CN101612396B - Canine distemper live vaccine and preparation method thereof - Google Patents
Canine distemper live vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN101612396B CN101612396B CN2009101579237A CN200910157923A CN101612396B CN 101612396 B CN101612396 B CN 101612396B CN 2009101579237 A CN2009101579237 A CN 2009101579237A CN 200910157923 A CN200910157923 A CN 200910157923A CN 101612396 B CN101612396 B CN 101612396B
- Authority
- CN
- China
- Prior art keywords
- canine distemper
- vaccine
- strain
- virus
- cdv
- 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
- 229960005486 vaccine Drugs 0.000 title claims abstract description 84
- 208000000655 Distemper Diseases 0.000 title claims abstract description 71
- 208000014058 canine distemper Diseases 0.000 title claims abstract description 70
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 241000700605 Viruses Species 0.000 claims abstract description 39
- 238000011081 inoculation Methods 0.000 claims description 49
- 231100000614 poison Toxicity 0.000 claims description 23
- 230000003612 virological effect Effects 0.000 claims description 19
- 239000002574 poison Substances 0.000 claims description 18
- 210000003501 vero cell Anatomy 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 15
- 201000010099 disease Diseases 0.000 claims description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 10
- 231100000915 pathological change Toxicity 0.000 claims description 10
- 230000036285 pathological change Effects 0.000 claims description 10
- 238000004108 freeze drying Methods 0.000 claims description 8
- 239000012531 culture fluid Substances 0.000 claims description 2
- 241000712083 Canine morbillivirus Species 0.000 abstract description 34
- 238000004519 manufacturing process Methods 0.000 abstract description 13
- 230000002238 attenuated effect Effects 0.000 abstract description 8
- 230000005847 immunogenicity Effects 0.000 abstract description 7
- 208000031295 Animal disease Diseases 0.000 abstract description 3
- 230000003053 immunization Effects 0.000 abstract description 2
- 238000002649 immunization Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 241000282472 Canis lupus familiaris Species 0.000 description 45
- 238000012360 testing method Methods 0.000 description 31
- 241000282485 Vulpes vulpes Species 0.000 description 27
- 241001465754 Metazoa Species 0.000 description 21
- 230000037213 diet Effects 0.000 description 17
- 235000005911 diet Nutrition 0.000 description 17
- 210000003608 fece Anatomy 0.000 description 17
- 230000036760 body temperature Effects 0.000 description 16
- 239000000273 veterinary drug Substances 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 13
- 239000013553 cell monolayer Substances 0.000 description 12
- 230000036039 immunity Effects 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 230000001575 pathological effect Effects 0.000 description 12
- 238000010790 dilution Methods 0.000 description 11
- 239000012895 dilution Substances 0.000 description 11
- 230000005856 abnormality Effects 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 230000033228 biological regulation Effects 0.000 description 8
- 238000007689 inspection Methods 0.000 description 8
- 238000007918 intramuscular administration Methods 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 8
- 230000012010 growth Effects 0.000 description 7
- 241000282421 Canidae Species 0.000 description 6
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 241000282487 Vulpes Species 0.000 description 6
- 244000309466 calf Species 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 238000005096 rolling process Methods 0.000 description 5
- 239000003440 toxic substance Substances 0.000 description 5
- 210000002845 virion Anatomy 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 241000287828 Gallus gallus Species 0.000 description 4
- 241000204003 Mycoplasmatales Species 0.000 description 4
- 241000772415 Neovison vison Species 0.000 description 4
- 241001482564 Nyctereutes procyonoides Species 0.000 description 4
- 210000003484 anatomy Anatomy 0.000 description 4
- 230000003203 everyday effect Effects 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000000384 rearing effect Effects 0.000 description 4
- 238000011076 safety test Methods 0.000 description 4
- 239000011265 semifinished product Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 241000700199 Cavia porcellus Species 0.000 description 3
- 241000204031 Mycoplasma Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000003340 mental effect Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000002356 single layer Substances 0.000 description 3
- 210000005239 tubule Anatomy 0.000 description 3
- 239000002435 venom Substances 0.000 description 3
- 231100000611 venom Toxicity 0.000 description 3
- 210000001048 venom Anatomy 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 206010000234 Abortion spontaneous Diseases 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 231100000645 Reed–Muench method Toxicity 0.000 description 2
- 208000019802 Sexually transmitted disease Diseases 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 238000004500 asepsis Methods 0.000 description 2
- 229940031567 attenuated vaccine Drugs 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000002405 diagnostic procedure Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 208000015994 miscarriage Diseases 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 235000011121 sodium hydroxide Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000000995 spontaneous abortion Diseases 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000710780 Bovine viral diarrhea virus 1 Species 0.000 description 1
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 101150034814 F gene Proteins 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 101150064138 MAP1 gene Proteins 0.000 description 1
- 241001430197 Mollicutes Species 0.000 description 1
- 241000712045 Morbillivirus Species 0.000 description 1
- 241001508687 Mustela erminea Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 208000025174 PANDAS Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 208000021155 Paediatric autoimmune neuropsychiatric disorders associated with streptococcal infection Diseases 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 241000282376 Panthera tigris Species 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- PPBOKXIGFIBOGK-BDTUAEFFSA-N bvdv Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)C(C)C)[C@@H](C)CC)C1=CN=CN1 PPBOKXIGFIBOGK-BDTUAEFFSA-N 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 230000009849 deactivation Effects 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000037920 primary disease Diseases 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 238000003307 slaughter Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000000859 sublimation Methods 0.000 description 1
- 230000008022 sublimation Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to a canine distemper live vaccine and a preparation method thereof. The preparation method takes a canine distemper virus natural attenuated strain CGMCC No.3201 with excellent immunogenicity and obtained by the inventor through field separation as a production strain to prepare the safe, effective and single-component canine distemper live vaccine. The canine distemper live vaccine effectively reduces the risk of importing other live viruses during canine distemper vaccine immunization and provides conditions for controlling the spread of fur-bearing animal diseases.
