CN102488894B - Fox encephalitis live vaccine and preparation method thereof - Google Patents
Fox encephalitis live vaccine and preparation method thereof Download PDFInfo
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- CN102488894B CN102488894B CN201110455840.3A CN201110455840A CN102488894B CN 102488894 B CN102488894 B CN 102488894B CN 201110455840 A CN201110455840 A CN 201110455840A CN 102488894 B CN102488894 B CN 102488894B
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Abstract
The invention relates to a fox encephalitis live vaccine and a preparation method thereof. According to the invention, a separated, identified, attenuated and preserved canine adenoviruses II type CAV-2RZ strain is adopted as a virus strain used for producing the vaccine. The virus strain is multiplied on MDCK cells. Steps such as collection and lyophilization are carried out, such that a safe and effective fox encephalitis live vaccine is obtained. With the live vaccine provided by the invention, the occurring of fox encephalitis can be effectively prevented, and a condition is provided for controlling fur animal diseases.
Description
Technical field
The present invention relates to a kind of fox encephalitis live vaccine and preparation method thereof, belong to veterinary biological product technical field.
Background technology
Epizootic fox encephalitis be caused by hepatitis infectiosa canis virus-I type virus with fox loss of appetite or useless absolutely, acute, the contagious infection disease of the feature such as drink wish increases, spirit is depressed, bradykinesia, fervescence, vomiting, body nihility power, nystagmus, paroxysmal tic, paralysis.Just reported epizootic fox encephalitis as far back as nineteen twenty-five Green, and the people such as nineteen thirty Greent and Dewey delivered its pathogen be viral evidence (list of references: body draws livestock epidemiology recklessly. Beijing: Science Press, 1963.).1989, epizootic fox encephalitis, first in the outburst of Shandong Province of China, was widely current in China thereafter and causes fox to occur higher death, has caused larger economic loss to China's fox rearing industry.
1962, Dithfield separates (list of references: Darai G during once breaking out canine infectious laryngotracheitis from dog respiratory tract, Rosen A, DepI-Hs H, Flugel RM.Characterization of the DNA of canine adenovirus byrestriction enzyme analysis[J] .Intervirology, 1985,23 (1): 23-8.) obtain the hepatitis infectiosa canis virus-II type virus that causes merely respiratory disease (laryngotracheitis) and do not start an inflammation of the liver, i.e. A26 strain (Toronto A26/61).Afterwards canine infectious hepatitis virus was called to 1 type adenovirus (CAV-1), A26 strain virus was called to canine infectious laryngotracheitis virus, be i.e. 2 type adenoviruss (CAV-2).Hepatitis infectiosa canis virus-II type strain aspect virulence, soluble antigen structure, cell infection scope and red cell agglutination scope all with some difference of type strain canine infectious hepatitis virus.But dog and the fox of the immunity of application hepatitis infectiosa canis virus-II type strain, but can to canine infectious hepatitis virus produce effective immunity (list of references: Yin Zhen. animal virology. the 2nd edition. Beijing: Science Press, 1997,1104-1130).1978, after the succedaneum listing of first commercial hepatitis infectiosa canis virus-II type attenuated vaccine strain as hepatitis infectiosa canis virus-I type attenuated vaccine strain, epizootic fox encephalitis vaccine used all replaced hepatitis infectiosa canis virus-I type viral vaccine with hepatitis infectiosa canis virus-II type virus in the world at present.
Summary of the invention
The object of the invention is to utilize the inventor to separate voluntarily, identify, cause the weak malicious CAV-2RZ strain of strain hepatitis infectiosa canis virus-II type weak, that preserve as the Strain of producing vaccine, inoculation mdck cell carries out virus breeding, through gathering in the crops, joining the step such as Seedling, lyophilizing, prepare safe and effective fox encephalitis live vaccine.
Detailed description of the invention
In order effectively to control epizootic fox encephalitis epidemic situation, the inventor utilized hepatitis infectiosa canis virus II type (Canine adenovirus type 2) the CAV-2RZ strain a little less than separating voluntarily, identify, causing to carry out the research of fox encephalitis live vaccine in 2003.Vaccine safety is tested and potency test result shows, our fox encephalitis live vaccine (CAV-2RZ strain) of development is safe and reliable, has no adverse reaction, and after immune fox, can produce strong immunity, can effectively prevent the generation of epizootic fox encephalitis.
1. production of vaccine strain
(1) viral source
Vaccine manufacture and inspection are hepatitis infectiosa canis virus II type (Canine adenovirus type 2) CAV-2RZ strain cell toxicant with seed culture of viruses, are separated, identified, cause weak, certainly and supply by the inventor.On November 17th, 2011, this strain virus was delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, was numbered: CGMCC No.5475.
