CN108018261A - Canine parainfluenza virus strain and its application - Google Patents

Canine parainfluenza virus strain and its application Download PDF

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Publication number
CN108018261A
CN108018261A CN201610946235.9A CN201610946235A CN108018261A CN 108018261 A CN108018261 A CN 108018261A CN 201610946235 A CN201610946235 A CN 201610946235A CN 108018261 A CN108018261 A CN 108018261A
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antigen
canine
parainfluenza virus
inactivation
virus
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CN108018261B (en
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田克恭
刘玉秀
刘彩红
孙进忠
张许科
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LUOYANG HUIZHONG BIOTECH Co.,Ltd.
Pulaike Biological Engineering Co Ltd
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Pulaike Biological Engineering Co Ltd
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    • C12N7/00Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525Virus
    • A61K2039/5252Virus inactivated (killed)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/55Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552Veterinary vaccine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/70Multivalent vaccine
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    • C12N2750/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011Details
    • C12N2750/14011Parvoviridae
    • C12N2750/14034Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/00011Details
    • C12N2760/18011Paramyxoviridae
    • C12N2760/18411Morbillivirus, e.g. Measles virus, canine distemper
    • C12N2760/18434Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011Details
    • C12N2760/18011Paramyxoviridae
    • C12N2760/18611Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18621Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2760/00011Details
    • C12N2760/18011Paramyxoviridae
    • C12N2760/18611Respirovirus, e.g. Bovine, human parainfluenza 1,3
    • C12N2760/18634Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The present invention relates to canine parainfluenza virus strain and its application, HeN0718 plants of the canine parainfluenza virus of the present invention has strong toxicity and good immunogenicity, the vaccine combination prepared using its inactivation antigen can make animal body quickly produce antibody after animal is immunized, and can effectively prevent and/or treat current dog parainfluenza relevant disease.The vaccine combination security is good, can be combined with other antigens.

Description

Canine parainfluenza virus strain and its application
Technical field
The invention belongs to veterinary biologics technical field, and in particular to canine parainfluenza virus strain and its vaccine combination Thing, preparation method and applications.
Background technology
Canine parainfluenza virus (Canine Parainfluenza virus, CPIV) is paramyxovirus section, and paramyxovirus is sub- Section, Rubulavirus member, be non-segmented negative sub-thread minus-stranded rna virus, CPIV particle shapes are changeable under Electronic Speculum, generally in Circle, diameter 80nm~200nm, but often because cyst membrane is damaged and form is irregular, show the virion of pleomorphism.
The host range of CPIV is wider, including dog, cavy, hamster, mouse, tiger, racoon dog, ermine etc., often causes the bronchitis of dog And Bronchopneumonia, damage airway epithelial cell.After dog infection CPIV, the disease can be detected from respiratory secretions Poison, virion often result in quick-fried by effective gas particle, foam and infection through air to Healthy Dogs, the larger dog group of density Hair, the dog of various kinds can infect, and young dog neurological susceptibility higher, drastically influence the development of countries in the world canine farming.
Vaccine is prevention and controls the sick major measure, and early in 1970, Emeay etc. developed the weak poison of CPIV D-008 Vaccine, muscle or subcutaneous vaccination can produce neutralizing antibody, and the incidence of disease can be reduced using the vaccine, mitigate clinical condition Shape, but cannot be fully against infection.Domestic and international You Duo companies include the vaccine of CPIV in offer at present, but in clinical practice It was found that there are the positive dog of part CPIV infection all once stringent inoculated CPIV vaccines histories.On the other hand, attenuated vaccine is using process It is middle to return strong and scattered malicious risk there are virulence, and the virulence decay in incubation of existing separated velogen strain is obvious, preparation Inactivated vaccine immune efficacy is not high, so that not there is related inactivated vaccine product to occur at present.Therefore, there is an urgent need for isolate immunogenicity Popular CPIV street strains of the good country, are prepared into efficient inactivated vaccine to prevent and/or treat dog parainfluenza epidemic disease Feelings are spread, and have far-reaching realistic meaning.
The content of the invention
To solve the deficiencies in the prior art, the present invention provides a kind of canine parainfluenza virus strain and its inactivation prepared Vaccine and preparation method thereof and application, the vaccine can effectively prevent the infection of canine parainfluenza virus.
The present invention relates to a kind of canine parainfluenza virus strain, which has strong toxicity and good immunogenicity.
The invention further relates to a kind of vaccine combination, the vaccine combination includes the canine parainfluenza virus of immune amount Strain antigen and/or veterinarily acceptable carrier;Wherein, the totivirus that the canine parainfluenza virus antigen includes inactivating resists It is former.The vaccine combination is prepared by velogen strain, has good immunogenicity, can provide dog protection completely.And the inactivation Vaccine Attenuate vaccine more of the prior art has significant biological safety.
The invention further relates to the vaccine combination answering in prevention and/or treatment dog parainfluenza relevant disease is prepared With.
The canine parainfluenza virus strain of the present invention is popular canine parainfluenza virus street strain, and vaccine is prepared into the strain Composition can prevent and/or treat dog parainfluenza epidemic situation sprawling, and the vaccine combination containing the strain animal is immunized after can make Animal body quickly produces antibody, or mixed infection independent to dog parainfluenza currently popular has good prevention and control to imitate Fruit, biological safety are good.
Brief description of the drawings
Fig. 1 is the cytopathy and identified by immunofluorescence figure that CPIV HeN0718 and rCPIV is produced on Vero cells;Figure 1A is blank control group Vero cytological maps;Figure 1B is the cytopathy that CPIV HeN0718 are produced on Vero cells;Fig. 1 C are The cytopathy that restructuring rCPIV is produced on Vero cells;Fig. 1 D are the identified by immunofluorescence figure of blank control group Vero cells; Fig. 1 E are the identified by immunofluorescence figure that CPIV HeN0718 are produced on Vero cells;Fig. 1 F produce for rCPIV on Vero cells Raw identified by immunofluorescence figure.
Embodiment
Hereinafter, embodiments of the present invention are illustrated.
Term " canine parainfluenza virus " (Canine Parainfluenza virus, CPIV) belongs to paramyxovirus section, and pair is glutinous Chordopoxvirinae, Rubulavirus member, be non-segmented negative sub-thread minus-stranded rna virus, the clinical symptoms triggered include hair Heat, runny nose and cough.Pathological change is characterized by coryza and bronchitis.
Term " canine parainfluenza virus strain (CPIV strains) " is if be in the present invention dog parainfluenza virus without specified otherwise Malicious street strain (CPIV street strains), canine parainfluenza virus velogen strain (CPIV velogen strains), wild type CPIV strains, it is interchangeable to make With.
Term " CDS " full name is coding sequence, refers to the sequence that one section of protein product is encoded in gene in the present invention Row, be with the one-to-one DNA sequence dna of protein sequence, and without the corresponding sequence of other non-protein among the sequence, Without considering the sequence variation during mRNA processing etc., it is completely corresponding with the codon of protein.
The present invention relates to a kind of canine parainfluenza virus strain, wherein, the canine parainfluenza virus strain full-length genome CDNA replaces with the sequence after its degenerate sequence for CDS in the sequence shown in SEQ ID No.1 or the SEQ ID No.1 sequences Row.
The present invention relates to a kind of canine parainfluenza virus strain, wherein, the canine parainfluenza virus strain is CPIV HeN0718 plants, the canine parainfluenza virus strain CPIV HeN0718 pnca gene groups full-length cDNA is shown in SEQ ID No.1 Sequence.
The separation of the canine parainfluenza virus strain includes:The animal viscera lesion for infecting canine parainfluenza virus is carried out Sampling, grinding or processing, then different lapping liquids or treatment fluid are inoculated in Vero cells and carry out virus purification, stabilization reaches appearance Cytopathy can freeze thawing receipts poison.
Wherein, the animal sources for infecting canine parainfluenza virus strain include but not limited to Canidae (Canidae) source, cat family (Felidae) source.The animal specimen of infection canine parainfluenza virus strain includes but not limited to lung, saliva, eye discharge, gas Pipe, nasal discharge, lymph node, spleen.
Term " vaccine combination " is also known as immunogenic composition, refers to a kind of preparation containing immunogene, including such as complete thin Born of the same parents, inactivation or attenuation, virus living or bacterium, or polysaccharide, or its combination, it, which is administered, carrys out costimulatory receptor to being present in The humoral and cellular immune response reaction of one or more antigens in immunogenic composition.Immunity inoculation is to apply vaccine combination simultaneously Immune or immunogenic response the process to antigen is stimulated in host, and preferably host is animal such as dog.
Term " immune amount " should be understood to " immune effective dose ", and also known as immunoprotection amount or generation immune response is effective Amount, is the amount of antigen that immune response can be effectively induced in recipient's body, which is enough to prevent or improves sign or the disease of disease Shape, including unfavorable health effect or its complication.The immune response may be enough to be used in diagnostic purpose or other experiments, or Prophylactic sign or symptom are likely to be suitable for, including caused unfavorable healthy result is infected as caused by pathogen Or its complication.Humoral immunity can be induced as both cell-mediated immunity or this.Animal is to immunogenicity group The immune response of compound can be for example, by measuring antibody titer, lymphocyte proliferation assays and indirect assessment, or with wild type Directly assessed after strain attack by monitoring sign or symptom, and measurement should can be passed through by the protective immunity that vaccine provides Such as the clinical symptom of the subject such as reduction of the death rate, incidence, temperature value, subject's general physiological state and totality is strong Health is assessed with performance.The immune response may include but be not limited to inducing cell and/or humoral immunity.
Term " canine parainfluenza virus antigen " refers at least contain a kind of any combinations of canine parainfluenza virus antigen forms Thing, the canine parainfluenza virus antigen can induce, stimulate or can resist the immune response of canine parainfluenza virus infection, the antigen Form includes but not limited to antigen inactivate, being attenuated or subunit.Wherein, the antigen of inactivation can include by various methods Chemical treatment, physical treatment (such as sonication, irradiation, heat) or any other common side for being enough to prevent organism from replicating or grow Method maintains its immunogenicity to realize inactivation, preferably inactivates pathogen after collection and optionally receives clarification purification, leads to Chemical treatment is crossed using such as formalin or formaldehyde, beta-propiolactone, aziridine, binary aziridine BEI, thimerosal; The method of inactivation be well known to a person skilled in the art, such as by beta-propiolactone (Plana-Duran, Vet.Microbiol., 1997,55:361-370) or pass through BEI processing (US5587164).