Description
Technical field
The present invention relates to a kind of canine distemper live vaccine and preparation method thereof, belong to the veterinary biological product technical field.
Background technology
Canine distemper (Canine Distemper; CD); be commonly called as the Canis familiaris L. pestilence; be by canine distemper virus (Canine Distemper virus; CDV) infecting a kind of acute, the deadly infectious diseases of rare animals such as fur-bearing animal such as the ermine cause, Nyctereutes procyonoides, fox and dog and panda, tiger, is at present dog industry, fur-bearing animal aquaculture and the conservation of wildlife to be supported by China already to endanger one of maximum eqpidemic disease.Mainly popular cold season in the Winter-Spring, be distribute, local popular or break out.Because the clinical manifestation of canine distemper is ever-changing, when clinical diagnosis, easily obscure mutually with canine distemper, brought certain difficulty to clinical diagnosis and experimentation.
Canine distemper virus belongs to Paramyxoviridae, and Morbillivirus is the ameristic RNA viruses of minus strand sub-thread of spiral shape symmetrical.Size 150～250nm, virion is most spherical in shape, and it is 15～17nm spiral type nucleocapsid that the center also has wide footpath, has shaft-like fibre prominent, only contains hemagglutinin, and the impassivity propylhomoserin.CDV is to warm and dry responsive, 50～60 ℃, gets final product deactivation in 30 minutes.Because CDV can not long-term surviving in hot season, so this disease is popular in Winter-Spring cold season.CDV is to ultraviolet and alkaline solution sensitivity, and visible light is easily with inactivation of virus, and available clinically 3% sodium hydroxide (Caustic soda), bleaching powder are as disinfectant.As far back as 1905, Carre with regard to the cause of disease that proposes primary disease be a kind of virus (Carr, Y H.Sur la maladie desjeunes chiens.Compt.Rend.Acad.d.Sc.1905,140:689-690).Nineteen fifty-one, Dedie has turned out CDV with the method for tissue culture first.Rockborn finds, CDV can form in syncytium, sternzellen and the nuclear on former generation Madin-Darby canine kidney(cell line) or intracytoplasmic inclusion (Xia Xianzhu. foster dog complete works. Changchun: the .1993.9:549-553 of the Jilin People's Press; Yin Zhen, Liu Jinghua. animal virology. second edition. Beijing: Science Press, 1997,10:789-801).China is the end of the seventies, priorities such as Hua Guoyin from the CDV attenuated vaccine of external import, be separated to the strain of many strain vaccines (Hua Guoyin, the research that the research I. of mink canine distemper immunity duplicates Embryo Gallus domesticus tissue culture attenuated vaccine. the poultry infectious disease, 1980,4:30-35).
Morbidity is anxious, characteristics such as be difficult to heal with medicine because canine distemper has; at present; the main canine distemper vaccine that relies on prevents; be used to have the vaccine product of multiple prevention canine distemper both at home and abroad; but in the practical application of disease prevention; because exist the immunogenicity of strain very different, problem such as antigen titre is not high, may there be the problem that can not produce effective immunoprotection in the vaccine of use and causes the situation of immuning failure to occur.Simultaneously, mostly multi-joint live vaccine in the canine distemper vaccine product both domestic and external, if plant is under the situation that disease beyond the canine distemper did not take place, use multi-joint live vaccine to prevent hastily, may exist and cause the canine distemper risk of disease in addition, this situation is particularly serious when using some to prepare nonstandard product.
The objective of the invention is to utilize the inventor to have good immunogenic canine distemper virus low virulent strain by the isolating strain in field, safety, canine distemper attenuated freeze dried vaccine are effectively prepared in the CDV-11 strain.Simultaneously, above-mentioned vaccine is a kind of single Seedling that can be used for canine distemper susceptible animals such as fox and mink, Nyctereutes procyonoides, dog, solved the problem that is not used in effective canine distemper list Seedling of animals such as fox on the market, fill up the blank on the market, and provide condition for the diffusion of control fur-bearing animal disease.
Summary of the invention
The objective of the invention is to utilize the inventor to be separated to such an extent that a strain has good immunogenic canine distemper virus nature attenuated strain as the Strain of producing vaccine by the field; be inoculated on the Vero cell; the cytopathy venom that results infect; add freeze drying protectant; carry out lyophilizing with the freeze-drying curve of suitable this strain, prepare safety, effectively, the canine distemper live vaccine of single composition.
The production strain of canine distemper live vaccine of the present invention is to be separated to such an extent that a strain has good immunogenic canine distemper virus nature attenuated strain from the field, therefore, those skilled in the art can expect that canine distemper virus nature attenuated strain of the present invention produces seed culture of viruses as live vaccine and can be used for producing the multi-joint live vaccine of canine distemper virus antigen preparation equally.
1 production of vaccine strain
(1) viral source
The inventor gathers the dog internal organs and the lymphoid tissue of body temperature one property crossed rising by in the doubtful sick dog of canine distemper, is separated to the doubtful virus of a strain canine distemper with the passage method, this virus has been carried out the virusology evaluation, and cell culture is inoculated pup.The virusology qualification result shows that PCR detects the CDV positive, and other physicochemical property and biological characteristics meet the characteristics of canine distemper virus; The pathological anatomy check result shows that the inoculation animal does not have the canine distemper clinical symptoms, does not all find the pathological change of canine distemper after the dissection yet, illustrates that the virus that is separated to is canine distemper attenuated, called after CDV-11 strain.It is that (this strain virus had been delivered China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on 07 15th, 2009, was numbered: CGMCC No.3201) in strain canine distemper nature attenuation strain CDV-11 strain that the present invention prepares used production virus.