(2) Strain characteristic
1) viral level
Seed culture of viruses is made to 10 times of serial dilutions with the cell nutrient solution (MEM) containing 2% new-born calf serum, get 10
-4, 10
-5, 10
-6, 10
-74 dilution factors, inoculation has grown up to the mdck cell 96 hole Microtitration plates of monolayer respectively, and each dilution factor is inoculated 8 holes, and every hole 100 μ l, establish simultaneously and do not connect malicious matched group, put 37 DEG C, 5%CO
2in incubator, cultivate and observe 96～120 hours, record the hole count of cytopathy (CPE).Press Reed-Muench method and calculate TCID
50(Chinese veterinary pharmacopoeia committee. three of People's Republic of China's veterinary drug allusion quotation two O mono-O versions. Chinese agriculture publishing house, 2011, the present invention is hereinafter to be referred as " Chinese veterinary pharmacopoeia "), every milliliter of viral level answers>=10
6.5tCID
50.
2) safety
By seed culture of viruses, through healthy susceptible fox of 2～10 monthly ages of intramuscular inoculation (hepatitis infectiosa canis virus NAT is not higher than 1: 4) 5,10ml/ only, observes 14, should be without abnormal response.
3) immunogenicity
With healthy susceptible fox of 2～10 monthly ages (hepatitis infectiosa canis virus NAT is not higher than 1: 4) 5, every intramuscular inoculation seed culture of viruses 1ml is (containing 10
4.5tCID
50), establish simultaneously and do not inoculate 5 of contrast foxes.Immunity blood sampling in latter 21 days, separation of serum, measures hepatitis infectiosa canis virus NAT, and immune fox hepatitis infectiosa canis virus NAT all should be not less than 1: 30, and contrast fox hepatitis infectiosa canis virus NAT all should be higher than 1: 4.
5) specificity
Seed culture of viruses is done to 1000 times of dilutions, mix with hepatitis infectiosa canis virus-II type specific antiserum of 10 times of dilutions of equivalent, do not neutralize contrast and add equal-volume MEM, in 37 DEG C and 60 minutes, inoculation has grown up to the mdck cell 24 porocyte culture plates of monolayer, neutralization group is inoculated 12 holes, does not neutralize contrast and cell and contrasts each 6 holes, every hole 0.5ml.Tissue Culture Plate is put to 37 DEG C, 5%C0
2in incubator, cultivate and observe 96～120 hours.Should not there is not CPE in neutralization group and cell matched group, not in and matched group should all there is CPE.
6) seed culture of viruses is preserved
-15 DEG C of following preservations, storage life is 24 months;-70 DEG C of following preservations, storage life is 60 months.
2. the production of fox encephalitis live vaccine (CAV-2RZ strain)
(1) well-grown mdck cell is gone down to posterity according to a conventional method, after cell grows up to monolayer, the growth-promoting media that inclines, changes with the maintenance medium containing 2% seed culture of viruses (containing the MEM of 2% new-born calf serum), put 37 DEG C and continue to cultivate, rolling bottle rotating speed is 8～10r/h.
(2) after inoculation, observe 1～2 cytopathy (CPE) every day, in the time that CPE reaches 80% left and right, can gather in the crops, freeze thawing 1 time, puts-15 DEG C of following Cryopreservations, checks for subsequent use.
(3) by the virus liquid being up to the standards, add 5% sucrose-skimmed milk used as stabilizers in the ratio of 1: 1, fully mix quantitative separating.
(4) after subpackage, carry out rapidly lyophilisation.
3. the inspection of fox encephalitis live vaccine (CAV-2RZ strain)
(1) character yellowish white Sponge Porosity agglomerate, easily departs from bottle wall, dissolves rapidly after adding diluent.
(2) steriling test is tested by the method for " Chinese veterinary pharmacopoeia " regulation, answers asepsis growth.
(3) mycoplasma inspection is tested by the method for " Chinese veterinary pharmacopoeia " regulation, should grow without mycoplasma.
(4) exogenous virus inspection, by the method performing check of " Chinese veterinary pharmacopoeia " regulation, should be polluted without exogenous virus.And the method specifying with reference to existing " Chinese veterinary pharmacopoeia ", carry out exogenous canine distemper virus, Canine Parvovirus, canine coronavirus and bovine viral diarrhea virus with Vero, F81, MDCK, BTC cell and check, should conform with the regulations.