Term " veterinarily acceptable carrier " refers to remove canine parainfluenza virus antigen in vaccine combination of the present invention Outside other all the components, do not stimulate body do not hinder using compound biological activity and characteristic carrier or dilution Agent, is preferably adjuvant.Term " adjuvant " may include aluminium glue adjuvant;Saponin(e (saponin), such as Quil A, QS-21 (Cambridge Biotech Incorporation,Cambridge MA)、GPI-0100(Galenica Pharmaceuticals Incorporation, Birmingham AL);Water-in-oil emulsion;Oil in water emulsion;W/O/W emulsion;Acrylic acid or first The polymer of base acrylic acid;The compound that the copolymer of maleic anhydride and alkenyl (alkenyl) derivative is selected.Term It is oily (European Pharmacopea types) that " emulsion " can be particularly based on light liquid paraffin;The class isoamyl produced by olefin oligomerisation Diene oil (isoprenoid oil), such as saualane (squalane) or squalene oil (squalene oil), especially isobutene Or certain herbaceous plants with big flowers alkene;Acid or alcohol the ester containing linear alkyl, more specifically vegetable oil, ethyl oleate, propane diols two-(caprylate/certain herbaceous plants with big flowers acid esters), Glycerine three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200;The ester of branched chain fatty acid or alcohol, especially isostearate.Oil with Emulsifier combination use is to form emulsion.The ester of emulsifying agent preferred nonionic surfactants, especially sorbitan, two contractings are sweet Reveal ester (such as anhydrous mannitol oleate), ester, the polyglycereol of aliphatic dihydroxy alcohol (glycol) of alcohol (mannide) (polyglycerol) ester, the ester of the ester of propane diols and oleic acid, the ester of isostearic acid, the ester or hydroxy stearate of castor oil acid The ester of acid, their optional ethoxylations, also have Pluronic L121, especially Pluronic products, especially It is L121.Write referring to Hunter etc.《The theory and practical application of adjuvants》 (Ed.by DES Stewart-Tull,John Wiley and Sons,New York,1995:51-94) write with Todd etc. 's《Vaccine》(1997,15:564-570).For example, it can be used what Powell M and Newman M write《Vaccine design,the Subunit and adiuvant approach》The SPT of (Plenum Press, 1995) description of page 147 Emulsion and the MF59 emulsions of the description of page 183.Term " polymer of acrylic or methacrylic acid " is preferably crosslinked propylene Acid or methacrylate polymer, the especially poly alkenyl ether or polyalcohols with sugared (sugar) are crosslinked, quilt known to these compounds Referred to as carbomer (Carbomer, trade name Carbopol) (Phameuropa, 1996,8 (2)).Those skilled in the art may be used also Referring to United States Patent (USP) US2909462, which depict this kind of acrylate copolymer, it is described with gathering hydroxylated compound crosslink Compound has at least three hydroxyl, preferably more than 8, and the hydrogen atom of wherein at least 3 hydroxyls is by former with least two carbon Unsaturated alkyl (aliphatic radical) substitution of son.Preferable group is the base that those contain 2-4 carbon atom Group, such as vinyl, pi-allyl and other ethylenically unsaturated groups (ethylenically unsaturated group).Institute Other substituents, such as methyl can be included by stating unsaturated group itself.These products are sold with the name of carbopol, (BF Goodrich, Ohio, USA) it is especially suitable.They with allyl sucrose or with Allyl pentaerythritol (allyl Pentaerythritol) it is crosslinked.It can be mentioned that carbopol 974P, 934P and 971P, most preferably with carbopol 971P among these. Term " copolymer of maleic anhydride and alkenyl derivative " also contemplates for the copolymer EMA of maleic anhydride and ethene (Monsanto), these polymer dissolve generation acid solution in water, neutralized, are preferably neutralized to physiological pH, to produce Assist agent solution, can mix immunogenicity, immunogenicity or vaccinal composition thereto in itself.Term " adjuvant " further includes, but Be not limited to, RIBI adjuvant systems (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A (monophosphoryl lipid A), Avridine lipids- Amine adjuvant, E.coli LT (restructuring is other), cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvants Deng.Preferably, the adjuvant includes aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion, propylene Acid or the polymer of methacrylic acid, the copolymer of maleic anhydride and alkenyl (alkenyl) derivative, RIBI adjuvants system System, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli are intolerant to warmheartedness poison One or more in element, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvants.
The invention further relates to a kind of vaccine combination, wherein, the vaccine combination includes the dog pair of immune amount Influenza virus strain or its culture inactivation antigen and veterinarily acceptable carrier.
As one embodiment of the present invention, in vaccine combination of the present invention, the canine parainfluenza virus Strain antigen is the inactivation antigen of HeN0718 plants of the canine parainfluenza virus strain CPIV or its culture, and the dog is secondary to flow The inactivation antigen content of HeN0718 plants of susceptible poison strain CPIV or its culture is before inactivation 104.0-107.0TCID50/ml。
As one embodiment of the present invention, in vaccine combination of the present invention, the canine parainfluenza virus poison The inactivation antigen content of HeN0718 plants of CPIV of strain or its culture is before inactivation 105.0-106.0TCID50/ml。
As one embodiment of the present invention, in vaccine combination of the present invention, the canine parainfluenza virus poison The inactivation antigen content of HeN0718 plants of CPIV of strain or its culture is before inactivation 106.0TCID50/ml。
As one embodiment of the present invention, the totivirus antigen of the inactivation is by by the canine parainfluenza virus The totivirus nutrient solution that strain is bred and obtained on cell, and inactivate gained through inactivator.
As one embodiment of the present invention, the inactivator includes but not limited to beta-propiolactone BPL, binary ethylenimine BEI, formalin, formaldehyde, N- acetylethylenimines AEI, Polyhaxemethylenguanidine Hydrochloride PHMG.
As one embodiment of the present invention, in vaccine combination of the present invention, the canine parainfluenza virus Strain culture is the culture in 1~18 generation of culture.
As one embodiment of the present invention, in vaccine combination of the present invention, the canine parainfluenza virus Strain culture is the culture in 6~18 generations of culture.
As one embodiment of the present invention, in vaccine combination of the present invention, the adjuvant includes:(1) aluminium Glue adjuvant, saponin(e, Avridine, DDA;(2) water-in-oil emulsion, oil in water emulsion, W/O/W emulsion;Or (3) acrylic acid Or the copolymer of the polymer of methacrylic acid, maleic anhydride and alkenyl derivative;And RIBI adjuvant systems, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, E.coli LT, One or more in cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvants;
Preferably, saponin(e is Quil A, QS-21, GPI-0100;
Preferably, emulsion is that SPT emulsions, MF59 emulsions, or emulsion are formed by oil with emulsifier combination, and emulsion can be based on light Saxol, isoprenoid oil (such as saualane or squalene oil, particularly alkene, isobutene because of olefin oligomerisation generation Or decene oligomerizationization produce oil), acid or alcohol containing linear alkyl ester (more specifically vegetable oil, ethyl oleate, propane diols two- (caprylate/certain herbaceous plants with big flowers acid esters), glycerine three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200), the ester of branched chain fatty acid or alcohol it is (outstanding Its isostearate);Emulsifying agent is nonionic surfactant (the especially ester of polyoxyethylated fatty acid (such as oleic acid), mountain The ester of pears glycan, the ester (such as anhydrous mannitol oleate) of mannide, the ester of aliphatic dihydroxy alcohol, the ester, poly- sweet of glycerine Ester, the ester and the ester of oleic acid, the ester of isostearic acid, the ester of castor oil acid or the ester of hydroxy stearic acid of propane diols of oil, it is above-mentioned Ester can be through ethoxylation, the ether of fatty alcohol and polyalcohol (such as oleyl alcohol), Pluronic L121 (especiallyParticularly L121));
Preferably, the polymer of acrylic or methacrylic acid is crosslinked acrylic or methacrylic acid polymer, especially It is with sugar poly alkenyl ether or the crosslinked compound carbomer of polyalcohols, be preferably carbopol 974P, 934P and 971P;
Preferably, the copolymer of maleic anhydride and alkenyl derivative is maleic anhydride and the copolymer of ethene EMA;
Preferably, the adjuvant is GEL A adjuvants;
The concentration range of the adjuvant is the preferably 5%V/V from 5% to 50%V/V.
As one embodiment of the present invention, in vaccine combination of the present invention, the vaccine combination into One step includes other antigens, and the antigen includes the group being made of the one or more in following further antigens:Canine distemper virus (Canine Distemper Virus, CDV) antigen, hepatitis infectiosa canis virus (Canine Adenovirus) I type, II type antigen, dog hook Leptospiral (Leptospira interrogans) antigen, canine coronavirus (Canine Coronavirus, CCV) antigen, Canine parvovirus (Canine Parvovirus, CPV) antigen, hydrophobin (Rabies virus, RV) antigen, dog influenza Viral (Canine Influenza virus, CIV) antigen, dog reovirus (Canine Reovirus, CRV) antigen, dog are pseudo- Rabies viruses (Pseudorabies) antigen, dog rotavirus (Rotavirus) antigen, canine alphaherpesvirus (Canine Herpervirus, CHV) antigen, canine viral papillomavirus (Canine Viral Papillomatosis) antigen, dog be micro- Small virus (Canine Minute Virus) antigen, dog mumps virus (Canine Mumps Virus) antigen, dog lymph are thin Born of the same parents' property choriomeningitis virus (Canine Lymphocytic Choriomeingitis Virus) antigen.
As a kind of preferred embodiment of the present invention, in vaccine combination of the present invention, the antigen include by The group of one or more compositions in following further antigens:Canine Parvovirus antigen, hepatitis infectiosa canis virus antigen, leptospira canicola antigen, Canine coronavirus antigen, canine parainfluenza virus antigen, Rabies Virus Antigen, canine influenza virus antigen.