(2) Strain characteristic
1) Virus culture 5% is received Vero cell (the Vero cell that the inventor preserves with virus sample by what keep liquid measure, nutritional solution is that the MEM that contains the MEM growth-promoting media of 10% new-born calf serum or contain 2% new-born calf serum keeps liquid, cultivation according to a conventional method) on the monolayer, puts 37 ℃, 5%CO
2Leave standstill cultivation in the incubator, observe CPE every day, observed continuously 96～120 hours, when cell swelling occurs, becomes and justify, to merge, come off, the change of cell fusion sexually transmitted disease (STD) reaches 80% o'clock results, puts-20 ℃ of preservations.CDV11 strain C1 is made malicious valency for venom measure, detect altogether three times, the result is respectively 10
5.63TCID
50/ ml, 10
6.0TCID
50/ ml and 10
5.5TCID
50/ ml.
2) Bing Du morphologic observation is done the negative staining of Electronic Speculum phosphotungstic acid to the culture fluid in CDV-11 strain C1 generation and is observed and (see Fig. 1 electromicroscopic photograph, the arrow place is a virion), as seen have among Fig. 1 based on pleomorphic type virion circular or oval, that differ in size, come in every shape, the a plurality of virion that have are assembled in heaps, be also shown in virus nucleocapsid silk in heaps or wall scroll, also can see the regular helical form nucleocapsid of arranging in bigger virion inside, also can see baculovirus particle.
3) nucleic acid based evaluation routinely method measure, the viral growth metabolism of CDV-11 strain is not suppressed by BUDR, proves that CDV-11 strain C1 is the RNA type for the nucleic acid of virus, and is consistent with the nucleic acid type of canine distemper virus.
4) blood clotting routinely method carry out, CDV-11 strain C1 does not all have compendency for virus to 1% chicken, Cavia porcellus, rabbit, swine erythrocyte.
5) physicochemical property detects virus according to a conventional method to different pH value, temperature, time with to the tolerance test of chemical reagent such as ether, chloroform, formaldehyde.The physicochemical property qualification result shows, preserves viral malicious valency more than 0 ℃ and descends very soon, and 37 ℃, behind the 60min, malicious valency has reduced by 10
3.5TCID
50, 56 ℃, behind the 30min, malicious valency has reduced by 10
4.0TCID
50The CDV low virulent strain is to ether, chloroform, formaldehyde chemical reagent sensitivity; PH 7.0～9.0 virus activities are stable, and the following or 9.0 above activity of pH7.0 all are subjected to very big influence.
5) Molecular Virology is identified the F gene conserved region sequence of delivering CDV according to Genbank, designs 1 couple of primer: P1:5 '-GCTGGTTGGAGAATAAGG-3 ', P2:5 '-CCAACTCCCA TAGCATAA-3 '.For extracting RNA the venom, carry out the RT-PCR amplification with above primer from CDV-11 strain C1 after reverse transcription, the result amplifies the specific nucleic acid band of 585bp, as figure.
6) Serological testing CDV-11 strain C1 (is given by professor Zhu Ruiliang of Shandong Agricultural University for viral liquid and canine distemper positive serum, SN tires and is 1:355) neutralization back inoculation Vero cell, found that CDV-11 strain C1 is suppressed by canine distemper positive serum specificity for the pathological changes interaction energy to the Vero cell, shows that the CDV-11 strain is a canine distemper virus.
7) safety
1. animal returns test
The pathological material of disease inoculation test is numbered healthy susceptible dog (the SN antibody≤1: 4) 4 of pathological material of disease 2～3 monthly ages of inoculation of acquired original respectively, every while collunarium 2ml, abdominal cavity 8ml, and the inoculation back was observed 21 days, detected body temperature every day, observation spirit, diet, feces etc.Wherein 1 dog occurs once raising at body temperature on the 12nd, recovers normal again on 2nd, and other 3 the fervescence phenomenon do not occur; 4 dog diet, feces character are normal, spiritual no abnormality seen.4 test dogs are checked through pathological anatomy, all do not found the pathological change of canine distemper.
The virocyte inoculation test is inoculated healthy susceptible dog (SN antibody≤1: 4) 4 of 2～3 monthly ages with CDV-11 strain C1 for viral liquid, number respectively, every while collunarium 2ml, abdominal cavity 8ml, the inoculation back was observed 21 days, detect body temperature every day, observe spirit, diet, feces etc.After the inoculation, fervescence does not all appear in the inoculation dog in 21 days, the results are shown in Table 3; Diet, feces character are normal, spiritual no abnormality seen.4 test dogs are checked through pathological anatomy, all do not found the pathological change of canine distemper.
By isolating virus is carried out electron microscopic observation, specificity, nucleic acid based, blood clotting, physicochemical property, Molecular Virology, the evaluation of pure property confirms that the virus that we are separated to meets canine distemper paramyxovirus characteristic, and is and pure.Return test through animal, only have 1 dog body temperature to occur once raising behind the healthy susceptible dog of pathological material of disease poison inoculation, all the other are all normal; The pathological material of disease poison is inoculated dog connecting biography on the cell after 11 generations with viral cultures, does not cause the dog fervescence.Not finding that all canine distemper pathology changes after all test dogs are dissected, isolating virus is described to the dog had no pathogenicity, is the weak poison of a strain.
2. stable
The CDV-11 strain was passed for 15 generations in cell, every milliliter of viral level is all 10
5.5TCID
50More than, prove the CDV-11 strain 15 generations with interior be stable, can be used as production of vaccine with kind the poison.
By the CDV-11 strain dog, Vulpes, Nyctereutes procyonoides, mink are carried out heavy dose of test of inoculating, canine distemper clinical symptoms and pathological change all do not appear in the inoculation animal, and the result of the test of living together shows that nonimmune animal is not infected.
