often criticize Seedling 6.1 20140511,20140520,20,140,528 3 batches of goatpoxes of preserving from preparation, sore mouth virus bigeminy cell weak-toxic vaccine and get three bottles at random, by commercial weight dilution mixture, measure median infective dose (TCID with bovine testicle primary cell
50), require sheep infective pustule virus TCID
50be not less than 10
6/ 0.1mL, goat capripoxvirus TCID
50be not less than 10
3.5/ 0.1mL.
6.2 often criticize vaccine immunity grows up sheep and lamb, adult goat and each 5 of lamb, and the sheep inoculation 0.4ml that grows up (oral mucosa streak inoculation 0.2ml/ only and root of the tail intradermal vaccination 0.2ml/), lamb reduces by half.Random choose sheep, each one of goat contrast with 0.4mL dose inoculation normal saline (sheep oral mucosa and root of the tail intradermal vaccination).Immunity was concentrated after 21 days, and together with the contrast sheep that condition is identical, random equivalent is divided into goatpox group and sore mouth virus group, and the strong poison of goatpox group goatpox cell the 16th generation (Goatpox virus HN-XY 2010F16) cell is malicious by force; The sore mouth virus group strong poison of sore mouth virus cell the 17th generation (Orf Virus HB-TS09F17), with the dosage of 100 ID50, carries out challenge test.Observe 10 ~ 15 days, result as shown in Tables 1 and 2.Potency test prove utilize goatpox low virulent strain (Goatpox virus HN-XY 2010 strain F43) and the bigeminy cell weak-toxic vaccine prepared by sore mouth virus cell weak-toxic strain (Orf Virus HB-TS09F65 strain) good to sheep and goat immune protective efficiency, the infection of useful clinical immunoprophylaxis sheep pox and sheep of virus.
Table 1 goatpox, sheep of virus bigeminy cell weak-toxic vaccine are grown up sheep efficacy test
Table 2 goatpox, sheep of virus bigeminy cell weak-toxic vaccine lamb efficacy test
Claims (6)
1. a goatpox, sheep of virus bigeminy cell weak-toxic vaccine, it is characterized in that effective ingredient comprises goat capripoxvirus passage and causes weak poison and sheep infective pustule virus passage causes weak poison, wherein, it is that goat capripoxvirus passage causes weak malicious Goatpox VirusHN-XY2010F43 strain that described goat capripoxvirus passage causes weak poison, be deposited in China typical culture collection center, the preservation time is on January 14th, 2015, deposit number is CCTCC NO:V201503, it is that sheep infective pustule virus passage causes weak malicious Orf Virus HB-TS09F65 strain that described sheep infective pustule virus passage causes weak poison, be deposited in China typical culture collection center, the preservation time is on March 19th, 2014, deposit number is CCTCC NO:V201406.
2. goatpox according to claim 1, sheep of virus bigeminy cell weak-toxic vaccine are preparing the application prevented and treated in sheep pox and sore mouth virus medicine.
3. prepare a method for goatpox according to claim 1, sheep of virus bigeminy cell weak-toxic vaccine, it is characterized in that comprising the following steps:
(1) preparation of virus antigen
Well-grown is selected to be covered with the new born bovine testis primary cell of monolayer and newborn Testis Caprae seu Ovis primary cell, incline nutritional solution, inoculate sheep infective pustule virus passage according to claim 1 respectively by the 1-10% of former nutritional solution volume and cause the virus liquid that weak and goat capripoxvirus passage causes weak poison, adsorb and add DMEM maintenance medium after 5-15 minute, adjust PH to 7.0 ~ 7.2, add penicillin, streptomycin each 100 units/ml, in 37 DEG C, cultivate in 5%CO2 concentration incubator; Cultivate 40 ~ 72h, results virus when cytopathy reaches more than 75%, put-20 DEG C of refrigerator multigelations twice, then steriling test, safety verification, mycoplasma inspection, exogenous virus inspection is carried out, reach the indices of regulation in " People's Republic of China's veterinary biological product quality standard ", and virus titer is not less than 10
-3.5tCID
50during/0.1ml, cause weak malicious antigen as goatpox, sheep infective pustule virus passage, for subsequent use;
(2) preparation of vaccine and lyophilizing
The goatpox that indices step (1) prepared is all qualified, sheep infective pustule virus passage cause weak malicious antigen and mix with volume ratio 1 ︰ 1; after mixing with the ratio of volume ratio 1 ︰ 1 with heat-resisting lyophilized protecting agent again; to be packaged in chain bottle under 2mL/ bottle aseptic condition; lyophilizing is carried out in low-temperature freeze-drying machine; be goatpox, sheep of virus bigeminy cell weak-toxic vaccine, it is preserved at 2 DEG C-8 DEG C.
4. method as claimed in claim 3, it is characterized in that, inoculate sheep infective pustule virus passage according to claim 1 respectively by 5% of former nutritional solution volume and cause the virus liquid that weak poison and goat capripoxvirus passage cause weak poison, adsorb and add DMEM maintenance medium after 10 minutes, adjust PH to 7.0 ~ 7.2, add penicillin, streptomycin each 100 units/ml, in 37 DEG C, 5%CO
2cultivate in concentration incubator.
5. method as claimed in claim 3; it is characterized in that; containing trehalose 30 ~ 70g/L in described heat-resisting lyophilized protecting agent; defatted milk powder 10 ~ 30g/L; polyvinylpyrrolidone 10 ~ 30g/L, gelatin 5 ~ 15g/L, arginine 0.5 ~ 4g/L; vitamin C 1 ~ 5g/L, residual components is ultra-pure water.
6. method as claimed in claim 3, is characterized in that, containing trehalose 50g/L, defatted milk 20g/L, polyvinylpyrrolidone 20g/L, gelatin 10g/L, arginine 1.5g/L, vitamin C 2.5g/L in described heat-resisting lyophilized protecting agent, residual components is ultra-pure water.
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| CN112126628A (en) * | 2020-07-20 | 2020-12-25 | 天康生物股份有限公司 | Goat pox virus propagation method, goat pox live vaccine, preparation method and application thereof |
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