The invention further relates to the preparation method of the vaccine combination, the preparation method includes:By the dog parainfluenza Virus stain breeds on cell and the canine parainfluenza virus cell liquid of the amplification cultivation is inactivated through inactivator, adds veterinary science Upper acceptable carrier mixes to obtain the final product.
As a kind of preferred embodiment of the present invention, before vaccine combination of the present invention includes content for inactivation 104.0-107.0TCID50HeN0718 plants of inactivation antigens of canine parainfluenza virus strain CPIV of/ml, content are before inactivation 104.0- 106.0TCID50CDVC/HN/001 plants of inactivation antigens of canine distemper virus and/or content of/ml are before inactivation 106.0- 108.0TCID50S0425 plants of inactivation antigens of canine parvovirus CPV of/ml, and 5%V/VGEL A adjuvants.
As a kind of preferred embodiment of the present invention, before vaccine combination of the present invention includes content for inactivation 106.0TCID50HeN0718 plants of inactivation antigens of canine parainfluenza virus strain CPIV of/ml, content are before inactivation 105.0TCID50/ C/HN/001 plants of inactivation antigens of canine distemper virus CDV and/or content of ml are before inactivation 107.0TCID50The canine parvovirus of/ml S0425 plants of inactivation antigens of CPV, and 5%V/VGEL A adjuvants.
In the vaccine combination of the present invention, other antigenic components generate association to the canine parainfluenza virus inactivation antigen of inactivation Same-action so that immune dog has only reached in shorter time to be enough to resist the neutralize antibody titers that strong poison attacks poison.
Term " prevention and/or treatment " refers to suppress answering for canine parainfluenza virus when being related to canine parainfluenza virus infection System, the propagation for suppressing canine parainfluenza virus prevent canine parainfluenza virus from being settled down in its host, and mitigate dog parainfluenza The disease of virus infection or the symptom of illness.If viral loads are reduced, illness mitigates and/or food ration and/or growth increase, It can so think that the treatment has reached therapeutic effect.
The invention further relates to the vaccine combination to prepare prevention and treatment canine parainfluenza virus infection relevant disease Medicine in application.
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more To be clear.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.Those skilled in the art It should be understood that the details and form of technical solution of the present invention can be carried out without departing from the spirit and scope of the invention Modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
Used chemical reagent is that analysis is pure in the embodiment of the present invention, purchased from Chinese medicines group.
To make the present invention easier to understand with reference to specific embodiments the present invention is further explained.It is of the present invention Experimental method, be conventional method if without specified otherwise;The biomaterial, if without specified otherwise, can be from business way Footpath obtains.
Separation, the identification of 1 canine parainfluenza virus of embodiment
CPIV street strains are separated from the animal of clinically canine parainfluenza virus infection, the clinical symptoms of infection animal are: Spirit is depressed, generate heat, nasal cavity flows out a large amount of mucus, opaque secretion, dry cough etc., as the standard of selected animal. Infection animal lung tissue is taken, is ground to obtain tissue homogenate with tissue refiner.
The lung homogenate liquid of sick dog is diluted to 10%W/V suspensions with serum-free DMEM nutrient solutions, with 0.22 μm of filter membrane Filtration sterilization, and by filtrate in 10%W/V ratios inoculation Vero cell monolayers (being purchased from the enzyme-linked detection technique Co., Ltd in Shanghai) Virus purification is carried out, 24~48 born of the same parents occur when small merges lesion, and about 96 hour cell lesions reach more than 80%, and freeze thawing is received poison and made For 1st generation virus liquid;The poison disease vaccination Vero cells for being obtained 1st generation with malicious method is synchronously connect, inoculation 24 it is small when after occur The cytopathy of born of the same parents' fusion, when inoculation 72~96 is small after cytopathy reach more than 80%, freeze thawing receives poison as 2nd generation virus Liquid;The poison disease vaccination Vero cells for being obtained 2nd generation with malicious method is synchronously connect, inoculation 24 it is small when after occur born of the same parents fusion cell Lesion, when inoculation 72~96 is small after cytopathy reach more than 80%, freeze thawing receives poison and is used as the 3rd generation virus liquid.By the 3rd generation virus Liquid carries out morphologic detection by electron microscopic observation, the results showed that:It can be seen that the virion with typical parainfluenza virus feature Son.Using Jiao Kuhua (development of Jiao Kuhua, Cen Hao, Zhang little Rong, Wu Yan great waves canine parainfluenza virus monoclonal antibodies and its characteristic Identify [J] .2009,31 (12):Indirect immunofluorescence 985-987.) carries out virus-specific identification to the 3rd generation virus liquid, The results show:There is specific green fluorescence in the cytopathy position that Vero cell inoculations virus forms cell fusion, shows spy It is different in nature good.
2 canine parainfluenza virus of embodiment is cloned
1st, the sequencing of canine parainfluenza virus full-length genome
With reference to the CPIV5 sequences (JQ743318.1) being had been filed in GenBank, 13 pairs of primers are designed, between amplified fragments Part is overlapped, and full-length genome can be covered by connecting, and primer sequence is shown in Table 1.
1 canine parainfluenza virus genome sequencing primer sequence table of table
With reference to the specification of RNA extracts kits, the total serum IgE of the separated CPIV virus liquids of embodiment 1 is extracted, is used respectively The anti-sense primer of P1~P13 carries out reverse transcription reaction, prepares the cDNA of virus, using obtained cDNA as template, uses design 13 pairs of primer amplification virus full-length genomes.PCR product through 1% agarose gel electrophoresis identification stripe size with expection be consistent after, PCR product is subjected to recovery purifying according to plastic recovery kit specification.DNA fragmentation after recycling is cloned into pEASY- Blunt carriers, are transformed into E.coli DH5 α competent cells, screening positive clone, and carry out sequencing analysis, and the result is shown in sequence Row 1.
2nd, the structure of full-length infectious clone pCMVTNT-FL
According to the analysis to restriction enzyme site in canine parainfluenza virus genome sequence 1 and transcription vector pCMVTNT, by gene Group is divided into 5 fragments, and purpose fragment is obtained through artificial synthesis, is connected into cloning vector pEasy-Blunt Simple respectively In, then by digestion, connection be connected into 5 fragments in transcription vector successively, finally obtain the transcription containing virus full length cDNA Carrier pCMVTNT-FL.And pCMVTNT-FL is sequenced, primer sequence is shown in Table 2.By aligned sequences and sequence 1 complete one Cause.
The full-length infectious clone's pCMVTNT-FL sequencing primer sequence tables of table 2
PV1-F 5’-CGACGCGTACCAAGGGGAAAATGAAGTGG-3’
PV1-R 5’-AGATAGGAGACCATCAGAAGTTG-3’
PV2-F 5’-CGACGCGT ATAGCTAGAATCACCAGCCTG-3’
PV2-R 5’-ACTGTACTGATAGAGCAGCTCTTACG-3’
PV3-F 5’-TCATTCTCGCTCACCAAGACACACTGGTG-3’
PV3-R 5’-TGACATAGCCCTTCAATTCCACCTCTGG-3’
PV4-F 5’-CTAGCTAGCTGGAGGTACCAGACAATTATCC-3’
PV4-R 5’-TGCAGCTGTTCAAGTGTGATGTTAACTC-3’
PV5-F 5’-CTAGCTAGCACCGTACATTGGCTCCAGG-3’
PV5-R 5’-ATAAGAATGCGGCCGCACCAAGGGGAAAACCA-3’
3rd, the structure of helper plasmid
Purpose fragment is obtained with NP, P the gene order artificial synthesis included in sequence 1, is cloned into pCI-neo respectively Carrier, is transformed into bacillus coli DH 5 alpha competent cell, screening positive clone, and digestion identifies that correct plasmid is named as pCI- NP, pCI-P, and carry out sequencing analysis.Primer sequence is shown in Table 3.
L1, L2 fragment are divided into the L genes included in sequence 1, purpose fragment is obtained through artificial synthesis, by what is obtained With after NheI and SalI double digestions, recycling L1 fragments are attached, turn L1 fragments with the pCI-neo carriers by same double digestion Change, obtain plasmid pCI-L1.After obtained L2 fragment XhoI and SalI double digestions, recycling L2 fragments are with passing through same double enzymes The pCI-L1 carriers cut are attached, convert, and obtain plasmid pCI-L12, i.e. pCI-L, and carry out sequencing analysis.Primer sequence is shown in Table 3.
3 helper plasmid sequencing primer sequence table of table
NP-F 5’-TATTGAATTCGCCACCATGTCATCCGTGCTCAAGGCATATG-3’
NP-R 5’-TATAGTCGACCTAGATGTCGAGATCGCCCAGTG-3’
P-F 5’-TATTGAATTCGCCACCATGGATCCCACTGATTTGAGC-3’
P-R 5’-TATAGTCGACTCAGATTGTACTGCGGATGATTGC-3’
L1-F 5’-TATTGCTAGCGCCACCATGGCTGGGTCTCGGGAGATA-3’
L1-R 5’-TATAGTCGACAGAGTATCTCCGAATGCCCAG-3’
L2-F 5’-TCCGTGTACCGTACATTGGCTCCAG-3’
L2-R 5’-TATAGTCGACTTAGATTTCCTCGCCATCGATC-3’
4th, the rescue of infection clones
Bhk cell is with 8 × 105The concentration in/hole is inoculated in 6 porocyte plates, during overnight growth to 70%-80% individual layers, Abandon supernatant nutrient solution, using 6 transfection reagents of FuGENE by the full length cDNA clone pCMVTNT-FL of recombinant virus genome and Helper plasmid pCI-NP, pCI-P and pCI-L are respectively with the amount cotransfection bhk cell of 1 μ g, 1 μ g, 1 μ g and 0.1 μ g, 5%CO2、 37 DEG C of cultures, concrete operations are carried out by kit specification.After transfection in 14~16h, cell plates seal 43 DEG C of water-bath 3h, and 5% CO2, after 37 DEG C of culture 48h, add 8 × 10 per hole transfectional cell5Vero cells.Continue observation culture until there is the secondary stream of typical dog Feel cytopathy (Fig. 1 C).Until lesion reaches more than 80%, recombinant virus is collected with multigelation method, is preserved as kind of a poison In -80 DEG C.The viral nomenclature of rescue is rCPIV, using HeN0718 plants of CPIV as positive control, what it was formed on Vero Typical dog parainfluenza cytopathy is shown in Figure 1B.Figure 1A is blank control group Vero cytological maps.