CDV-11 strain C5 is passed through animal body for poison passed for 3 generations continuously,, gather internal organs after slaughtering and detect per generation poison inoculation fox, when the 1st, 2 generations, can detect has CDV to exist in the animal body, repeat the 2nd generation and escalated dose inoculation 4,5 generations test again, does not all detect CDV.Result of the test shows that the CDV-11 strain is safe to dog, Vulpes, Nyctereutes procyonoides, mink, and does not have the anti-strong phenomenon of virulence, is the desirable strain that is used to prepare canine distemper attenuated Seedling.
8) immunogenicity
For the vaccine research of CDV-11 strain, the immunogenicity test has been carried out in the CDV-11 strain, animals such as immune fox, dog detect neutralizing antibody and carry out counteracting toxic substances, and the result shows that the CDV-11 strain has good immunogenicity.Relatively CDV11 strain C5, C15, C20 for immunogenicity after, the result there is no too big-difference, therefore chooses C5～C15 for planting poison as the basis, produces and is no more than for 3 generations with the seed subculture, can guarantee the immunogenicity of vaccine.
The production preparation of 2 canine distemper live vaccines
The production that (1) will prepare is with kind of a poison, adopts asynchronously to connect malicious method and be inoculated into Vero cell monolayer on the rolling bottle, then the rolling bottle cell put 37 ℃ of cultivations, and rotating speed is 8～10r/h.
(3) can gather in the crops viral liquid when cytopathy reaches 80% left and right sides, freeze thawing 1 time places-15 ℃ to freeze to preserve, and this is semi-finished product, and is to be checked standby.
(4) through the semi-finished product acceptance tests qualified after; with conventional freeze drying protectant (Chinese veterinary drug allusion quotation committee. People's Republic of China's veterinary drug allusion quotation; version was three ones in 2005. Chinese agriculture publishing house; 2006; to call " Chinese veterinary drug allusion quotation " in the following text) mix according to 1: 1 with the volume ratio of viral liquid, carry out lyophilizing.After lyophilizing is finished, labeling, warehouse-in is preserved.
The method of inspection of 3 canine distemper live vaccines
(1) steriling test is answered asepsis growth.Test by the regulation in " Chinese veterinary drug allusion quotation ".
(2) the mycoplasma check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have the mycoplasma growth.
(3) diagnostic test does 10 with vaccine
-2Dilution is with equivalent 10
-1The canine distemper positive serum of dilution mixes, and in 37 ℃ and 30 minutes, 4 bottles in inoculation Vero cell is established 2 bottles of virus control groups simultaneously, puts 37 ℃ of cultivations, observes CPE96～120 hour continuously.
(4) the exogenous virus check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have exogenous virus.
(5) after safety verification dissolves every part vaccine with the 1ml normal saline, 5 of 2～3 monthly age of intramuscular inoculation susceptible dogs (SN antibody titer≤1: 4), 10 parts/only, observed 21.
(6) efficacy test dissolves vaccine with the MEM that contains 2% new-born calf serum, makes every 1ml vaccine contain 1 part, does 10 times of dilutions of going forward one by one then, gets 10
-2, 10
-3, 10
-4, 10
-54 dilution factors, inoculation has grown up to the Vero cell 96 holes trace Tissue Culture Plate of monolayer respectively, each 8 hole of dilution factor inoculation, every hole 100 μ l, establish simultaneously and do not connect poison contrast, put in 37 ℃, 5% CO2 gas incubator and cultivate, observed 96～120 hours, record cytopathy (CPE) hole count is pressed the Reed-Muench method and is calculated TCID
50, every part vaccine virus content 〉=10
4.5TCID
50
(7) residual moisture is measured every batch of freeze-dried products and is appointed and take out 4 samples, and each sample residual moisture all should not surpass 4%.If have above the time, can heavily examine 1 time, heavily inspection back surpasses regulation if any 1 sample, this batch goods should be judged to defective (" Chinese veterinary drug allusion quotation ").
(8) vacuum is measured vacuum before measuring packing, and the goods of no vacuum should be rejected and be scrapped, and must not heavily find time to sell (" Chinese veterinary drug allusion quotation ").
Description of drawings
The normal Vero cell monolayer of Figure 11-1; The Vero cell monolayer showed cell pathological changes that the 1-2CDV-11 strain virus infects,
The electromicroscopic photograph of Fig. 2 virus, the arrow place is a CDV-11 strain virus particle.
Fig. 3 CDV11 strain virus pcr amplification product cleavage map 1 road M, 2 road CDV-11 strain virus, the specific nucleic acid band of the 585bp of demonstration amplified production
Advantage of the present invention
The present invention utilizes the inventor to be separated to such an extent that a strain has good immunogenic canine distemper virus nature attenuated strain as producing strain by the field, prepare safety, effectively, the canine distemper live vaccine of single composition.The present invention effectively reduces when immune canine distemper vaccine, introduces the risk of other poison of living, for the diffusion of controlling the fur-bearing animal disease provides condition.
Embodiment
Following examples further specify the present invention, but not as limitation of the present invention.
The pure property check of virus seed culture of viruses
1 steriling test is inoculated each 2 of T.G, G.P tubule and G.A slant mediums respectively with viral cultures, and every 0.2ml puts 37 ℃ of cultivations for one; Put 25 ℃ of cultivations for one, observed 3, T.G, G.P tubule and the G.A slant medium of the viral cultures inoculation after the transplanting are observed 5 average daily no antibacterials, fungus growth.Have or not antibacterial, fungus growth.