(5) vaccine is diluted to 1 part/ml by diagnostic test, then do 100 times of dilutions, mix with hepatitis infectiosa canis virus-II type specific antiserum of 10 times of dilutions of equivalent, do not neutralize contrast and add equal-volume MEM, in 37 DEG C, with 60 minutes, inoculation grew up to the mdck cell 24 porocyte culture plates of monolayer, and neutralization group is inoculated 12 holes, do not neutralize contrast and cell and contrast each 6 holes, every hole 0.5ml.Tissue Culture Plate is put to 37 DEG C, 5%CO
2in incubator, cultivate and observe 96～120 hours.Should not there is not CPE in neutralization group and cell matched group, not in and matched group should all there is CPE.
(6) safety verification indicates head part by label vaccine is diluted to every milliliter containing 5 parts, healthy susceptible fox of 2～10 monthly ages of intramuscular inoculation (hepatitis infectiosa canis virus NAT is not higher than 1: 4) 5,2ml/ only, observes 14, should all be good for and live.
(7) viral level is measured
Vaccine is diluted to 1 part/ml, remakes 10 times of serial dilutions, get 10
-4, 10
-5, 10
-6, 10
-74 dilution factors, inoculation has grown up to the mdck cell 96 hole Microtitration plates of monolayer respectively, and each dilution factor is inoculated 8 holes, and every hole 100 μ l, establish simultaneously and do not connect malicious control wells, put 37 DEG C, 5%CO
2in incubator, cultivate and observe 96～120 hours, record CPE hole count, calculate TCID by Reed-Muench method
50, every part vaccine virus content answers>=10
4.5tCID
50.
(8) residual moisture is measured and is measured by existing " Chinese veterinary pharmacopoeia " annex, should meet the regulation of veterinary biologics general rule.
(9) vacuum is measured by the method for " Chinese veterinary pharmacopoeia " regulation and is measured, and should conform with the regulations.
The microorganism information the present invention relates to
Microorganism fungus kind involved in the present invention is: hepatitis infectiosa canis virus II type (Canine adenovirus type 2) CAV-2RZ strain, this strain virus on November 17th, 2011 this strain virus delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, be numbered: CGMCC No.5475.
Advantage of the present invention
The present invention utilizes the inventor to separate voluntarily, identify, cause a strain weak, that preserve to have good immunogenic hepatitis infectiosa canis virus CAV-2RZ strain as producing strain, through gathering in the crops, joining the steps such as Seedling, prepares safe and effective fox encephalitis live vaccine.The production strain that the present invention adopts can, for current Prevalence of diseases, effectively prevent the generation of epizootic fox encephalitis, for the control of fur-bearing animal disease creates conditions.
Embodiment
Following examples further illustrate the present invention, but not as limitation of the present invention.
Embodiment 1
The weak malicious CAV-2RZ strain safety test of hepatitis infectiosa canis virus II type
For the safety of malicious CAV-2RZ strain to fox a little less than checking seedling use strain hepatitis infectiosa canis virus-II type, the inventor inoculates fox for virus liquid through intramuscular routes heavy dose (10ml) with the weak malicious CAV-2RZ strain C1 of hepatitis infectiosa canis virus II type, by clinical observation, histopathologic examination, all there is not laryngotracheitis clinical symptoms and pathological change in inoculation fox, shows that hepatitis infectiosa canis virus-II type CAV-2RZ strain seed culture of viruses of Attenuation is safe to fox.
Embodiment 2
The Study On Immunogenicity of hepatitis infectiosa canis virus-II type CAV-2RZ strain
Whether strain has the important evidence that good immunogenicity is screening vaccine strain; therefore; the inventor to hepatitis infectiosa canis virus II type a little less than malicious CAV-2RZ strain immunogenicity be studied; and NAT and counteracting toxic substances protection dependency after vaccine immunity fox are compared; determine the marginal value of antibody protection, for method for testing efficacy and the standard of formulating vaccine provide foundation.
Result of the test is as follows:
Table 1CAV-2RZ strain Study On Immunogenicity result
Result of the test shows, hepatitis infectiosa canis virus-II type weak malicious CAV-2RZ strain C5, C10 and C20 are for malicious immunogenicity no significant difference, when immunizing dose>=10
4.5tCID
50/ time, immunity after counteracting toxic substances protective rate all reach 100%.
Embodiment 3
The production of fox encephalitis live vaccine (CAV-2RZ strain)
(1) well-grown mdck cell is gone down to posterity according to a conventional method, after cell grows up to monolayer, the growth-promoting media that inclines, changes with the maintenance medium containing 2% seed culture of viruses (containing the MEM of 2% new-born calf serum), put 37 DEG C and continue to cultivate, rolling bottle rotating speed is 8～10r/h.