Afterwards by 48h after infection clones rCPIV and former female poison CPIV vero cells infections, using indirect immunofluorescence Virus-specific identification is carried out, as a result sees Fig. 1 E, 1F respectively, Fig. 1 E, 1F are shown:RCPIV vero cells infections form born of the same parents' fusion Cytopathy position there is specific green fluorescence, compared with original mother poison CPIV, it is unchanged.Fig. 1 D are blank control group health The immunofluorescence figure of cell.
The pathogenicity of 3 canine parainfluenza virus of embodiment
The Healthy Dogs 15 at CPIV antigen-antibody feminine gender 2-3 monthly ages are chosen, are randomly divided into 3 groups, 5/group, the 1st group passes through Nasal cavity carries out 1 separated CPIV virus liquids 2ml (viral levels 10 of virus inoculation embodiment5.0TCID50/ ml), the 2nd group passes through Nasal cavity carries out the rCPIV virus liquids 2ml (viral levels 10 of the clone of virus inoculation embodiment 25.0TCID50/ ml), the 3rd group of conduct Blank control group, does not attack poison, isolated rearing.Observation 14 days, whether main detection experimental dog spirit, appetite, body temperature, nose have stream The Clinical signs such as tears, cough, limb motion harmony.It the results are shown in Table 4.
The pathogenicity test results of 4 canine parainfluenza virus of table
Group Number of animals Dosage of inoculation Morbidity number
1 1-5 2ml/ is only 5/5
2 6-10 2ml/ is only 5/5
3 10-15 2ml physiological saline 0/5
As shown in Table 4:The rCPIV that 1 separated CPIV of embodiment and embodiment 2 are cloned can cause experimental animal to fall ill, and It is pathogenic consistent.
By above-mentioned CPIV separation strains and clone strain in cytopathy, immunofluorescence, this pathogenicity, gene sequencing etc. Experimental Comparison, phenotype is identical between the two, it is believed that is same Strain, and is named as HeN0718 plants.
The HeN0718 plants of toxicity stabitity experiments of 4 canine parainfluenza virus of embodiment
Naturally, pass on HeN0718 plants of CPIV is continuous on Vero cells, take respectively P1 generations, P5 generations, P10 generations, P15 for, P18 carries out the experiment of this pathogenicity for virus liquid.The Healthy Dogs 30 at CPIV antigen-antibody feminine gender 2-3 monthly ages are chosen, at random It is divided into 6 groups, 5/group, 4-8 groups carry out virus inoculation P1 generations, P5 generations, P10 generations, P15 generations, P18 generation viruses by nasal cavity respectively Liquid 2ml (viral levels 105.0TCID50/ ml), the 9th group is used as blank control group, does not attack poison, isolated rearing.Observation 14 days, it is main Want observation experiment dog spiritual, appetite, body temperature, whether nose have runny nose, cough, the Clinical signs such as limb motion harmony.As a result It is shown in Table 5.
HeN0718 plants of pathogenicity test results of canine parainfluenza virus of the different generations of table 5
As shown in Table 5, experimental animal falls ill during HeN0718 plants of equal energy of the canine parainfluenza virus of different generations, and pathogenic Unanimously.
Illustrate that HeN0718 plants of virulence of canine parainfluenza virus of the present invention are stablized, it is malicious without occurring in cell cultivation process Power attenuation.
The preparation of 5 HeN0718 plants of inactivated vaccines of canine parainfluenza virus of embodiment
HeN0718 plants of cultures of CPIV are inoculated in Vero cells, the Vero to form individual layer is accessed according to MOI=0.01 In cell culture, 37 DEG C of cultures are placed in, when cytopathy reaches 80%, harvest toxic cell culture fluid, through 2 freeze thawing Afterwards, poison is received, measures malicious valency.Final concentration of 0.025%V/V BPL (beta-propiolactone) are added to be inactivated, and according to《Chinese veterinary drug Allusion quotation》(the Chinese veterinary drug committee, version three in 2010, Chinese agriculture publishing house, 2010) method is to the dog parainfluenza virus after inactivation Poison carries out steriling test, mycoplasma is examined, exogenous virus is examined, the results showed that:After HeN0718 plants of inactivations of CPIV, it is not affected by thin Bacterium, the pollution of mould, are also not affected by the infection of mycoplasma and exogenous virus, and pure property is good.
By the virus liquid after inactivation with fully being mixed purchased from the Gel A adjuvants of Seppic companies, up to inactivated vaccine 1,2, 3、4.Specific proportioning is shown in Table 6.
HeN0718 plants of inactivated vaccine proportionings of 6 CPIV of table
Title Inactivate provirus content (TCID50/ml) Gel A adjuvants contents (V/V%)
Vaccine 1 104.0 5%
Vaccine 2 105.0 5%
Vaccine 3 106.0 5%
Vaccine 4 107.0 5%
The safety testing of 6 HeN0718 plants of inactivated vaccines of canine parainfluenza virus of embodiment
1st, the safety testing of a single dose inoculation
Using the healthy susceptible canine 25 of 50 ages in days or so, 5 groups are randomly divided into, 5/group, wherein 10-13 groups pass through respectively The 1,2,3,4, the 14th group of hypodermic injection 1ml physiological saline of 1ml/ inactivated vaccine of subcutaneous vaccination, as blank control.The same terms Lower raising, is observed 14 days, and whether the appetite of observation dog, spirit, body temperature, eye nose are normal daily, and whether injection portion and whole body have Adverse reaction.As a result (7 are shown in Table):After single dose inoculation susceptible canine, the state of mind of dog is good, diet is intended to normally, body temperature Normally, anophthalmia nasal discharge produces, and injection site and whole body have no adverse reaction, and all dogs are strong to live.
Safety testing result of 7 inactivated vaccine of table to single dose inoculation of dog
2nd, the safety testing of single dose repeated inoculation
Using the healthy susceptible canine 25 of 50 ages in days or so, 5 groups are randomly divided into, 5/group, wherein 15-18 groups pass through respectively 1ml/ inactivated vaccine of subcutaneous vaccination first immunisation 1,2,3,4, and booster immunization 1ml/ when the 14th day after first immunisation, the 19 groups of hypodermic injection 1ml physiological saline, as blank control.Raise, observe 14 days under the same terms, observe the food of dog daily Whether desire, spirit, body temperature, eye nose are normal, and whether injection portion and whole body have adverse reaction.As a result (8 are shown in Table):Single dose repeats After being inoculated with susceptible canine, the state of mind of dog is good, diet is intended to normally, and body temperature is normal, and injection site and whole body have no adverse reaction, All dogs are strong to live.
Safety testing result of 8 inactivated vaccine of table to dog single dose repeated inoculation
3rd, the safety testing of an overdose inoculation
Using the healthy susceptible canine 25 of 50 ages in days or so, 5 groups are randomly divided into, 5/group, wherein 20-23 groups pass through respectively The 1,2,3,4, the 24th group of hypodermic injection 4ml physiological saline of 4ml/ inactivated vaccine of subcutaneous vaccination, as blank control.The same terms Lower raising, is observed 14 days, and whether the appetite of observation dog, spirit, body temperature, eye nose are normal daily, and whether injection portion and whole body have Adverse reaction.As a result (9 are shown in Table):After overdose inoculation susceptible canine, the state of mind of dog is good, diet is intended to normally, body temperature Normally, injection site and whole body have no adverse reaction, and all dogs are all strong to live.
Safety testing result of 9 inactivated vaccine of table to overdose inoculation of dog
Result above shows:It is safe that inactivated vaccine 1,2,3,4 prepared by the present invention, which is used to dog be immunized,.
The Study On Immunogenicity of 7 HeN0718 plants of inactivated vaccines of canine parainfluenza virus of embodiment
Using the healthy susceptible canine 25 of 50 ages in days or so, 5 groups are randomly divided into, 5/group, wherein 25-28 groups pass through respectively The 1,2,3,4, the 29th group of hypodermic injection 1ml physiological saline of 1ml/ inactivated vaccine of subcutaneous vaccination, as blank control.The same terms Lower raising, 14 days appetite for finding dog, spirit, body temperature, eye noses of observation are normal, and injection portion and whole body have no adverse reaction.
21st day after immune, CPIV HeN0718 strain virus liquid 2ml (viral levels 10 are used5.0TCID50/ ml) attack Hit experiment dog, attack after poison whether the appetite of observation dog, spirit, body temperature, eye nose normal daily, and injection site and whole body whether There is adverse reaction, and calculate and attack malicious protective rate, the results are shown in Table 10.
The Study On Immunogenicity result of 10 inactivated vaccine of table
As shown in Table 10:The healthy susceptible canine of inactivated vaccine 1,2,3,4 pairs of 50 ages in days or so is immunized respectively, 1ml/ Only, 21 days after being immunized, carry out attacking poison for HeN0718 plants with canine parainfluenza virus, vaccine 1, vaccine 2, vaccine 3 and vaccine 4 are to dog Protective rate is 100%, shows that inactivated vaccine 1,2,3,4 has good protecting effect.
The preparation of 8 canine distemper antigen of embodiment
By C/HN/001 plants of canine distemper virus (during Canine Distemper Virus, strain C/HN/001 are preserved in State's Type Tissue Collection, preserving number are CCTCC NO.V201604, and preservation address is Wuhan, China Wuhan University, are protected The Tibetan date is on March 29th, 2016) in Vero/DongSLAM-1F5 plants of monoclonal cell system Vero/DogSLAM cell lines (Vero/DogSLAM Cell line, strain Vero/DongSLAM-1F5 are preserved in China typical culture collection center, Preserving number is CCTCC NO.C201611, and preservation address is Wuhan, China Wuhan University, and preservation date is 03 month 2016 29 Day) on expand, harvest virus liquid, after 2 freeze thawing, receive poison, measure malicious valency.Add final concentration of 0.025%V/V BPL (β- Propiolactone) inactivated, and according to《Chinese veterinary pharmacopoeia》(the Chinese veterinary drug committee, version three in 2010, Chinese agriculture are published Society, 2010) method to CDV C/HN/001 carry out steriling test, mycoplasma is examined, exogenous virus is examined, the results showed that:CDV After C/HN/001 plants of inactivations, the pollution of bacterium, mould is not affected by, is also not affected by the infection of mycoplasma and exogenous virus, pure property Well.