Viral cultures 5ml is got in the check of 2 mycoplasmas, is inoculated into the vial of liquid mycoplasma culture medium, gets 0.8ml again and be transplanted to 2 small test tube liquid mycoplasma culture mediums and 2 pairs of agar solid plates respectively from the bottle suspension.Liquid culture is placed 37 ℃ of cultivations, and solid culture is placed on 37 ℃ and contains 5%CO
2Environment is cultivated.Cultivated 5,10,15 days, and got 0.6～0.8ml culture respectively and transplant in to 2 small test tube fluid mediums (0.2ml/ props up) from bottle, 2 pairs of solid mediums (0.1～0.2ml/ pays) so repeat 3 times, and all transplanting cultures are all cultivated and observed 14 days.In the observation period, do not find that significant change appears in bottle and tubule culture color, the liquid culture of transplanting does not have " fried egg " shape mycoplasma bacterium colony on solid medium.
The check of 3 exogenous viruses
1) after cytopathogenic effect exogenous virus check neutralizes viral cultures and CDV positive serum fully, (every bottle of cell bottle floor space is 5cm * 7cm), and every bottle graft kind 1.0ml connects and passed for 2 generations to inoculate 3 bottles of F81, DK cell monolayer respectively, in per generation, observed 7, CPE all do not occur.
2) the fluorescent antibody inspection neutralizes viral cultures fully with the canine distemper positive serum, (every bottle of cell bottle floor space is 2cm * 3cm) to mixture inoculation Vero cell monolayer, cultivated 4, go down to posterity, with the 2nd generation culture through acetone fixed, detect the microscopy cell surface with fluorescence method and all do not find RV, BVDV fluorescent grain.
3) after hemadsorbing virus detected viral cultures and CDV positive serum neutralized fully, (every bottle of cell bottle floor space was that 2cm * 3cm), 37 ℃ of cultivations were tested on the 7th to inoculation Vero cell monolayer.Wash cell monolayer to be checked with the PBS buffer, rinsing 3 times, 0.2% guinea-pig red blood cell, people " O " type erythrocyte and the chicken red blood cell equivalent mixed liquor that add an amount of new preparation, jiggle, make it evenly to cover whole cell monolayer surface, select 2 cell monolayers, one bottle places 4 ℃, another bottle places 25 ℃ to cultivate 30min, wash with PBS, all do not adsorb chicken, Cavia porcellus, people " O " type erythrocyte with microscopic examination cell monolayer to be checked, show that the exogenous virus that does not have the erythrocyte characterization of adsorption pollutes.
The manufacturing of vaccine
The preparation of 1 cell nutrient solution
Adopt conventional cell growth medium (containing 10% nascent new-born calf serum) and keep liquid (containing 2% nascent new-born calf serum) and use as cell culture.
2 produce with kind of the preparation of poison
With the canine distemper that is separated to nature low virulent strain, the Vero cell monolayer on the inoculation rolling bottle, connect the poison amount for to keep 2% of liquid measure, when reaching 80%, cytopathy can gather in the crops, and in 1 generation of repeated transmission, preparation is produced with kind malicious.
The breeding of 3 viral liquid
With producing with kind of a poison, be inoculated into the Vero cell monolayer on the rolling bottle, connect the poison amount and be 2% of cell maintenance medium.Then the rolling bottle cell is put 37 ℃ of cultivations, rotating speed is 8～10r/h.
After the inoculation, every day, observation of cell pathological changes situation can be gathered in the crops when cytopathy reaches 80% left and right sides, and freeze thawing 1 time is put-15 ℃ and freezed to preserve, and this is semi-finished product.
5 lyophilizing
After the acceptance test of process semi-finished product is qualified, mixed according to 1: 1 with the volume ratio of viral liquid with conventional freeze drying protectant (" Chinese veterinary drug allusion quotation "), be distributed into the 2ml/ bottle, carry out lyophilizing according to the freeze-drying curve that designs, that is :-65 ℃, keep making the vaccine quick freezing in 2 hours, evacuation then is when vacuum reaches 13.3Pa, it is dry to begin to heat up, the sublimation stage product temperature is-20～-10 ℃, and shelf temperature is 8 ℃, and the distillation time is 10 hours; Resolution temperature is 30 ℃, 2 hours, all was freeze-drying time approximately? hour.
Warehouse-in is preserved after 6 labelings.
Embodiment 3
The product inspection of vaccine.
(1) steriling test is answered asepsis growth.Test by the regulation in " Chinese veterinary drug allusion quotation ".
(2) the mycoplasma check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have the mycoplasma growth.
(3) diagnostic test does 10 with vaccine
-2Dilution is with equivalent 10
-1The canine distemper positive serum of dilution mixes, and in 37 ℃ and 30 minutes, 4 bottles in inoculation Vero cell is established 2 bottles of virus control groups simultaneously, puts 37 ℃ of cultivations, observes CPE96～120 hour continuously.
(4) the exogenous virus check is tested by the regulation in " Chinese veterinary drug allusion quotation ", should not have exogenous virus.
(5) after safety verification dissolves every part vaccine with the 1ml normal saline, 5 of 2～3 monthly age of intramuscular inoculation susceptible dogs (SN antibody titer≤1: 4), 10 parts/only, observed 21.0303001 batch, 0303002 batch of three batches of vaccine and 0303003 batch of equal safety verification of inventor's trial production are qualified.
(6) efficacy test dissolves vaccine with the MEM that contains 2% new-born calf serum, makes every 1ml vaccine contain 1 part, does 10 times of dilutions of going forward one by one then, gets 10
-2, 10
-3, 10
-4, 10
-54 dilution factors, inoculation has grown up to the Vero cell 96 holes trace Tissue Culture Plate of monolayer respectively, each 8 hole of dilution factor inoculation, every hole 100 μ l, establish simultaneously and do not connect poison contrast, put in 37 ℃, 5% CO2 gas incubator and cultivate, observed 96～120 hours, record cytopathy (CPE) hole count is pressed the Reed-Muench method and is calculated TCID
50
Tiring of three batches of vaccines of inventor's trial production: 0303001 batch is 10
5.19TCID
50/ head part, 0303002 batch be 10
5.0TCID
50/ head part, 0303003 batch be 10
5.12TCID
50/ head part.