(2) after inoculation, observe 1～2 cytopathy (CPE) every day, in the time that CPE reaches 80% left and right, can gather in the crops, freeze thawing 1 time, puts-15 DEG C of following Cryopreservations, checks for subsequent use.
(3) by the virus liquid being up to the standards, add 5% sucrose-skimmed milk used as stabilizers in the ratio of 1: 1, fully mix quantitative separating.
(4) after subpackage, carry out rapidly lyophilisation.
Embodiment 4
Fox encephalitis live vaccine safety test
3 batches of fox encephalitis live vaccines (CAV-2RZ strain) of preparing by method provided by the invention with the inventor, carry out respectively minimum and use 1 single dose inoculation of age in days fox, single dose repeated inoculation, 1 overdose inoculation, 1 overdose inoculation of conceived bitch and 1 overdose of dog is inoculated to safety test.
1. pair minimum single dose inoculation safety testing of age in days fox that uses
Fox encephalitis live vaccine (CAV-2RZ strain) single dose inoculation susceptible fox, 1 part/only, observation 14 days after inoculation, observes its spirit, diet and feces and changes.
Result shows, all foxes are all strong living of duration of test, and immune fox spirit, diet, injection site, feces, body temperature etc. are all normal, and contrast Vulpes no significant difference, the 14th day time, all foxes catched and killed, and has no the variation of fox laryngotracheitis pathology.
2. single dose repeated inoculation safety testing
Fox encephalitis live vaccine (CAV-2RZ strain) single dose inoculation susceptible fox, 1 part/only, inoculate latter 14 days booster immunizations once, 1 part/only, two exempt from rear observation 14 days, measure body temperature every day, observe its spirit, diet and feces and change.
Result shows, all foxes are all strong living of duration of test, and immune fox spirit, diet, feces, body temperature etc. are showed no extremely, and contrasts no significant difference, the 14th day time, all foxes catched and killed, and has no the variation of fox laryngotracheitis pathology.
3. overdose inoculation safety testing
Fox encephalitis live vaccine (CAV-2RZ strain) overdose inoculation susceptible fox, 10 part/only, observation 14 days after inoculation, observes its spirit, diet and feces and changes.
Result shows, all foxes are all strong living of duration of test, and immune fox spirit, diet, feces, body temperature etc. are all normal, and contrast Vulpes no significant difference, the 14th day time, all foxes catched and killed, and has no the variation of fox laryngotracheitis pathology.
4. the overdose inoculation safety test of the conceived bitch of healthy susceptible
The conceived healthy susceptible bitch of fox encephalitis live vaccine (CAV-2RZ strain) overdose inoculation, 10 part/only, observation 14 days after inoculation, observes its spirit, diet and feces and changes.
Result demonstration, all foxes are all strong living of duration of test, and immune fox spirit, diet and feces etc. are all without abnormal; Bitch is farrowing smoothly in expected date of confinement, and farrowing is normal, and contrasts Vulpes no significant difference.
5. dog overdose inoculation safety testing
Fox encephalitis live vaccine (CAV-2RZ strain) overdose is inoculated healthy susceptible dog, and 10 part/only, observation 14 days after inoculation, observes its spirit, diet and feces and change.
Result shows, all dogs are all strong living of duration of test, and immune dog spirit, diet, feces, body temperature etc. are all normal, and contrasts dog no significant difference.The 14th day time, test dog is catched and killed, had no dog laryngotracheitis pathology and change.
Embodiment 5
Fox encephalitis live vaccine antibody horizontal detects
The fox encephalitis live vaccine (CAV-2RZ strain) of preparing by the method for the invention with the inventor carries out immunity to immune group and two groups of foxes of matched group respectively, after 21 days, immune group and control animals are taken a blood sample, separation of serum, measures NAT by fixed virus diluted blood therapy for clearing away heat.
Result is as follows:
Table 2 fox TPPA result
Claims (1)
1. the preparation method of a fox encephalitis live vaccine, it is characterized in that by preserving number being 37 DEG C of cultivations of hepatitis infectiosa canis virus II type (Canine adenovirus type2) inoculation mdck cell of CGMCC No.5475, in the time connecing poison cell and have 80% to occur pathological changes, results virocyte culture fluid adds conventional freeze drying protectant, makes freeze-dried live vaccine through lyophilization; The hepatitis infectiosa canis virus II type of this CGMCCNo.5475 on November 17th, 2011 this strain virus delivered No. 3 China Committee for Culture Collection of Microorganisms of the Institute of Microorganism, Academia Sinica common micro-organisms center preservations in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
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