The preparation of 9 Canine Parvovirus antigen of embodiment
By S0425 plants of canine parvovirus, (Canine Parvovirus, Strain S0425 are preserved in Chinese Typical Representative culture Thing collection, preserving number are CCTCC NO.V201634, and preservation address is Wuhan, China Wuhan University, and preservation date is On June 14th, 2016) expanded on F81 cells, virus liquid is harvested, after 2 freeze thawing, receives poison, measures malicious valency.Add final concentration Inactivated for 0.025%V/V BPL (beta-propiolactone), and according to《Chinese veterinary pharmacopoeia》(the Chinese veterinary drug committee, version in 2010 Three, Chinese agriculture publishing house, 2010) method carries out CPV steriling test, mycoplasma is examined, exogenous virus is examined, as a result table It is bright:After S0425 plants of inactivations of CPV, the pollution of bacterium, mould is not affected by, is also not affected by the infection of mycoplasma and exogenous virus, it is pure Net property is good.
The preparation of the vaccine combination of 10 antigen containing canine parainfluenza virus of embodiment
CDV C/HN/001 plants of antigens, realities prepared by HeN0718 plants of antigens of CPIV prepared by embodiment 5, embodiment 8 Apply that example 9 prepares S0425 plants of antigens of CPV and the GEL A adjuvants of Seppic companies are matched somebody with somebody according to component and content shown in table 11 System.
The vaccine combination content proportioning of 11 antigen containing canine parainfluenza virus of table
The application of the vaccine combination of 11 antigen containing canine parainfluenza virus of embodiment
1st, the safety testing of the vaccine combination of the antigen containing canine parainfluenza virus
Choose CPIV, CDV antigen, negative antibody, CPV antigen negatives, antibody HI≤1: healthy age at 4 2~3 monthly ages compares lattice Dog 15, is randomly divided into 3 groups, 5/group, vaccine 5-7 prepared by embodiment 10 is immunized respectively, every beasle dog injects 10 The vaccine of dosage, is observed 14.As a result:Immunity inoculation beasle dog spirit, diet are normal;Body temperature is without significant change, In the range of normal 38.5 DEG C -39.5 DEG C;There are not swelling, necrosis, no systemic adverse reactions in inoculation position.Show:Vaccine The safety testing of 5-7 is qualified.
2nd, the Study On Immunogenicity of the vaccine combination of the antigen containing canine parainfluenza virus
Choose CPIV, CDV antigen, negative antibody, CPV antigen negatives, antibody HI≤1: 4 2~3 monthly age health beasle dogs 55,11 groups are randomly divided into, 5/group, the 30th, 31 group of immune vaccine 5, the 32nd, 33 group of immune vaccine 6, the 34th, 35,36 group is exempted from Epidemic disease vaccine 7,1ml/ is only.Another to set the 37th, 38,39 group as malicious control is attacked, the 40th group is used as blank control, is not immunized, and isolation is raised Support.Take a blood sample within 14 days, 21 days before immune and after immune, detection HI antibody titers (detection of HI antibody titers according to:Yin Shake, Liu Jing China animal virology second edition Beijing:Science Press, 1997:204-437).21 days after immune, to the 30th, 32nd, 34,37 groups of all dogs are respectively by the way that by nasal challenge canine parainfluenza virus HeN0718 strain virus liquid 2ml, (viral level is 105.0TCID50/ ml), observe 14.It the results are shown in Table 12.
Animal test results after the vaccine combination of 12 antigen containing CPIV of table attacks CPIV
As shown in Table 12:(1) antibody titer after being immunized, vaccine 5-7 can produce higher CPIV SN antibody after dog is immunized Potency, when showing that canine parainfluenza virus inactivation antigen is used in mixed way with canine distemper virus, the weak malicious antigen of canine parvovirus, does not influence The SN antibody titers of CPIV;(2) malicious protection is attacked, vaccine 5-7 attacks poison with CPIV velogen strains after being immunized and do not occur dog parainfluenza virus The classical symptom of viral disease, and protective rate is 100%.
With reference to group 27, group 30, group 32, the as shown by data for organizing 34 in table 10,12, in same amount CPIV inactivation antigens (106.0TCID50/ ml) vaccine combination in, to produce identical neutralize antibody titers, only single seedling containing CPIV inactivation antigens The time longer compared to needs compared with the connection seedling (also containing CPV and/or CDV inactivation antigens) containing CPIV inactivation antigens;Either The same time after immune, compared with the connection seedling (also containing CPV and/or CDV inactivation antigens) containing CPIV inactivation antigens, only contain The neutralize antibody titers of single seedling generation of CPIV inactivation antigens are lower.That is the presence of other antigenic components promotes CPIV The immune efficacy of HeN718 inactivation antigens, generates synergistic effect.
21 days after immune, S0425 plants of canine parvovirus is attacked by oral route respectively to the 31st, 35,38 group of all dogs Virus liquid 6ml (viral levels 107.0TCID50/ ml), observe 14.It the results are shown in Table 13.
Animal test results after the vaccine combination of 13 antigen containing CPIV of table attacks CPV
As shown in Table 13:(1) antibody titer after being immunized, vaccine 5,7 can produce higher CPV HI antibody after dog is immunized Potency, when showing that canine parvovirus inactivation antigen is used in mixed way with canine parainfluenza virus, canine distemper virus antigen, does not influence CPV HI antibody titers;(2) malicious protection is attacked, attacking poison with the velogen strain of CPV prevalences after vaccine 5,7 is immune does not occur canine parvovirus disease Symptom, and protective rate is 100%.
After 21 days immune, collunarium 1ml and intraperitoneal injection 2ml approach attack dogs are passed through respectively to the 33rd, 36,39 group of all dogs Distemper virus CDV C/HN/001 strain virus liquid (viral levels 105.0TCID50/ ml), observe 14, the results are shown in Table 14.
Animal test results after the vaccine combination of 14 antigen containing CPIV of table attacks CDV
As shown in Table 14:(1) antibody titer after being immunized, vaccine 6,7 can produce higher CDV SN antibody after dog is immunized Potency, when showing that canine distemper virus inactivation antigen is used in mixed way with canine parainfluenza virus, Canine Parvovirus antigen, does not influence CDV SN antibody titers;(2) malicious protection is attacked, attacking poison with the velogen strain of CDV prevalences after vaccine 6,7 is immune does not occur canine distemper virus disease Symptom, and protective rate is 100%.
In summary:There is not appetite after being immunized in the vaccine combination of the antigen containing canine parainfluenza virus prepared by the present invention Decline, the typical clinical symptoms of the canine parainfluenza virus disease such as body temperature rise, thin nasal discharge, vomiting, cough, spasm, twitch, it is right Other contained antigens do not have an immune interference effect in vaccine combination, other contained antigens can also secondary to dog flow in vaccine combination Synergistic function occurs for Influenza Virus antigen.
The above is only the preferred embodiment of the present invention, and limitation in any form is not done to the present invention, though So the present invention is disclosed above with preferred embodiment, but is not limited to the present invention, any to be familiar with this professional technology people Member, in the range of technical solution of the present invention is not departed from, when the technology contents using the disclosure above make a little change or repair The equivalent embodiment for equivalent variations is adornd, as long as being the content without departing from technical solution of the present invention, the technology according to the present invention is real Any simple modification, equivalent change and modification that confrontation above example is made, still falls within the scope of technical solution of the present invention It is interior.
SEQUENCE LISTING
<110>Pulaike Biological Engineering Co., Ltd.