(7) residual moisture is measured every batch of freeze-dried products and is appointed and take out 4 samples, and each sample residual moisture all should not surpass 4%.If have above the time, can heavily examine 1 time, heavily inspection back surpasses regulation if any 1 sample, this batch goods should be judged to defective (" Chinese veterinary drug allusion quotation ").
(8) vacuum is measured vacuum before measuring packing, and the goods of no vacuum should be rejected and be scrapped, and must not heavily find time to sell (" Chinese veterinary drug allusion quotation ").
Embodiment 4
The vaccine safety test
The 1 minimum safety test of using an age in days dog and a single dose inoculation of fox
Each 10 of two kinds of experimental animals (SN antibody≤1: 4 the 50 age in days left and right sides pups and back 1 all foxes of weaning) are divided into 2 groups again, 5 every group, 1 part of 0302003 batch of vaccine of the 1st group of intramuscular inoculation (inventor's manufacturing)/only, do not inoculate in contrast for the 2nd group.Isolated rearing detects body temperature, observes the mental status, diet and feces character.And in the time of 21 days every group extract 2 and catch and kill and do the pathology inspection.
After dog inoculates pup with canine distemper live vaccine, do not see any CDV symptom through clinical observation, body temperature is all in 38.2 ℃～39.2 ℃ normal range, and spirit, diet, feces character are all normal, and matched group is no abnormality seen also.Catch and kill 2 for every group in the time of the 21st day, through pathologic finding, no abnormality seen; After canine distemper live vaccine is inoculated young fox, do not see any CDV symptom through clinical observation, body temperature is all in 38.7 ℃～39.9 ℃ normal range, and spirit, diet, feces character are all normal, and matched group is no abnormality seen also.Catch and kill 2 with every group in the time of the 21st day, through pathologic finding, no abnormality seen.
Behind the dog and fox in single dose inoculation wean 1 week of back of canine distemper live vaccine (CDV-11 strain) freeze-dried vaccine, all dogs and fox are all normal.Illustrate that this vaccine is safe, the 2 week beginning immunity of animal wean back is safe and reliable in actual applications.
The safety test of the 2 pairs of dogs and fox single dose repeated inoculation
Each 10 of two kinds of experimental animals (dog and the foxes at 2～3 monthly ages of SN antibody≤1: 4) are divided into 2 groups, 5 every group, 0303003 batch of vaccine (10 of the 1st group of intramuscular inoculation
5.12TCID
50/ head part) 1 part/only, at interval 2 week back repeated inoculations 1 time, do not inoculate in contrast for the 2nd group.Isolated rearing detects body temperature, observes the mental status, diet and feces character.And when inoculating back 21 days the last time every group extract 2 and catch and kill and do the pathological anatomy inspection.
Canine distemper live vaccine is through 2 vaccinizations, and 10 pups do not see any CDV symptom through clinical observation, and body temperature is all in 38.2 ℃～39.4 ℃ normal range, and spirit, diet, feces character are all normal, and matched group is no abnormality seen also.In the time of the 21st day dog is catched and killed, through pathologic finding, no abnormality seen, instruction book dosage vaccinization vaccine is safe to pup.
Canine distemper live vaccine is through 2 vaccinizations, and 10 young Vulpes do not see any CDV symptom through clinical observation, and body temperature is all in 38.7 ℃～39.9 ℃ normal range.Spirit, diet, feces character are all normal, and matched group is no abnormality seen also.In the time of the 21st day fox is catched and killed, through pathologic finding, no abnormality seen.
By above evidence canine distemper live vaccine (CDV11 strain) freeze-dried vaccine single dose repeated inoculation dog and fox is safe.
The safety test of the 3 pairs of dogs and overdose of fox (10 part/only) inoculation
With each 20 of two kinds of experimental animals (dog and the foxes at 2～3 monthly ages of SN antibody≤1: 4), be divided into 4 groups, 5 every group.0303001 batch of vaccine (10 of the 1st group of intramuscular inoculation
5.19TCID
50/ head part) 10 parts/only, 0303002 batch of vaccine (10 of the 2nd group of intramuscular inoculation
5.0TCID
50/ head part) 10 parts/only, 0303003 batch of vaccine (10 of the 3rd group of intramuscular inoculation
5.12TCID
50/ head part) 10 parts/only, do not inoculate in contrast for the 4th group.4 group isolated rearings respectively detect body temperature, observe the mental status, diet and feces character.And every group of dog of inoculation randomly drawed 2 and catches and kills and do the pathology inspection in the time of 21 days.
All inoculation dogs were through 21 days clinical observation, and spirit, diet, feces are all normal, the canine distemper symptom do not occur.Body temperature is all in 38.2 ℃～39.4 ℃ normal range.In the time of the 21st day animal is catched and killed, do not seen any canine distemper pathological changes through dissecting.
All inoculation foxes were through 21 days clinical observation, and spirit, diet, feces are all normal, the canine distemper symptom do not occur.Body temperature is all in 38.7 ℃～39.9 ℃ normal range.In the time of the 21st day animal is catched and killed, do not seen any canine distemper pathological changes through dissecting.
The result shows: overdose of canine distemper live vaccine (CDV11 strain) (10 part/only) inoculation is safe to dog and fox.
The safety testing of the 4 pairs of conceived dogs and fox
Each 10 of conceived about 40 days dog of test usefulness and foxes.0303003 batch of vaccine (10 of the 1st group of 5 intramuscular inoculations
5.12TCID
50/ head part) 10 parts/only, do not inoculate in contrast for the 2nd group 5 are observed spirit on the 21st, diet, feces character, follow-up investigation farrowing situation (because pregnant animal may cause the machinery miscarriage in capture-process, so not detecting body temperature).
Through clinical observation on the 21st, inoculation dog spirit, diet, feces were all normal, and farrowing does not have obvious difference with matched group; Inoculation Vulpes spirit, diet, feces are all normal, and farrowing does not have obvious difference with matched group.Presentation of results canine distemper live vaccine (CDV11 strain) heavy dose of (10 parts) conceived dog of inoculation and fox are safe.
By canine distemper live vaccine to dog and fox carry out that minimum age in days single dose, single dose repeat, overdose inoculation and to the female beastly inoculation test of pregnancy, the result shows that canine distemper live vaccine (CDV-11 strain) is safe to dog and fox.Owing to may cause the machinery miscarriage in the capture-process, generally pregnant animal does not recommend to carry out vaccination in actual production for pregnant animal.
Embodiment 5
Canine distemper live vaccine (CDV-11 strain) potency test
Test is 3 batches of canine distemper (lyophilizing) live vaccine (CDV-11 strain) that the inventor prepares with vaccine: 0303001 (10
5.19TCID
50/ head part), 0303002 (10
5.0TCID
50/ head part), 0303003 (10
5.12TCID
50/ head part) tests.Before immunity, vaccine is diluted with normal saline, make every milliliter of vaccine contain 10
4.0TCID
50Canine distemper virus, carry out immunity after the dilution immediately.
With 45 young Vulpes of canine distemper SN antibody≤1: 4, observe through the health in a week, under all normal situation of body temperature, spirit, diet, feces, these young Vulpes are divided into 3 batches at random, 15 every batch, every batch is further divided into 3 groups, 5 every group.The 1st batch every (contains 2 * 10 through intramuscular injection 2ml respectively
4.0TCID
50) 0303001,0303002,0303003 batch of vaccine; The 2nd crowd every difference intramuscular injection 1ml (contains 10
4.0TCID
50) 0303001,0303002,0303003 batch of vaccine; The 3rd batch every respectively intramuscular injection 0.5ml (0.5 * contain 10
4.0TCID
50) 0303001,0303002,0303003 batch of vaccine; The 4th batch 5 are not in contrast immune.Isolated rearing, in back 21 days of inoculation, the SN antibody titer was measured in blood sampling respectively.
Immune 1ml (is contained 10
4.0TCID
50) fox and the matched group fox of dosage carried out counteracting toxic substances in back 21 days in immunity, attacks 100ID for every
50By dog preparation CDV/Q3 generation strong poison (to the ID of fox
50Be 10
-1.70/ ml), observed 21 behind the counteracting toxic substances.
At immune 0.5ml (0.5 * 10
4.0TCID
50) in the fox of canine distemper live vaccine, have at least 93.3% (toatl proportion 14/15) to reach more than 1: 50 at back 21 days SN antibody of immunity; Immunity 1ml (10
4.0TCID
50) and 2ml (2 * 10
4.0TCID
50) fox SN antibody 100% all more than 1: 50, wherein all obtain protection behind the fox counteracting toxic substances of immune 1ml.Immunity 0.5 * 10 is described
4.0TCID
50Above vaccine can make the protection immunity fox more than 90% resist the attack of strong poison.Concrete outcome sees Table 1.
Table 13 batch canine distemper live vaccine (CDV-11 strain) is to the potency test of fox
Above result shows: when immunity 0.5 * 10
4.0TCID
50/ only above canine distemper live vaccine (CDV-11 strain), the SN antibody of back 21 days 90% above foxes of immunity 〉=1: 50.This experimental result shows that the minimum immune dosage of vaccine is 0.5 * 10
4.0TCID
50/ only; Immunity 10
4.0TCID
50/ canine distemper live vaccine (CDV-11 strain) only, the counteracting toxic substances protective rate is 100%.Therefore the immunizing dose of fox is 10
4.0TCID
50/ only; The standard of dispatching from the factory of vaccine is decided to be: every part viral level 〉=10
4.5TCID
50
Sequence table
＜110〉Qilu Animal Health Products Co., Ltd.
＜120〉a kind of canine distemper live vaccine and preparation method thereof
<160>2
<170>
<210>1
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: canine distemper virus PCR primer P1
<400>1
GCTGGTTGGA?GAATAAGG 18
<210>2
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉to the description of artificial sequence: canine distemper virus PCR primer P1
<400>2
CCAACTCCCA?TAGCATAA 18
Claims (2)
1. a canine distemper live vaccine is characterized in that this vaccine contains the canine distemper live virus that preserving number is CGMCC No.3201.