<120>Canine parainfluenza virus strain and its application
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 15246
<212> DNA
<213>Canine parainfluenza virus
<400> 1
accaagggga aaatgaagtg gtgacccaaa tcatcgaaga ccctcgagat tacataggtc 60
cggaacctat ggccttcgtg accgacctcg agtcagagta gttcaataag gacctatcaa 120
gtttgggcaa ttttttgtct ccgacacaaa aatgtcatcc gtgctcaagg catatgagcg 180
attcacgctc actcaagaac tgcaagatca gagtgaggaa ggtacaatcc cacctacaac 240
actaaaaccg ataatcaggg tatttatact aacctctaat aacccagagc taagatcccg 300
gcttcttcta ttctgcctac ggattgttct cagtaatggc gcaagggatt ctcatcgctt 360
tggagcatta cttacaatgt tttcactacc atcagccaca atgctcaatc atgtcaaatt 420
agctgaccag tcaccagaag ccgatatcga aagggtagag atcgatggtt ttgaggaggg 480
atcattccgc ttaatcccca atgctcgttc aggcatgagc cgtggagaga tcaatgccta 540
tgctgcactt gcagaagatc tacctgacac actaaaccat gaaacacctt ttgttgattc 600
cgaagtcgag gggactgcat gggatgagat tgagactttc ttagatatgt gttacagtgt 660
cttaatgcag gcatggatag tgacttgcaa gtgtatgact gcgccagacc aacctgctgc 720
ctctattgag aaacgcctgc aaaaatatcg tcagcaaggc aggatcaacc caagatatct 780
cctgcaaccg gaggctcgac gaataatcca gaatgtaatc cggaagggga tggtggtcag 840
acatttcctc accttcgaac tgcagcttgc ccgagcacaa agccttgtat caaataggta 900
ttatgctatg gtaggggatg ttgggaagta tatagagaat tgtggaatgg gaggcttctt 960
tttgacacta aaatatgcat taggaaccag atggcccaca cttgccctag ctgcattttc 1020
aggagagcta acaaagctaa agtccctcat ggcattatac cagacccttg gtgagcaggc 1080
ccgatacttg gccctattgg agtcaccaca tttgatggat tttgctgcag caaactaccc 1140
actgctatac agctatgcta tgggaatagg ctatgtgtta gatgtcaaca tgaggaacta 1200
tgctttctcc agatcataca tgaataagac atatttccaa ttaggaatgg aaactgcaag 1260
aaaacaacag ggtgcagttg acatgaggat ggcagaagat ctcggtctaa ctcaagccga 1320
acgcaccgag atggcaaata cacttgccaa attgaccaca gcaaatcgag gggcagacac 1380
caggggagga gtcaaccctt tctcatctgt caccgggaca actcaggtgc ccgctgcggt 1440
aacaggtgac acattcgaga gctacatggt cgcggatcga ctgaggcaga gatatgctga 1500
tgcaagcacc cacgatgatg agatgccacc actggaagag gaggaagaag acgatacatc 1560
tgcaggtcca cgcactggac caactctcga acaagtggcc cttgacatcc agaacacagc 1620
agttggagct ccaatccata cagatgactt gaatgccgca ctgggcgatc tcgacatcta 1680
gacaattcag atctcaatcc aaaatcgaca cacccaagta accagctaga tggaaccaca 1740
gtggactcca caaggttcct gcccactaca cgcttttaag aaaaaaatag gcccggacgg 1800
gttagtaaca agtgatcgcc gatgtcaata acacaatcca caatctacaa tggatcccac 1860
tgatttgagc ttctccccag atgagatcaa caagctcata gagacaggcc tgaacactgt 1920
ggagtatttt acttcccagc aagtcacagg aacatcctct cttggaaaga atacaatacc 1980
accaggggtt acaggactac taaccaatgc tgcagaggca aagatccaag agtcagccaa 2040
ccatcagaag gttccagttg atgggggtgc aaaatcaaag aaaccgcgac caaaaattgc 2100
cattgtgcca gcagatgaca aaacggtgcc cgggaagccg atcccaaacc ctctattagg 2160
tctggactcc accccaagca cccaaactgt gcttgatcta agtgggaaaa cattaccatc 2220
aggatcctat aaaggggtta agcttgcgaa atttggaaaa gaaaatctga tgacacggtt 2280
catcgaggaa cccagagaga atcctatcgc aaccagttcc ccaaccgatt ttaagagggg 2340
cagggatacc ggagggttcc atagaaggga gtactcaatt ggatgggtgg gagatgaagt 2400
caaggtcact gagtggtgca atccatcctg ttctccaatc accgctgcag caaggcgatt 2460
tgaatgcact tgtcaccagt gtccagtcac ttgctctgaa tgtgaacgag atacttaata 2520
cagtgagaaa tctggactct cggatgaatc aactggagac aaaagtagat cgcattctct 2580
catctcagtc tctaatccag accatcaaga atgacatagt tggactcaaa gcagggatgg 2640
ctactttaga aggaatgatt acaactgtga aaattatgga cccgggagtt cccagcaatg 2700
ttactgtgga agatgtgcgc aagaaattaa gtaaccatgc tgttgttata ccagaatcat 2760
tcaatgatag tttcttgact caatctgaag atgtaatttc acttgatgag ttggcccgac 2820
caactgcaac aagtgttaag aagattgtca ggaaggttcc tcctcagaag gatctgactg 2880
gattgaagat cacactagag caattggcaa aggattgcat cagtaaaccg aagatgaggg 2940
aagagtatct ccttaaaatc aaccaggctt ccagtgaggc ccagctactt gacctcaaga 3000
aagcaatcat ccgcagtaca atctgatcta gaaacaccca attacaccac actggcatga 3060
cactgcacca accctgaggg ctttagaaaa aacgatcgat aataaataag cccgaacact 3120
atacaccacc cgaggcagcc atgccatcca tcagcatccc tgcagaccct accaatccac 3180
gtcaatcaat aaaagcgttc ccaattgtga ttaacagtaa tgggggtgag aaaggccgct 3240
tagttaaaca actacgcaca acctacttga atgacctaga tactcatgag ccactggtga 3300
cgttcgtaaa tacttatgga ttcatctacg aacagactcg gggaaatacc attgtcggag 3360
aggatcaaca tgggaagaaa agagaggctg tgactgctgc aatggttact cttggatgtg 3420
ggcctaatct accatcatta gggaatgtcc tgggacaact aagtgaattc caggtcattg 3480
ttagaaagac atccagcaaa gcagaagaga tgatctttga aattgttaag tatccgagaa 3540
tatttaggag tcatacatta atccagaaag gactagtctg tgtctccgca gaaaaatttg 3600
ttaagtcacc agggaaagta caatctggaa tggactatct cttcattccg acatttttgt 3660
cagtgactta ctgtccagct gcaatcaaat ttcaggtacc tggccctatg ttaaagatga 3720
gatcaagata cactcagagc ttacaacttg aactaatgat aagaatcctg tgtaagcccg 3780
attcgccact tatgaaggtc cacatccctg acaaggaagg aagaggatgt cttgtatcag 3840
tttggttgca tgtatgcaac atctacaaat caggaaacaa gaatggcagt gagtggcagg 3900
aatactggat gagaaagtgt gctaacatgc aacttgaagt gtcgattgca gacatgtggg 3960
gaccaaccat cataattcat gccaggggtc acattcccaa aagtgctaag ttgtttttcg 4020
gaaagggtgg atggagctgc catccacttc atgaagttgt tccaagtgtc accaaaacac 4080
tatggtctgt aggctgtgag attacaaagg ctaaggcaat catacaagag agtagcatct 4140
ctcttctcgt ggagactact gacatcataa gtccaaaagt caaaatttca tctaagcatc 4200
gtcgctttgg gaaatcaaat tggggtctgt tcaggaaaac cagatcacta cccaacctga 4260
cgaagctgga atgactgacc tctaatcgag attacaccac ctcaaactat agatgggtgg 4320
tacctaagtg atcaatcttg taagtactga tcgtaggcta caacacatta atattattcg 4380
ggttagatag ctcaattagc tctttattaa taataacact actattccaa tagctagaat 4440
caccagcctg atttatctcc aaaatgattc aaagaaaaca agtcatatta agactatctt 4500
aagcacgaac ccatatcgtc cttcaaatca tgggtactag aattcaactt ctgatggtct 4560
cctatctatt ggcaggaaca ggcagccttg atccagcagc cctcatgcaa atcggtgtca 4620
ttccaacaaa tgtccggcaa cttatgtatt atactgaggc ttcatcagca ttcattgttg 4680
tgaagttaat gcctacaatt gactcgccga ttagtgggtg taatataaca tccatttcaa 4740
gctataatgc aacaatgaca aaacttctac agccgatcgg tgagaattta gagacgatta 4800
ggtaccagtt gattccaact cggaggagac gccggtttgt aggggtggtg attggattgg 4860
ccgcattagg agtagctact gcagcacagg ttactgccgc agtagcacta gtaaaggcga 4920
ataaaaatgc tgcggctata ctcaatctca aaaatgcaat ccaaaaaaca aatgcagcag 4980
ttgcagatgt ggttcaggcc acacaatcac taggaacggc agttcaagca gttcaagatc 5040
acataaatag tgtggtaagt ccagcaatta cagcagccaa ttgcaaagcc caagatgcta 5100
tcattggctc aattctcaat ctttatttga ccgagttgac aactatcttc cataatcaaa 5160
ttacaaaccc cgcattgagt cctattacaa ttcaagcttt aaggatccta ctagggagca 5220
ccttgccgac cgtggtcaga aaatctttca atacccagat aagtgcagct gagcttctct 5280
catcagggtt attgacaggc cagattgtgg gattagattt gacctacatg cagatggtca 5340
taaaaattga gctgccaact ttaactgtac aacctgcaac tcagatcata gatctagtca 5400
ccatttctgc attcattaac aatcgagaag ttatggccca attaccaaca cgtgttattg 5460
tgactggcag cttgatccaa gcctatcccg catcgcaatg tactatcacc cccaacactg 5520
tgtattgtag gtataatgat gcccaagtac tctcagataa tacgatggct tgcctccaag 5580
gtaacttgac aagatgcacc ttctctccgg tggttgggag ctttcttact cgattcgtgc 5640
tgtttgatgg aatagtttat gcaaattgca ggtcgatgtt gtgcaagtgc atgcagcctg 5700
ctgctgttat cctacagcca agttcatccc ctgtaactgt cattgacatg cacaagtgtg 5760
tgagtctgca gcttgacaat ctcagattca ccatcactca attggccaat ataacctaca 5820
atagcaccat caagcttgaa acatctcaga tcttgcctat tgatccgttg gatatatccc 5880
agaatctagc tgcggtgaat aagagtctaa gtgatgcact acaacactta gcacaaagtg 5940
acacatacct ttctgcaatc acatcagcta cgactacaag tgtattatcc ataatagcaa 6000
tctgtcttgg atcgttaggt ttaatattaa tagtcttgat cagtgtagtt gtgtggaagt 6060
tattgaccat tgtcgctgct aatcgaaata gaatggagaa ttttgtttat cataattcag 6120
catcccacca ctcacggtct gatctcagtg agaaaaatca acctgcaact cttggaacaa 6180
gataagacag tcatctatta ataattttaa agaaaaaaac gataggaccg aaactagtat 6240
tgaaagaacc gtctcggtca atccaggcaa tcaagccgac accgtctcgg aaagctcaaa 6300
tcatgctgcc cgatccggaa gatccggaga gcaaaaaaac tacaaggaga acaggaaacc 6360
caatcatccg ctttccattc accctccctc tatttgtaaa cttcactgtc ccaactccaa 6420
gacacccact gtcccaacac ttgccatagg ccatctacta tatcatctct cctgccatac 6480
ttcctattca catcatatct attttaaaga aaacataggc ccgaacacta atcgtgccgg 6540
cagtgccact gcacacacaa cactacacgt acaatacact acaatggttg cagaagatgc 6600
ccctgttagg ggcacttgcc gagtattatt tcgaacaata actttaattt ttctatgcac 6660
actactagca ttaagcatct ctatccttta tgagagttta ataacccaag agcaaataat 6720
gagccacgca ggctcaactg gatctaattc tcgattagga agtatcattg atcttcttaa 6780
taatattctc tctgtcgcaa atcagattat atataactct gcagtcgctc tacctctaca 6840
attggacact cttgaatcaa cactccttac agccattaag tctcttcaaa caagtgacaa 6900
gctagaacag aactgctcat ggggtgctgc actgattaat gataatagat acattaatgg 6960
catcaatcag ttttatttct caattgctga gggtcgcaat ctggcacttg gcccacttct 7020
taatatacct agtttcattc caactgccac gacaccagag ggctgcacca ggatcccatc 7080
attctcgctc accaagacac actggtgtta tacacacaat gttatcctga atggatgcca 7140
ggatcatgta tcctcaaatc aatttgtttc catgggaatc attgagccca cttctgccgg 7200
gtttccatcc tttcgaaccc taaagactct atatctcagc gatggggtca atcgtaagag 7260
ctgctctatc agtacagttc cggggggttg tatgatgtat tgtttcgtct ctactcaacc 7320
agagagggat gactaccttt ctaccgctcc tccagaacaa cgaattatta taatgtacta 7380
taatgataca atcgtggagc gcataattaa tccacccgga gtactagatg tatgggcaac 7440
attgaaccca ggaacaggaa gcggggtata ttatttaggt tgggtactct ttccaatata 7500
tggcggcgtg attaaaaaga cgagtttatg gaataatcaa gcaaataaat actttatccc 7560
ccagatggtt gctgctctct gctcacaaaa ccaggcaact caagtcaaaa atgctaagtc 7620
atcatactat agcagctggc tcggcaatcg aatgattcag tctggaatcc tggcatgtcc 7680
tcttcaacag gatctaacta atgagtgttt agttctgccc ttttctaatg atcaggtgct 7740
tatgggtgct gaagggagat tatacatgta tggtgactcg gtgtattact accaaagaag 7800
caatagttgg tggcctatga ccatgctgta taaggtaacc ataacgttca ctaatggtca 7860
gccatctgct atatcagctc agaatgtgcc cacacagcag gtccctagac ctgggacaag 7920
aaactgctct gcaacaaata gatgtcccgg tttttgcttg aaaggagtgt atgctgatgc 7980
ctggttactg accaacccct cgtctaccag cacatttgga tcggaagcaa ccttcactgg 8040
ctcttatctc aacgcggcaa ctcagcgtat caatccgacg atgtatatcg cgaacaacac 8100
acagatcata agctcacagc aatttggatc aagcggtcaa gaagcagcat atggtcacac 8160
aacttgtttt agggacacag gctctgttat ggtatactgt atctatatca ttgaactgtc 8220
atcatccctc ttaggacaat ttcagatagt tccatttatc cgtcaggtga cactatccta 8280
aaggcaaaag catccaggtc tgacccagca aatcaaggca ttataccaga ccatgaaatg 8340
tataccaaac attattaaca ctaatgacac acaaaaatgg ttttaagaaa aaccaagaga 8400
acaataggcc agaatggctg ggtctcggga gatattactc cctgaagtcc atctcaattc 8460
gccaattgta aagcataagc tatactatta cattctactt ggaaacctcc caaatgagat 8520
cgacattgac gatttaggtc cattacataa tcaaaattgg aatcagatag cacatgaaga 8580
gtctaactta gcccaacgct tggtaaatgt aagaaatttt ctaattactc acatccctga 8640
tcttagaaag ggccattggc aagagtatgt caatgtaata ctgtggccgc gaattcttcc 8700
cttgattccg gattttaaaa tcaatgacca attacctcta ctcaaaaatt gggacaagct 8760
agttaaagaa tcatgttcag taatcaatgc gggtacttcc cagtgtattc agaatctcag 8820
ctatggactg acaggtcgtg ggaacctctt tacacgatca cgtgaactct ctggtgaccg 8880
cagggatatt gatcttaaga cggttgtggc ggcatggcat gactcagact ggaaaagaat 8940
aagtgatttt tggattatga tcaaattcca gatgagacaa ttaattgtta ggcaaacaga 9000
tcataatgat cctgatttaa tcacatatat cgaaaacaga gaaggcataa tcatcataac 9060
tcctgaactg gtagcattat ttaacactga gaatcataca ctaacatata tgacctttga 9120
aattgtactg atggtttcag atatgtacga aggtcgtcac aacattttat cactatgcac 9180
agttagcact tacctgaatc ccctgaagaa aagaataaca tatttattga gccttgtaga 9240
taacttggct tttcagatag gtgatgctgt atacaacata attgctttgc tagaatcctt 9300
tgtatatgca cagttgcaaa tgtcagatcc catcccagaa ctcagaggac aattccatgc 9360
attcgtatgt tctgagattc ttgatgcact aaggggaact aatagcttca cccaagatga 9420
atcaagaaat gtgacaacca atttgatatc cccattccaa gatctgaccc cagatcttac 9480
ggctgaattg ctctgtataa tgaggctttg gggacacccc atgctcactg ccagtcaagc 9540
tgcgggaaag gtacgcgagt ctatgtgtgc tggaaaggtg ttagactttc caaccattat 9600
gaaaacacta gcctttttcc atactattct gatcaatggt tacaggagga agcatcatgg 9660
agtatggcca cccttgaact taccgggtaa tgcttcaaag ggtctcatag aacttatgaa 9720
tgacaatact gagataagct atgaattcac acttaagcat tggaaggaag tctctcttat 9780
aaaattcaag aaatgttttg atgcagacgc aggtgaggaa ctcagtatat ttatgaaaga 9840
taaagcaatt agtgcaccaa aacaagactg gatgagtgtg tttagaagaa gcctaatcaa 9900
acagcgccat cagcatcatc aggtccccct acctaattca ttcaatcgac ggctattgct 9960
aaactttctt ggcgatgaca aattcgaccc gaatgtggag ctacagtatg taacatcagg 10020
tgagtatcta catgatgaca cgttttgtgc atcatattca ctgaaagaga aggaaattaa 10080
acctgatggt cgaatttttg caaagttgac taaaagaatg agatcatgtc aagttatagc 10140
agaatctctt ttagcgaacc atgctgggaa gttaatgaaa gagaatggtg ttgtaatgaa 10200
tcagctatca ttaacaaaat cactattaac aatgagtcag attggaataa tatccgagaa 10260
agctagaaaa tcgactcggg ataacataaa tcaacctggt tttcagaata tccagagaaa 10320
taaatcacgt cactccaagc aagtcaacca gcgagatcca agtgatgact ttgaattggc 10380
agcatctttt ttaactactg atctcaaaaa atattgttta caatggaggt accagacaat 10440
tatcccattt gctcaatcat taaacagaat gtatggttat cctcatctct ttgagtggat 10500
tcacttacgg ctaatgcgta gtacacttta cgtgggggat cccttcaacc caccagcgga 10560
taccagtcaa tttgatctag ataaagtgat taatggagat atctttattg tatcacccag 10620
aggtggaatt gaagggctat gtcaaaaggc ttggacaatg atatctatct ctgtgataat 10680
tctatctgcc acagagtctg gcacacgagt aatgagtatg gtgcagggag ataatcaagc 10740
aattgctgtc accacacgag taccaaggag cttgccgact cttgagaaaa agactattgc 10800
ttttagatct tgtaatctat tctttgagag gttaaaatgt aataattttg gattaggtca 10860
ccatttgaaa gaacaagaga ctatcattag ttcccacttc tttgtttata gcaagagaat 10920
attctatcag gggaggattc taacacaagc cttaaaaaat gctagtaagc tctgcttgac 10980
agctgatgtc ctaggagaat gtacccaatc atcatgttct aatcttgcaa ctactgtcat 11040
gagattaact gagaatggtg ttgaaaaaga tatctgtttc tatttgaata tttatatgac 11100
catcaaacag ctctcctatg atatcatctt ccctcaagtg tcaattcctg gagatcagat 11160
cacattagaa tacataaata atccacatct ggtatcacga ttggctcttc tgccatctca 11220
gctaggaggt ctaaactacc tgtcatgtag taggctgttc aatcgaaaca taggcgaccc 11280
ggtggtttcc gcagttgcag atcttaagag attaattaac tcaggatgta tggattactg 11340
gatcctttat aatttattag gtagaaaacc gggaaacggc tcatgggcta ctttagcagc 11400
tgacccgtac tcaataaaca tagagtatca atacccccca actacagctc ttaagaggca 11460
cacccaacaa gctctgatgg aactcagtac aaatccaatg ttacgtggca tattctctga 11520
caatgcacag gcagaagaaa ataatcttgc tagatttctc ctggataggg aggtgatctt 11580
tccacgtgta gctcacataa tcattgagca aaccagtgtc gggaggagaa aacagattca 11640
aggatatttg gattcaacta gatcgataat gaggaaatca ctagaaatta agcccttgtc 11700
caataggaag cttaatgaaa tactagatta caacatcaat tacctagctt acaatttggt 11760
attactcaag aatgctattg aacctccgac ttatttgaag gcaatgactc ttgaaacatg 11820
tagcatcgac attgcaagga gcctccggaa gctttcctgg gccccactct tgggtgggag 11880
aaatcttgaa ggattagaga caccagatcc cattgaaatt actgcaggag cattaattgt 11940
tggatcaggc tactgtgaac agtgtgctgc aggagacaat cgattcacat ggtttttctt 12000
gccatctggt atcgagatag gaggggatcc ccgtgataat cctcctatcc gtgtaccgta 12060
cattggctcc aggactgatg agaggagggt agcctcaatg gcatacatca ggggtgcctc 12120
gagtagccta aaagcagctc ttagactggc aggagtgtac atctgggcat tcggagatac 12180
tctggaaaat tggatagatg cactggattt gtctcatact agagttaaca tcacacttga 12240
acagctgcaa tccctcaccc cacttccaac ctctgccaat ctaacccatc ggttggatga 12300
tggcacaact accctaaagt ttactcctgc gagctcttat accttttcaa gtttcactca 12360
tatatcaaat gatgagcaat acctgacaat taatgacaaa actgcagatt caaatataat 12420
ctaccaacag ttaatgatca ctggactcgg aatcttagaa acatggaata atcctccaat 12480
caatagaaca ttcgaagaat ctaccctaca tttgcacact ggtgcatcat gttgtgtccg 12540
acctgtggac tcctgcatca tctcagaagc attaacagtc aagccacata ttacagtacc 12600
gtacagcaat aaatttgtat ttgatgaaga cccgctatct gaatatgaga ctgcaaaact 12660
ggaatcgtta tcattccaag cccagttagg caacattgat gctgtagata tgacaggtaa 12720
attaacatta ttgtcccaat tcactgcaag gcagattatt aatgcaatca ctggactcga 12780
tgagtctatc tctcttacta atgatgccat tgttgcatca gactatgtct ccaattggat 12840
cagtgaatgc atgtatacca aattagatga attatttatg tattgtgggt gggaactact 12900
attagaacta tcctatcaaa tgtattatct gagggtagtt gggtggagta acatagtgga 12960
ttattcttac atgatcctaa gaaggatacc tggtgcagcg ttaaacaatc tggcatctac 13020
attaagtcat ccaaaacttt tccgacgggc tatcaactta gatatagttg cccccttaaa 13080
tgctcctcat tttgcatctc tggactacat caagatgagt gtggatgcaa tactctgggg 13140
ctgtaaaaga gtcatcaatg tgctctccaa tggaggggac ttagaattag ttgtgacatc 13200
tgaagatagt cttattctca gtgaccgatc catgaatctc attgcaagga aattaacttt 13260
attatcactg attcatcata atggtttgga attaccaaag attaaggggt tctcacctga 13320
tgagaagtgt ttcgctttga cagaattttt gaggaaagtg gtgaactcag ggttgagttc 13380
aatagagaac ctatcaagtt ttatgtacaa tgtagagaac ccacggcttg cagcattcgc 13440
cagcaataat tactacctga ccagaaaatt atcgaattca atacgagata ctgagtcggg 13500
tcaagtagcg gtcacctcat attatgaatc attagaatat attgatagtc ttaagctaac 13560
cccacatgtg cctggtacct catgcattga ggatgatagt ctatgtacaa atgattacat 13620
aatctggatc atagagtcca atgcaaactt ggagaagtat ccaattccaa atagccctga 13680
ggatgattcc aatttccata actttaagtt gaatgctcca tcgcaccata ccttacgccc 13740
attagggtta tcatcaactg cttggtataa gggtataagc tgttgcaggt accttgagcg 13800
attaaagcta ccacagggtg atcatttata tattgcagaa ggtagtggtg ccagtatgac 13860
aatcatagaa tacttattcc caggaagaaa gatatattac aattctttat ttagtagtgg 13920
tgacaatccc ccacaaagaa attatgcacc aatgcctact cagttcattg agagtgtccc 13980
atacaagctc tggcaagcac acacagatca atatcccgag atttttgagg atttcatccc 14040
tctatggaac ggaaatgccg ccatgactga cataggaatg acagcttgtg tagaatttat 14100
catcaataga gtcggcccaa ggacttgcag tttagtacat gtagatttgg agtcaagtgc 14160
aagcttaaat caacaatgcc tgtcaaagcc gataattaat gctattatca ctgctacaac 14220
tgttttgtgc cctcatgggg tgcttattct gaaatatagt tggttgccat ttactagatt 14280
tagtactttg atcactttct tatggtgcta ctttgagaga atcactgttc ttaggagcac 14340
atattctgat ccagctaatc atgaggttta tttaatttgt atccttgccg acaactttgc 14400
attccagact gtctcgcagg caacaggaac ggcgatgact ttaaccgatc aagggtttac 14460
tttgatatca cctgaaagaa taaatcagta ttgggatggt cacttgaagc aagaacgtat 14520
cgtagcggaa gcaattgata aggtggttct aggagaaaat tctctattca attcgagtga 14580
taatgaatta atcctcaaat gtggagggac accaaatgca cggaatctta tcgatatcga 14640
gccagtcgca actttcatag aatttgaaca actgatctgc acaatgttga caacccactt 14700
gaaggaaata attgatataa caaggtctgg aacccaggat tatgaaagtt tattactcac 14760
tccttacaat ttaggtcttc ttggtaaaat cagtacgata gtgagattat taacagaaag 14820
gattctaaat catactatca ggaattggtt gatcctccca ccttcgctcc ggatgatcgt 14880
gaagcagaac ttggaattcg gcatattcag gattacttcc atcctcaact ctgatcgatt 14940
cctgaagctt tctccaaata ggaaatactt gattacacaa ttaactacag gctacattag 15000
aaaattgatt gagggggatt gtaatatcga actaactaga cctatccaaa agcaaatctg 15060
gaaagcatta ggttgtgtag tctattgtca cgatccagta gatcaaaggg aatcaacaga 15120
gtttattgat ataaatatta atgaagaaat agaccgcggg atcgatggcg aggaaatcta 15180
aatatatcaa gaatcagaat tagtttaaga aaaaataaga ggattaatct tggttttccc 15240
cttggt 15246

Claims (10)

1. a kind of canine parainfluenza virus strain, wherein, the canine parainfluenza virus strain Genomic full_length cDNA is SEQ ID CDS replaces with the sequence after its degenerate sequence in sequence or the SEQ ID No.1 sequences shown in No.1.
2. canine parainfluenza virus strain according to claim 1, wherein, the canine parainfluenza virus strain is CPIV HeN0718 plants, the canine parainfluenza virus strain CPIV HeN0718 pnca gene groups full-length cDNA is shown in SEQ ID No.1 Sequence.
3. a kind of vaccine combination, wherein, the vaccine combination includes the dog parainfluenza virus described in the claim 1 of immune amount Poison strain or its culture inactivation antigen and veterinarily acceptable carrier.
4. vaccine combination according to claim 3, wherein, the canine parainfluenza virus strain antigen is the dog The inactivation antigen of HeN0718 plants of parainfluenza virus poison strain CPIV or its culture, the canine parainfluenza virus strain CPIV The inactivation antigen content of HeN0718 plants or its culture is before inactivation 104.0-107.0TCID50/ml;10 before preferably inactivating5.0- 106.0TCID50/ml;10 before more preferably inactivating6.0TCID50/ml。
5. vaccine combination according to claim 3, wherein, the canine parainfluenza virus strain culture is culture 1 The culture in~18 generations;Preferably, the canine parainfluenza virus strain culture is the culture in 6~18 generations of culture.
6. vaccine combination according to claim 3, wherein, the veterinarily acceptable carrier is adjuvant, institute Stating adjuvant includes:(1) aluminium glue adjuvant, saponin(e, Avridine, DDA;(2) water-in-oil emulsion, oil in water emulsion, W/O/W Emulsion;Or the copolymer of the polymer of (3) acrylic or methacrylic acid, maleic anhydride and alkenyl derivative;And RIBI adjuvant systems, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli One or more in heat-labile toxin, cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvants;
Preferably, saponin(e is Quil A, QS-21, GPI-0100;
Preferably, emulsion is that SPT emulsions, MF59 emulsions, or emulsion are formed by oil with emulsifier combination, and emulsion can be based on light liquid Paraffin oil, isoprenoid oil (such as saualane or squalene oil, particularly alkene, isobutene or the last of the ten Heavenly stems because of olefin oligomerisation generation Alkene oligomerization produce oil), acid or alcohol containing linear alkyl ester (more specifically vegetable oil, ethyl oleate, propane diols two-(octanoic acid Ester/certain herbaceous plants with big flowers acid esters), glycerine three-(caprylate/certain herbaceous plants with big flowers acid esters) or Rikemal PO 200), the ester of branched chain fatty acid or alcohol it is (especially different Stearate);For nonionic surfactant, (ester, the sorb of especially polyoxyethylated fatty acid (such as oleic acid) gather emulsifying agent The ester of sugar, the ester (such as anhydrous mannitol oleate) of mannide, the ester of aliphatic dihydroxy alcohol, the ester of glycerine, polyglycereol Ester, the ester of the ester of propane diols and oleic acid, the ester of isostearic acid, the ester of the ester of castor oil acid or hydroxy stearic acid, above-mentioned ester can Through ethoxylation, the ether of fatty alcohol and polyalcohol (such as oleyl alcohol), Pluronic L121 (especiallyParticularly L121));
Preferably, the polymer of acrylic or methacrylic acid is crosslinked acrylic or methacrylic acid polymer, especially With sugared poly alkenyl ether or the crosslinked compound carbomer of polyalcohols, preferably carbopol 974P, 934P and 971P;
Preferably, the copolymer of maleic anhydride and alkenyl derivative is maleic anhydride and the copolymer EMA of ethene;
Preferably, the adjuvant is GEL A adjuvants;
The concentration range of the adjuvant is the preferably 5%V/V from 5% to 50%V/V.
7. vaccine combination according to claim 3, wherein, the vaccine combination further comprises other antigens, The antigen includes the group being made of the one or more in following further antigens:Canine distemper virus antigen, canine adenovirus Ⅰ, II type Antigen, leptospira canicola antigen, canine coronavirus antigen, Canine Parvovirus antigen, Rabies Virus Antigen, canine influenza virus Antigen, dog reovirus antigen, dog Pseudorabies virus antigen, dog wheel virus antigen, canine alphaherpesvirus antigen, canine viral breast Head tumor virus antigen, dog parvovirus antigen, dog Mumps virus antigens, dog lymphocytic choriomeningitis virus resist It is former;Preferably, the antigen includes the group being made of the one or more in following further antigens:Canine Parvovirus antigen, dog adenopathy Malicious antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, Rabies Virus Antigen, dog influenza Viral antigen.
8. vaccine combination according to claim 7, wherein, the vaccine combination includes content for before inactivation 104.0- 107.0TCID50CPIVHeN0718 plants of inactivation antigens of canine parainfluenza virus strain of/ml, content are before inactivation 104.0- 106.0TCID50C/HN/001 plants of inactivation antigens of canine distemper virus CDV and/or content of/ml are before inactivation 106.0- 108.0TCID50S0425 plants of inactivation antigens of canine parvovirus CPV of/ml, and 5%V/VGEL A adjuvants.
9. according to vaccine combination according to claim 8, wherein, before the vaccine combination includes content for inactivation 106.0TCID50HeN0718 plants of inactivation antigens of canine parainfluenza virus strain CPIV of/ml, content are before inactivation 105.0TCID50/ C/HN/001 plants of inactivation antigens of canine distemper virus CDV and/or content of ml are before inactivation 107.0TCID50The canine parvovirus of/ml S0425 plants of inactivation antigens of CPV, and 5%V/VGEL A adjuvants.
10. prevention and treatment canine parainfluenza virus infection is being prepared according to claim 3~9 any one of them vaccine combination Application in the medicine of relevant disease.
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