2. the preparation method of a canine distemper live vaccine; it is characterized in that with preserving number being that the weak naturally viral disease strain of CGMCC No.3201 is by inoculation Vero cell; 37 ℃ of cultivations; when connecing poison cell and have 80% pathological changes to occur; results virocyte culture fluid adds freeze drying protectant commonly used, makes freeze-dried live vaccine through lyophilization.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101579237A CN101612396B (en) | 2009-07-17 | 2009-07-17 | Canine distemper live vaccine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101579237A CN101612396B (en) | 2009-07-17 | 2009-07-17 | Canine distemper live vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101612396A CN101612396A (en) | 2009-12-30 |
| CN101612396B true CN101612396B (en) | 2011-12-07 |
Family
ID=41492405
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009101579237A Active CN101612396B (en) | 2009-07-17 | 2009-07-17 | Canine distemper live vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101612396B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914503B (en) * | 2010-07-30 | 2012-04-18 | 中国农业科学院哈尔滨兽医研究所 | Canine distemper attenuated vaccine strain and application thereof |
| CN102618503B (en) * | 2012-03-26 | 2013-06-12 | 江苏省农业科学院 | Hybrid tumor cell strain 1D7 capable of secreting high neutralizing activity canine distemper virus monoclonal antibody |
| TWI511743B (en) * | 2013-07-26 | 2015-12-11 | Antiserum, neutralizing antibody, and pharmaceutical composition containing the same | |
| CN104587460B (en) * | 2014-12-25 | 2016-09-21 | 华威特（江苏）生物制药有限公司 | Mink viral enteritis, canine distemper bigeminal live vaccine and its preparation method and application |
| CN107236715B (en) * | 2017-05-16 | 2020-01-14 | 中国农业科学院特产研究所 | Raccoon dog distemper virus attenuated vaccine strain and application thereof in preparation of raccoon dog distemper live vaccine |
| CN107158370B (en) * | 2017-05-16 | 2020-04-17 | 中国农业科学院特产研究所 | Raccoon dog canine distemper freeze-dried live vaccine and preparation method and application thereof |
| CN107115525B (en) * | 2017-05-16 | 2019-11-26 | 中国农业科学院特产研究所 | Racoon dog canine distemper live vaccine and its preparation method and application |
| CN109280649B (en) * | 2017-12-20 | 2022-04-12 | 吉林特研生物技术有限责任公司 | Method for preparing mink canine distemper antigen-protein complex, antigen-protein complex and application of antigen-protein complex |
| CN112316128B (en) * | 2020-10-20 | 2021-08-24 | 辽宁益康生物股份有限公司 | Canine distemper virus and canine parvovirus infectious hepatitis virus triple live vaccine |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3080291A (en) * | 1960-06-10 | 1963-03-05 | Jensen Salsberg Lab Inc | Serial passage of distemper virus in tissue cultures of chick embryo and canine tissue and vaccine therefrom |
| US3138531A (en) * | 1961-07-12 | 1964-06-23 | Lilly Co Eli | Canine distemper vaccine |
| CN1117081A (en) * | 1995-03-16 | 1996-02-21 | 高云 | Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method |
| CN1679931A (en) * | 2004-03-11 | 2005-10-12 | 科米药物国际株式会社 | Canine distemper virus separated in korea and recombined vaccine therefrom |
| CN1876181A (en) * | 2006-04-21 | 2006-12-13 | 中国人民解放军军事医学科学院军事兽医研究所 | Weak toxicity united vaccine formulation for canidae animal important disease series and preparation process |
-
2009
- 2009-07-17 CN CN2009101579237A patent/CN101612396B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3080291A (en) * | 1960-06-10 | 1963-03-05 | Jensen Salsberg Lab Inc | Serial passage of distemper virus in tissue cultures of chick embryo and canine tissue and vaccine therefrom |
| US3138531A (en) * | 1961-07-12 | 1964-06-23 | Lilly Co Eli | Canine distemper vaccine |
| CN1117081A (en) * | 1995-03-16 | 1996-02-21 | 高云 | Triple live vaccine and toxin vaccine for distemper, rabies and pavovirus and its preparing method |
| CN1679931A (en) * | 2004-03-11 | 2005-10-12 | 科米药物国际株式会社 | Canine distemper virus separated in korea and recombined vaccine therefrom |
| CN1876181A (en) * | 2006-04-21 | 2006-12-13 | 中国人民解放军军事医学科学院军事兽医研究所 | Weak toxicity united vaccine formulation for canidae animal important disease series and preparation process |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101612396A (en) | 2009-12-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101612396B (en) | Canine distemper live vaccine and preparation method thereof | |
| CN105412921B (en) | A kind of I group of 4 type aviadenovirus vaccine | |
| CN103041385B (en) | Porcine transmissible gastroenteritis and epidemic diarrhea combined live vaccine and preparation method thereof | |
| CN102174476A (en) | Inactivated vaccine for preventing duck virus hepatitis and preparation method thereof | |
| CN103599533A (en) | Chicken new castle disease-infectious bronchitis trivalent combined vaccine and preparation method thereof | |
| CN101797380A (en) | Method for preparing hogcholera vaccine | |
| CN105031639A (en) | Mink distemper-canine parvovirus enteritis bivalent vaccine and its preparation method and use | |
| CN102220287A (en) | Avian infectious bronchitis cold adaptation attenuated vaccine strain and application thereof | |
| CN104258386B (en) | A kind of mink viral enteritis inactivated vaccine-canine distemper live vaccine combination | |
| CN101099863B (en) | Preparation method of triple inactivated vaccine for preventing Newcastle disease, infectious bronchitis and egg production drop syndrome | |
| CN111073863B (en) | Porcine epidemic diarrhea and porcine delta coronavirus bivalent attenuated vaccine and preparation method thereof | |
| CN103143007B (en) | Method for preparing infectious bursal disease virus live vaccine by utilizing passage chicken embryo fibroblast | |
| CN108465107B (en) | Duck type 2 adenovirus and Muscovy duck parvovirus disease combined inactivated vaccine | |
| CN103710314A (en) | Avian Encephalomyelitis virus attenuated vaccine strain | |
| CN101235363B (en) | Pig transmissible gastroenteritis virus vaccine strain and application thereof | |
| CN104888213A (en) | Preparation method of classical swine fever spleen-lymph-sourced compound living vaccine | |
| CN103784951B (en) | Prevent and treat antigen composition of respiratory disease of scabies secondary infection of pig and its preparation method and application | |
| CN101380470B (en) | Pig parvovirus live vaccine | |
| CN103127497B (en) | Porcine circovirus 2 type, mycoplasma pneumoniae bivalent inactivated vaccine and preparation method thereof | |
| US4472378A (en) | Live vaccine for the prevention of salmonellosis in water fowl, a process for making and applying the same | |
| CN102166355B (en) | Quadruple inactivated vaccine for preventing chicken diseases | |
| CN101766814A (en) | Swine pasteurellosis bivalent inactivated vaccine and preparation method thereof | |
| CN113384692A (en) | Duck reovirus and duck circovirus bivalent inactivated vaccine and preparation method thereof | |
| CN108939063B (en) | Muscovy duck triple inactivated vaccine | |
| CN103800900B (en) | Staphylococcus aureus and the mammitis of cow vaccine that its deactivation is obtained |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |