CN107537033A

CN107537033A - Vaccine combination, kit and its application

Info

Publication number: CN107537033A
Application number: CN201610492950.XA
Authority: CN
Inventors: 田克恭; 吴洪超; 孙进忠; 张许科
Original assignee: Pulaike Biological Engineering Co Ltd
Current assignee: Pulaike Biological Engineering Co Ltd
Priority date: 2016-06-23
Filing date: 2016-06-23
Publication date: 2018-01-05
Anticipated expiration: 2036-06-23
Also published as: CN107537033B

Abstract

The present invention relates to a kind of Canine Parvovirus antigen containing immune amount and the vaccine combination of other antigens of immune amount, the kit of other antigens of Canine Parvovirus antigen and effective dose containing effective dose is further related to, prepared vaccine combination and kit can be effectively used to the medicine for preparing prevention and/or treatment Canine parvovirus infection relevant disease.The vaccine combination and kit of the present invention can effectively prevent existing popular canine parvovirus street strain, and also create synergy with the incomplete antigen in other antigens, further improve the immune effect of connection seedling.

Description

Vaccine combination, kit and its application

Technical field

The invention belongs to veterinary biologics field, and in particular to a kind of epidemic disease containing Canine Parvovirus antigen and other antigens Seedling composition, kit and its preparation method and application.

Background technology

The disease incidence that canine parvovirus (Canine Parvovirus, CPV) is triggered is suddenly, the course of disease is short, the death rate is high, Infectiousness is strong, can infect various ages, sex, the dog of kind, fox, mink, recoon dog and wolf, special easy infection young animal, and 8 Animal death rate of the onset in week old is up to 70%, and the mortality of animals in 1 years old is also up to 30%.

CDV (Canine distemper virus, CDV) be existing a kind of high incidence in world wide, High mortality viral disease, along with the evolution of the change of ecological environment and animal and virus, its host is by traditional dog Section (including dog, fox, recoon dog etc.), Procyonidae and Mustelidae (mink etc.) animal expand to all 8 sections of Carnivora and Artiodactyla The many animals such as Suidae, Primates Macaca and clasper mesh Phocidae, and the trend constantly expanded is presented.

Hepatitis infectiosa canis virus (Canine Adenovirus, CAV) is pathogenic most strong one kind disease in mastadenovirus Poison, there is 2 serotypes, and wherein I types adenovirus (CAV-I) causes canine infectious hepatitis (ICH) and epizootic fox encephalitis (FE)；II type glands Viral (CAV-II) then causes the infectious laryngotracheitis and enteritis of dog.

The main host of hydrophobin (Rabies Virus, RV) is the animals such as dog, cat, is infectious diseases common to human beings and animals, Most human rabies are propagated with the band poison dog of mankind's close contact or cat, and its harm has caused government and the height of the people Degree concern.

The respiratory tract of canine parainfluenza virus (Canine Parainfluenza Virus, CPIV) main infection dog, it is clinical Performance heating, runny nose and cough, pathological change is characterized by coryza and bronchitis.

Canine coronavirus (Canine Coronavirus, CCV) is in acute gastroenteritis syndrome, causes anorexia, abdomen Rush down, vomit, be that canine farming endangers one of larger disease with fox rearing industry.

Leptospira canicola (Leptospira canicola, Lep) has more than ten of serotype, is responsible for falling ill, and be Zoonosis；It is domestic often to cause acute hemorrhagic yellow with icterohemorrhagic form and the pathogenicity highest of dog type, wherein icterohemorrhagic form Subcutaneous ulcer syndrome, dog type is based on ephritis symptom.

CPV strains involved by the tiny vaccine of dog of commercialization at present are the CPV-2 types occurred early stage, and are flowed at present Capable CPV strains differ greatly with it on amino acid levels, and especially these commercialization import vaccines are from being introduced the country Canine parvovirus is individually in clinical case or mixed infection is not significantly inhibited or reduced, still to pet dog, canine farming High risks and serious loss are caused with cultivation industries such as fur economic animals.

Clinically individually infection accounts for 30% or so to CPV, mixed with other viral (including CDV, CCV, CAV, RV, CPIV) Infection up to more than 50% is closed, especially low 70% of the case cure rate of mixed infection than simple infection.

Therefore, need badly to provide and contain Canine Parvovirus antigen and various other anti-for currently a popular CPV street strains Former vaccine combination, to meet needs clinically.

The content of the invention

In order to solve the deficiencies in the prior art, the invention provides a kind of Canine Parvovirus antigen containing immune amount and other The vaccine combination of antigen, the vaccine combination can prevent and/or treat the infection of canine parvovirus etc., and it is for stream at present Capable CPV street strains, can effectively prevent Canine parvovirus infection.

The present invention relates to a kind of vaccine combination, the vaccine combination includes the Canine Parvovirus antigen of immune amount, exempted from Other antigens and veterinarily acceptable carrier of epidemic disease amount；Wherein, the Canine Parvovirus antigen is canine parvovirus S0425 Strain antigen, canine parvovirus S0425 strains preserving number is CCTCC NO.V201634.

The invention further relates to the preparation method of the vaccine combination.

The invention further relates to the vaccine combination to prepare prevention and/or treat Canine parvovirus infection relevant disease Application in medicine.

The invention further relates to a kind of kit, the Canine Parvovirus antigen of the kit including effective dose, effective dose Other antigens and veterinarily acceptable carrier, the Canine Parvovirus antigen are canine parvovirus S0425 strain antigens, dog Parvovirus S0425 strains preserving number is CCTCC NO.V201634；Wherein, in the kit Canine Parvovirus antigen and other Antigen is placed or together placed respectively.

The invention further relates to the preparation method of the kit.

The invention further relates to the kit to prepare the medicine of prevention and/or treatment Canine parvovirus infection relevant disease In application.

Dog tiny independent or mixed infection of the vaccine combination, kit of the present invention to current popular has good prevention And control effect, it can be achieved with protecting completely, the live seedling of its immune effect more commercially is quite even more merely through primary immune response It is excellent, and duration of immunity is grown, the primary immune response duration is more than 18 months.

Embodiment

Hereinafter, embodiments of the present invention are illustrated.

Term " vaccine combination " is also known as immunogenic composition, refers to a kind of preparation containing immunogene, including such as albumen Matter, peptide, full cell, the virus of inactivation, subunit or attenuation work or bacterium, or polysaccharide, or its combination, it, which is administered, comes Costimulatory receptor is reacted the humoral and cellular immune response for being present in one or more antigens in immunogenic composition.Immunity inoculation is Stimulate using vaccine combination and in host immune or immunogenic response the process to antigen.Preferable host is lactation Animal such as dog.

Term " immune amount " should be understood to " immune effective dose " that 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, and the amount is enough to prevent or improved 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, the incidence of disease, Temperature numerical, 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 parvovirus " refers to " canine parvovirus " (Canine Parvovirus, CPV) category Parvoviridae, carefully Small virus category, the clinical symptoms triggered include enteritis symptom and myocarditis symptom, and the common symptoms of enteritis type are vomitted for serious Tell and serious hemorrhagic diarrhea.Myocarditis type can cause pup breathing and cardiovascular exhaustion.

Term " Canine Parvovirus antigen " refers at least contain a kind of any combinations thing of Canine Parvovirus antigen form, institute The immune response that Canine Parvovirus antigen can induce, stimulate or can resist Canine parvovirus infection is stated, the antigen forms include But inactivation, attenuation work or subunit antigen is not limited to, the subunit antigen is to include VP2 albumen or its domain Antigen.Attenuation and ablation method are well known to those skilled in the art.Method of attenuating includes but is not limited in cell culture In continuous passage (virus and some bacteriums), the continuous passage (bacterium), ultraviolet in broth culture in the cell line of verification Line irradiation (virus and bacterium) and mutagenesis (virus and bacterium).The ablation method of virus or bacterium includes but is not limited to use good fortune That Malin, beta-propiolactone (betapropriolactone, BPL) or binary ethylenimine (Binary ethylenimine, BEI) Processing or other method well known by persons skilled in the art.

Term " CDV antigen " at least containing a kind of CDV (Canine Distemper Virus, CDV) any combinations thing of antigen forms, the CDV antigen can induce, stimulate or can resist CDV infection Immune response, the antigen forms include but is not limited to inactivating, living or subunit antigen.The CDV resists Original includes the CDV R-20/8 strains of the antigen living such as canine penton live vaccine of Wuxing Animal Health Pharmaceutical Plant, Jilin Prov.'s production, and Shandong is moved 11 plants of the CDV of the canine distemper live vaccine of thing health products Co., Ltd production, the dog of Yang Ling Lv Fang bioengineering Co., Ltd production The CDV XN112 strains of 5-linked live vaccine, the CDV Snyder Hill strains of the multi-joint seedling of dog of Pfizer's production, Intervet company The CDV Onderstepoort strains of the dog quadruple vaccine of production, the CDV BA5-Vero of the multi-joint seedling of dog of Cimmeria company production 150 plants, the CDV Lederle strains of the multi-joint seedling of dog of Hai Bolai companies production, CDV YBR strains are (referring to Chinese patent CN101914503A), CDV CC12-30 strains (referring to Chinese patent CN102839158A)；The antigen of inactivation such as Chinese patent CDV YB strains disclosed in CN101914503A, CDV NJ01 strains are (referring to Bi Zhenwei etc., atypia CDV nucleocapsid protein Gene sequencing and its expression China Preventive Veterinary Medicine report in Escherichia coli, 2011,33 (8):625-629), CDV TM-CC strains (Li Weike wild-type canine distemper virus strain TM-CC reverse genetics systems and expression canine parvovirus VP2 protein Protein reconstitution dog The structure Northeast Forestry University thesis for the doctorate of the hot low virulent strain of pest, 2014), the CDV LIU strains (such as He Hongbin, Li Jinzhong, summer salty post Sequence analysis China animal doctor's journal of CDV street strain H protein gene, 1999,19 (4):404-407), CDV 93039 strain virus liquid (the elegant tinkling of pieces of jade of reference chance, Tian Kegong dog experimental infections CDV pathological research China livestock and poultry infectious disease, 1998,20(2):76-78)；The antigen of subunit is such as the H protein of antigenic protein containing CDV, F protein, M albumen or its domain Antigen；And the mixing of these antigen forms components.

Term " hepatitis infectiosa canis virus II types antigen " refer at least containing a kind of hepatitis infectiosa canis virus (Canine Adenovirus-II, CAV-II) any combinations thing of antigen forms, the hepatitis infectiosa canis virus II types antigen can induce, stimulates or can resist hepatitis infectiosa canis virus sense The immune response of dye, the antigen forms include but is not limited to antigen inactivating, living or subunit.The hepatitis infectiosa canis virus II Type antigen includes the CAV-II YCA18 strains of the antigen living such as canine penton live vaccine of Wuxing Animal Health Pharmaceutical Plant, Jilin Prov.'s production, The CAV-II XN3 strains of the canine penton live vaccine of Yang Ling Lv Fang bioengineering Co., Ltd production, the dog of Pfizer's production are multi-joint The CAV-II NL-CPI strains of seedling, the CAV-II Manhattan LPV3 strains of the dog quadruple vaccine of Intervet company production, Mei Li The CAV-II of the multi-joint seedling of dog of CAV-II CGF2004/75 strains, the production of Hai Bolai companies of the multi-joint seedling of dog of sub- company's production Manhattan strains, CAV YCA18 strains (referring to Chinese patent CN1876181A)；The antigen of inactivation such as Ditchfield etc. (Ditchfield et al, Can.Vet.J., 3:238-247,1962) described, the antigen such as the egg of antigenicity containing CAV of subunit The antigen of white E3 albumen or its domain；And the mixing of these antigen forms components.

Term " rabies virus antigen " refers to appointing at least containing a kind of rabies viruses (Rabies Virus, RV) antigen forms What composition, the rabies virus antigen is inducible, stimulates or can resist the immune response of rabies virus infection, the antigen shape Formula includes but is not limited to antigen inactivating, living or subunit.It is great that the rabies virus antigen includes antigen such as universe member living The RV Flury strains of the mad dog live vaccine of biotech firm's production, RV Rb/E3 strains (referring to Chinese patent CN1876181A)；Inactivation Antigen for example Jilin decent job company production rabies inactivated vaccine RV Flury LEP strains, Shanghai Hai Li companies production it is mad The RV SAD strains of dog disease inactivated vaccine, the RV for the rabies inactivated vaccine that Academy of Military Medicine, PLA develops The RV CTN-1 strains of the rabies inactivated vaccine of production such as CVS-11 strains, Tangshan Yi An bio-engineering corporations, the life of Intervet company The RV HCP-SAD of the rabies inactivated vaccine of RV Pasteur RIV strains, the Pfizer's production of the rabies inactivated vaccine of production The RV of the rabies inactivated vaccine of RV G52 strains, Virbac's production of the rabies inactivated vaccine that strain, Cimmeria company produce VP12 strains；The antigen of the subunit such as antigen of the G-protein of antigenic protein containing RV or its domain；And these antigen forms components Mixing.

Term " canine parainfluenza virus antigen " refers at least contain a kind of canine parainfluenza virus (Canine Parainfluenza virus, CPIV) antigen forms any combinations thing, the canine parainfluenza virus antigen can induce, stimulate Or the immune response of canine parainfluenza virus infection can be resisted, the antigen forms include but is not limited to inactivating, living or sub- single The antigen of position.The canine parainfluenza virus antigen include antigen such as CY-c strains living (preserving number is CGMCC No.5268, referring to Chinese patent CN102343088A), A-20/8 strains (referring to Chinese patent CN1876181A), XN-4 strains (Jinchang moral, Li Liujin, The separation of the canine parainfluenza virus low virulent strains such as Li Qin and identification Journal of the Fourth Military Medical University, 2000,21 (6):722-724), Solvary strains (the separation identification China livestock and poultry infectious disease of the dog parainfluenza prevalence strains such as Jinchang moral, Li Liujin, Lou Qinglin, 1997(1):24-27), withKC-Plus present work attenuated canine parainfluenza virus D008 strains, with Canixin The work attenuated canine parainfluenza virus Manhattan strains of DHPPi/L presentations, the work attenuated canine sidestream presented with Duramune DAPPi Influenza Virus FDL strains, with Vanguard 7 present work attenuated canine parainfluenza virus N L-CPI-5 strains, withAnd/orThe work attenuated canine parainfluenza virus Cornell strains that KC is presented, with Wuxing Animal Health Pharmaceutical Plant, Jilin Prov.'s production The canine parainfluenza virus A-20/8 strains of canine penton live vaccine, and/or come from Naramune^TM、 Univac 2、Univac 5、Kennel-Jec^TM、The canine parainfluenza virus strain of Plus 5 attenuation living；Such as canine parainfluenza virus D008 strains of the antigen of inactivation (U.S. ATCC is preserved, preserving number VR-399), XN931 strains (Jinchang moral, Li Liujin, apply the such as new formal plan canine parainfluenza viruses prevalence The separation of strain and identification Journal of the Fourth Military Medical University, 2000,21 (6):719-721), XN934 strains (Jinchang moral, Li Liujin, The separation of the canine parainfluenza virus low virulent strains such as Li Qin and identification Journal of the Fourth Military Medical University, 2000,21 (6):722-724), 78238 plants of (Harbin Veterinary Medicine Inst., China Academy of Agriculture chief editor veterinary microbiology Beijing:Chinese agriculture publishing house, 1998:414-415), yz0506 strains (separation of Cen Hao canine parainfluenza viruses and the preparation Yangzhou University masters of monoclonal antibody Academic dissertation, 2007)；Subunit antigen such as the F of immunogenic protein containing CPIV (fusion protein, i.e. fusion protein), HN (hemagglutinin-neuraminidase, i.e. hemagglutinin neuraminidase), M (matrix protein, i.e. matrix egg In vain), NP (nucleocapsid protein, i.e. nucleocapsid protein), P (phosphopritein, i.e. phosphoprotein) and/or L The antigen of (Large protein, i.e. large protein) albumen or its domain；And the mixing of these antigen forms components.

Term " canine coronavirus antigen " refer at least containing a kind of canine coronavirus (Canine Coronavirus, CCV) any combinations thing of antigen forms, the canine coronavirus antigen can induce, stimulates or can resist canine Coronavirus Infection Immune response, the antigen forms include but is not limited to inactivating, living or subunit antigen.The canine coronavirus resists Original includes antigen such as CCV YS1/V60 strains living (referring to Chinese patent CN1876181A)；The antigen of inactivation such as Pfizer gives birth to The CCV NL-18 strains of the multi-joint seedling of dog of production；The subunit antigen such as antigen of the S protein of immunogenic protein containing CCV or its domain； And the mixing of these antigen forms components.

Term " leptospira canicola antigen " refers at least contain a kind of leptospira canicola (Leptospira Canicola, Lep) antigen forms any combinations thing, the leptospira canicola antigen can induce, stimulates or can resist dog hook The immune response of Leptospiral infection, the antigen forms include but is not limited to antigen inactivating, living or subunit.It is described Leptospira canicola antigen includes antigen such as outer membrane protein (Outer membrane protein, OMPs) living, and inactivation resists Lep dog type C-51 strains, the icteric NADL11403 strains of the former multi-joint seedling of dog produced such as Pfizer, the production of Intervet company Lep dog type CA-12-000 strains, the hook end type 820K strains of the multi-joint seedling of dog；Subunit antigen such as immunogenic protein containing Lep, outer membrane The antigen of albumen (OMPs) or its domain；And the mixing of these antigen forms components.

Term " dog " is commonly known as dog person, but also includes other members such as wolf, recoon dog, the fox of Canidae family.

Term " TCID₅₀" refer to tissue culture infection dose, and it has been defined as the given batch of infection 50% Viral dilution needed for inoculating cell culture.Various methods can be used for calculating TCID₅₀, including Sperman-Karber methods are (such as BW Mahy,HO Kangro.Virology methods manual,1996:25-46), Reed-Muench methods.In this hair In bright when antigenic content is inactivation antigen content, the antigenic content before inactivation is represented.

Term " CFU " is CFU (Colony Forming Units) abbreviation, and it is represented：Micro- life can be produced Microorganism, microbial spores/spore (spore), the gemma of individual developmental of microorganism or the number of vegetative cell of thing bacterium colony. In the present invention when antigenic content is inactivation antigen content, the antigenic content before inactivation is represented.

Term " veterinarily acceptable carrier " refer to vaccine combination species of the present invention except CDV antigen it Other outer all the components, body is not stimulated not hinder carrier or the dilution of biological activity and characteristic using compound Agent, 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 is as can be used Powell M and Newman M Write《Vaccine design,the Subunit and adiuvant approach》(Plenum Press, 1995) The SPT emulsions and the MF59 emulsions described of page 183 of the description of page 147；W/O/W emulsion；Acrylic or methacrylic acid Polymer；The compound that the copolymer of maleic anhydride and alkenyl (alkenyl) derivative is selected.

It is oily (European Pharmacopea types) that term " emulsion " can be particularly based on light liquid paraffin；Because of olefin oligomerisation Caused isoprenoid is oily (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 Its isostearate.Oil is with emulsifier combination use to form emulsion.Emulsifying agent preferred nonionic surfactants, especially mountain The ester of pears glycan, the ester (such as anhydrous mannitol oleate) of mannide (mannide), aliphatic dihydroxy alcohol (glycol) Ester, the ester of polyglycereol (polyglycerol), the ester of propane diols and the ester of oleic acid, the ester of isostearic acid, the ester of castor oil acid Or the ester of hydroxy stearic acid, their optional ethoxylations, also Pluronic L121, especially Pluronic products, particularly 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.《Vaccine》(1997,15:564-570).

Term " polymer of acrylic or methacrylic acid " is preferably the acrylic or methacrylic acid polymer of crosslinking, Poly alkenyl ether or polyalcohols especially with sugared (sugar) are crosslinked, these compounds be known as carbomer (Carbomer, Trade name Carbopol) (Phameuropa, 1996,8 (2)).Those skilled in the art are referring also to United States Patent (USP) US2909462, which depict this kind of acrylate copolymer, and it has extremely with gathering hydroxylated compound crosslink, the compound Few 3 hydroxyls, preferably more than 8, the hydrogen atom of wherein at least 3 hydroxyls is by the unsaturated lipid with least two carbon atom Alkyl (aliphatic radical) substitutes.Preferable group is the group that those contain 2-4 carbon atom, such as vinyl, Pi-allyl and other ethylenically unsaturated groups (ethylenically unsaturated group).The unsaturated group is certainly Body can include other substituents, such as methyl.These products are sold with the name of carbopol, and (BF Goodrich, Ohio, USA) is special It is unsuitable.They are crosslinked with allyl sucrose or with Allyl pentaerythritol (allyl pentaerythritol).Among these may be used Refer to carbopol 974P, 934P and 971P, most preferably with carbopol 971P.

Term " copolymer of maleic anhydride and alkenyl derivative " also contemplates for maleic anhydride and ethene Copolymer EMA (Monsanto), these polymer dissolve in water produces acid solution, neutralized, is preferably neutralized to physiological pH, To produce assist agent solution, immunogenicity, immunogenicity or vaccinal composition can be mixed thereto in itself.Term " adjuvant " is also Include, but 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 Lipid-amine adjuvant, E.coli LT (restructuring is other), cholera toxin, IMS 1314, muramyl dipeptide, Gel Adjuvant etc..Preferably, the adjuvant include aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion, The polymer of acrylic or methacrylic acid, the copolymer of maleic anhydride and alkenyl (alkenyl) derivative, RIBI assistants Agent system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli are thermo-labile One or more in enterotoxin, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvants.

The present invention relates to a kind of vaccine combination, the vaccine combination includes the Canine Parvovirus antigen of immune amount, exempted from Other antigens and/or veterinarily acceptable carrier of epidemic disease amount；Wherein, the Canine Parvovirus antigen is canine parvovirus S0425 strain antigens, canine parvovirus S0425 strains preserving number are CCTCC NO.V201634.

The canine parvovirus S0425 strains (Canine Parvovirus, Strain S0425) are preserved in Chinese Typical Representative training Thing collection is supported, preserving number is CCTCC NO.V201634, and preservation address is Wuhan, China Wuhan University, and preservation date is On June 14th, 2016.

As one embodiment of the present invention, canine parvovirus S0425 strain antigens described in the vaccine combination is The totivirus antigen of inactivation, the content of the totivirus antigen of the inactivation is before inactivation 10⁵-10⁹TCID₅₀/ml。

As a kind of preferred embodiment of the present invention, the canine parvovirus S0425 strains antigen is the totivirus of inactivation Antigen, the content of the totivirus antigen of the inactivation is before inactivation 10⁶-10⁸TCID₅₀/ml。

As a kind of preferred embodiment of the present invention, the canine parvovirus S0425 strains antigen is the totivirus of inactivation Antigen, the content of the totivirus antigen of the inactivation is before inactivation 10⁷TCID₅₀/ml。

As one embodiment of the present invention, other described antigens include CDV antigen, hepatitis infectiosa canis virus I types resist Original, hepatitis infectiosa canis virus II types antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, rabies disease Malicious antigen, canine influenza virus antigen, dog reovirus antigen, dog Pseudorabies virus antigen, dog wheel virus antigen, canine herpesvirus disease Malicious antigen, canine viral papilloma virus antigens, dog parvovirus antigen, dog Mumps virus antigens, dog lymphatic arteries and veins One or more in network clump meningitis virus antigen, bordetella branchiseptica antigen.

As a kind of preferred embodiment of the present invention, other described antigens include CDV antigen, the coronal disease of dog It is malicious antigen, canine parainfluenza virus antigen, hepatitis infectiosa canis virus I types antigen, hepatitis infectiosa canis virus II types antigen, leptospira canicola antigen, mad One or more in dog disease viral antigen.

As one embodiment of the present invention, the veterinarily acceptable carrier includes adjuvant, the adjuvant bag Include：(1) aluminium glue adjuvant, saponin(e, Avridine, DDA；(2) water-in-oil emulsion, oil in water emulsion, W/O/W emulsion；Or (3) copolymer of the polymer of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative；And RIBI adjuvants System, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli are intolerant to warmheartedness One or more in 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 Saxol, because caused by olefin oligomerisation isoprenoid oil (such as saualane or squalene oil, particularly alkene, isobutene Or it is oily caused by decene oligomerization), 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 the acrylic or methacrylic acid polymer of crosslinking, especially It is the compound carbomer being crosslinked with the poly alkenyl ether of sugar or polyalcohols, is preferably carbopol 974P, 934P and 971P；

Preferably, the copolymer of maleic anhydride and alkenyl derivative is the copolymer of maleic anhydride and 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, the CDV antigen includes CDVR-20/8 strains antigen, CDV 11 Strain antigen, CDV XN112 strains antigen, CDV Snyder Hill strains antigen, CDV Onderstepoort strain antigens, CDV BA5- 150 plants of antigens of Vero, CDV Lederle strain antigens, CDV YBR strains antigen, CDV CC12-30 strains antigen, CDV YB strains resist Original, 93039 plants of CDV NJ01 strains antigen, CDV TM-CC strains antigen, CDV LIU strains antigen, CDV antigens, H protein, F protein, M The subunit antigen of albumen or its domain, and the mixing of these antigen forms components；

The hepatitis infectiosa canis virus II types antigen includes CAV-II YCA18 strains antigen, CAV-II XN3 strains antigen, CAV-II NL-CPI strains antigen, CAV-II Manhattan LPV3 strain antigens, CAV-II CGF2004/75 strains antigen, CAV-II The subunit antigen of Manhattan strains antigen, CAV YCA18 strain antigens, E3 albumen or its domain, and these antigen forms The mixing of component；

The rabies virus antigen include RV Flury strains antigen, RV Rb/E3 strain antigens, RV Flury LEP strains antigen, RV SAD strains antigen, RV CVS-11 strains antigen, RV CTN-1 strains antigen, RV Pasteur RIV strains antigen, RV HCP-SAD strains The antigen of antigen, RV G52 strains antigen, RV VP12 strain antigens, RV G subunit proteins antigens or its domain, and these are anti- The mixing of original shape formula component；

The canine parainfluenza virus antigen includes CY-c strains antigen, A-20/8 strains antigen, Solvary strains antigen, D008 strains Antigen, Manhattan strains antigen, FDL strains antigen, NL-CPI-5 strains antigen, Cornell strains antigen, A-20/8 strain antigens, Naramune^TM、 Univac 2、Univac 5、Kennel-Jec^TM、Plus 5 canine parainfluenza virus strain antigen；D008 strains Antigen, XN931 strains antigen, XN934 strains antigen, 78238 plants of antigens, yz0506 strains antigen, subunit antigen CPIV F subunits Proteantigen, HN subunit proteins antigen, M subunit proteins antigen, NP subunit proteins antigen, P subunit proteins antigen or The antigen of L subunit proteins antigen or its domain, and the mixing of these antigen forms components；

The canine coronavirus antigen includes CCV YS1/V60 strains antigen, NL-18 strains antigen, CCV S subunit proteins and resisted Former or its domain antigen, and the mixing of these antigen forms components；

The leptospira canicola antigen includes Lep dog type C-51 strains antigen, icteric NADL11403 strains antigen, Lep dogs Type CA-12-000 strains antigen, hook end type 820K strains antigen, Lep subunit proteins antigen, outer membrane protein or its domain it is anti- Original, and the mixing of these antigen forms components.

As one embodiment of the present invention, the CDV antigen is the weak poison of CDV Onderstepoort strains Strain, content 10⁵-10⁶TCID₅₀/ head part；The hepatitis infectiosa canis virus II types antigen is CAV-II YCA18 strain Natural Avirulent Strains, is contained Measure as 10⁵TCID₅₀/ head part.

The vaccine combination of the present invention can be directly applied to intramuscular, nasal cavity or subcutaneous, for the suitable of parenteral administration Method includes intramuscular, collunarium and hypodermic injection.As a kind of preferred embodiment of the present invention, it is administered under the drug percutaneous Medicine.

The invention further relates to the preparation method of the vaccine combination, wherein, the preparation method includes：Step (1) point The Canine Parvovirus antigen, other described antigens are not prepared；And step (2) will be made described in the step (1) in proportion Standby Canine Parvovirus antigen, other antigens are placed, and add veterinarily acceptable carrier.

Term " prevention and/or treatment " refers to suppress the duplication of canine parvovirus, suppression when being related to Canine parvovirus infection The propagation of canine parvovirus processed prevents canine parvovirus from being settled down in its host, and mitigates the disease of Canine parvovirus infection The symptom of disease or illness.If viral loads are reduced, illness mitigates and/or food ration and/or growth increase, then can is recognized Therapeutic effect is reached for the treatment.

The invention further relates to a kind of kit, the Canine Parvovirus antigen of the kit including effective dose, effectively Other antigens and veterinarily acceptable carrier, the Canine Parvovirus antigen of amount are canine parvovirus S0425 strain antigens, Canine parvovirus S0425 strains preserving number is CCTCC NO.V201634, wherein, in the kit Canine Parvovirus antigen and its He places or together placed antigen respectively.

As one embodiment of the present invention, in kit of the invention, the canine parvovirus S0425 strain antigens are The totivirus antigen of inactivation, the content of the totivirus antigen of the inactivation is before inactivation 10⁵-10⁹TCID₅₀/ml。

As one embodiment of the present invention, in kit of the invention, the canine parvovirus S0425 strain antigens are The totivirus antigen of inactivation, the content of the totivirus antigen of the inactivation is before inactivation 10⁶-10⁸TCID₅₀/ml。

As one embodiment of the present invention, in kit of the invention, the content of the totivirus antigen of the inactivation For before inactivation 10⁷TCID₅₀/ml。

As one embodiment of the present invention, in kit of the invention, Canine Parvovirus antigen in the kit When with other antigens being inactivation antigen and/or subunit antigen form, Canine Parvovirus antigen is together placed with other antigens；Institute It is inactivation antigen form to state Canine Parvovirus antigen, when including active antigen form composition in other described antigens, the active antigen Form antigenic component is placed respectively with inactivation antigen form antigenic component.

As one embodiment of the present invention, other antigens described in the kit include CDV antigen, Hepatitis infectiosa canis virus I types antigen, hepatitis infectiosa canis virus II types antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus Antigen, Rabies Virus Antigen, canine influenza virus antigen, dog reovirus antigen, dog Pseudorabies virus antigen, dog rotavirus Antigen, canine alphaherpesvirus antigen, canine viral papilloma virus antigens, dog parvovirus antigen, dog Mumps virus antigens, dog One or more in lymphocytic choriomeningitis virus antigen, bordetella branchiseptica antigen.

As a kind of preferred embodiment of the present invention, other antigens resist including CDV described in the kit Original, canine coronavirus antigen, canine parainfluenza virus antigen, hepatitis infectiosa canis virus I types antigen, hepatitis infectiosa canis virus II types antigen, dog hook end spiral shell Revolve the one or more in body antigen, Rabies Virus Antigen.

As one embodiment of the present invention, the CDV antigen includes CDV R-20/8 strains antigen, CDV 11 plants of antigens, CDV XN112 strains antigen, CDV Snyder Hill strains antigen, CDV Onderstepoort strain antigens, CDV 150 plants of antigens of BA5-Vero, CDV Lederle strain antigens, CDV YBR strains antigen, CDV CC12-30 strains antigen, CDV YB strains 93039 plants of antigen, CDV NJ01 strains antigen, CDV TM-CC strains antigen, CDV LIU strains antigen, CDV antigens, H protein, F eggs In vain, the subunit antigen of M albumen or its domain, and the mixing of these antigen forms components；

As a kind of preferred embodiment of the present invention, in the kit, the CDV antigen is CDV Onderstepoort strain low virulent strains, content 10⁵-10⁶TCID₅₀/ head part；The hepatitis infectiosa canis virus II types antigen is CAV-II YCA18 strain Natural Avirulent Strains, content 10⁵TCID₅₀/ head part.

As one embodiment of the present invention, active antigen part also includes freeze drying protectant in the kit, described Inactivation antigen and/or subunit antigen part also include adjuvant；The freeze drying protectant include skim milk, gelatin, trehalose, Bovine serum albumin(BSA), polyvinylpyrrolidone, sorbierite, sodium glutamate, Vc, sucrose, glycine and/or arginine；

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 the polymer of (3) acrylic or methacrylic acid, maleic anhydride and alkenyl derivative are total to Polymers；And RIBI adjuvant systems, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, One or more in E.coli LT, cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvants；

Preferably, saponin(e is Quil A, QS-21, GPI-0100；

Preferably, the adjuvant is GEL A adjuvants；

The invention further relates to the preparation method of the kit, wherein, the preparation method includes：Step (1) is made respectively The standby Canine Parvovirus antigen, other described antigens；And canine parvovirus of the step (2) in proportion by the preparation resists Former, other antigens are placed, and add veterinarily acceptable carrier.

The invention will now be further described with reference to specific embodiments, and advantages of the present invention and feature 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 embodiment, the present invention is expanded on further.It is of the present invention Experimental method, be conventional method if without specified otherwise；Described biomaterial, if without specified otherwise, can be from business way Footpath obtains.

Separation, identification and the assay of the canine parvovirus of embodiment 1

The separation and identification of 1.1 canine parvovirus

CPV street strains are separated from the animal of clinically Canine parvovirus infection, the clinical symptoms of infection animal are：Detest Food, spirit is depressed, body temperature rise, vomiting, and passage of loose stools, viscous just even catsup sample bloody stool, morbidity later stage, will in dewatering symptom etc. Its standard as selected animal.

The intestinal contents of sick dog is diluted to 10%V/V suspensions with serum-free RPMI-1640 nutrient solutions, with 0.22 μm of filter membrane Filtration sterilization, and filtrate is inoculated with F81 cell monolayers (being purchased from the enzyme-linked detection technique Co., Ltd in Shanghai) in 10%V/V ratios and entered Row virus purification, there is within 24~48 hours the cytopathy of wire drawing, about 96 hour cell lesions reach more than 80%, and poison is received in freeze thawing As 1st generation virus liquid；The poison disease vaccination F81 cells for being obtained 1st generation with malicious method is synchronously connect, inoculation occur after 24 hours The cytopathy of wire drawing, cytopathy reaches more than 80% after being inoculated with 72~96 hours, and freeze thawing receives poison and is used as 2nd generation virus liquid； There is the cytopathy of wire drawing after 24 hours in the poison disease vaccination F81 cells for being obtained 2nd generation with malicious method is synchronously connect, inoculation, Cytopathy reaches more than 80% after being inoculated with 72~96 hours, and freeze thawing receives poison and is used as the 3rd generation virus liquid.3rd generation virus liquid is led to Electron microscopic observation is crossed to carry out morphologic detection, the results showed that：It can be seen that the virion with typical parvovirus feature.

Identification and the agriculture of immunogenicity research China (are separated referring to Kang Hongtao Raccoon dog parvovirus using IIF Industry academy of sciences master thesis, 2012) virus-specific identification is carried out to the generation virus liquid of S0425 strains the 3rd, as a result show：F81 There is specific green fluorescence in the cytopathy position that cell virus inoculation forms wire drawing, shows specific good.

With CPV P21:5’-AGAGACAATCTTGCACCAAT-3’、CPV P22:5’- ATGTTAATATAATTTTCTAGGTGCT-3 ' is primer, according to (Zhang R, Yang S, the Zhang W, et such as Zhang R al.Phylogenetic analysis of the VP2 gene of canine parvoviruses circulating in China.Virus genes,2010,40(3):The VP2 genes of method amplification CPV 397-402), amplification purpose fragment connect Competent escherichia coli cell is converted after connecing carrier T, selects positive colony, extraction plasmid send Invitrogen (Shanghai) trade limited Company is sequenced, and is compared with CPV VP2 gene orders in GenBank, the results showed that：S0425 strains belong to CPV-2b types, with The homology of Pfizer vaccine strains (EU914139) VP2 gene orders is 90%.

Result above further confirms that separation strains are canine parvovirus street strain, is named as CPV S0425 strains, submits and protects Hide.

The measure of 1.2 canine parvovirus poison strain contents

CPV S0425 strains are expanded on F81 cells, by the CPV S0425 strains after amplification according to Reed-Muench methods (referring to Yin Zhen animal virology Beijing:Science Press, 1997) carry out viral level measure, the results showed that：The strain Content is 10^7.0TCID₅₀/ml。

The preparation of the vaccine combination of embodiment 2

The preparation of 2.1CPV inactivated whole virus antigens

The CPV S0425 strains of embodiment 1 are inactivated through final concentration of 0.025%V/V BPL (beta-propiolactone), and pressed According to《Chinese veterinary pharmacopoeia》(the Chinese veterinary drug committee, version three in 2010, Chinese agriculture publishing house, 2010) method is carried out to CPV Steriling test, mycoplasma are examined, exogenous virus is examined, the results showed that：After CPV S0425 strains inactivation, bacterium, mould are not affected by Pollution, is also not affected by the infection of mycoplasma and exogenous virus, pure property is good.

The preparation of 2.2 vaccine combinations

The CPV S0425 strains antigen that is prepared embodiment 2.1 with normal saline solution, other antigens of the prior art with And PET GEL A adjuvants (French SEPPIC companies are purchased from, it is final concentration of 5%) to be prepared according to component and content shown in table 1. Wherein, when the CPV S0425 strains antigen of inactivation, the antigen of work form vaccine combination (including vaccine 1,2,3), by inactivation CPV S0425 strains antigen, PET GelA adjuvants carry out mixed preparing by final concentration shown in table 1 with PBS solution and obtain mixed liquor, and Carrying out character inspection respectively to each mixed liquor, (it is colourless clear liquid to be long placed in rear upper strata, and lower floor is pale precipitation, is in after shaking Homogenous suspension), steriling test is (by existing《Chinese veterinary pharmacopoeia》Test, equal asepsis growth)；And to active antigen progressive Shape is examined and (for the spongy treehole agglomerate of milky, is easy to bottle wall disengaging), steriling test is (by existing《Chinese veterinary pharmacopoeia》Examined Test, equal asepsis growth).It is immunized in use, diluting living resist using the mixed liquor of the CPV S0425 strains antigen containing inactivation, adjuvant Original, make the final concentration of antigen living as shown in table 1.

Component and content contained by vaccine combination of the table 1 containing Canine Parvovirus antigen

Numbering	S0425(TCID₅₀/ head part)	CDV(TCID₅₀/ head part)	CAVⅡ(TCID₅₀/ head part)
				Vaccine 1	10⁷	10⁵	0
Vaccine 2	10⁷	10⁶	0
				Vaccine 3	10⁷	10⁶	10⁵

Note：CDV antigens in vaccine 1,2,3 are weak malicious Onderstepoort strains (referring to Maia O B, Gouveia A M G.Serologic response of maned wolves(Chrysocyon brachyurus)to canine distemper virus and canine parvovirus vaccination[J].Journal of Zoo and Wildlife Medicine,2001,32(1):78-80.)；The antigens of CAV II in vaccine 3 are weak malicious YCA18 strains (referring to the summer The experimental immunization study China veterinary drug magazine of the naturally weak malicious YCA18 strains of the type hepatitis infectiosa canis viruses of the such as salty post, model spring, Hu Rongliang II, 2001,35(2):1-4)。

The application of the vaccine combination of embodiment 3

The safety testing of 3.1 vaccine combinations

Choose CDV antigens, negative antibody, CPV antigen negatives, antibody HI≤1: 4 and CAV antigens, the 2~3 of negative antibody Monthly age health beasle dog 15,3 groups are randomly divided into, 5/group, vaccine 1-3 prepared by embodiment 2, every beasle dog are immunized respectively The vaccine of 10 dosages is injected, is observed 14.As a result：Immunity inoculation beasle dog spirit, diet are normal；Body temperature is without bright Aobvious 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 1-3 safety testing is qualified.

The animal experiment of the Canine Parvovirus antigen of 3.2 vaccine combinations

CPV antigen negatives, antibody HI≤1: 4 2~3 monthly age health beasle dogs 25 are chosen, is randomly divided into 5 groups, 5/ Group, the 1st, 2,3 group of difference immune vaccine 1-3,1 part/only.The 4th group of immune canine parvovirus disease work epidemic disease purchased from Pfizer (Wei Jia -5, as existing vaccine 1, it contains canine parvovirus NL-35-D weakening strains at least 10 to seedling^7.0TCID₅₀/ head part), 1 Part/only.Separately setting the 5th group is used as blank control, is not immunized, isolated rearing.Adopted within 14 days, 21 days after immune preceding and immune Blood, detection HI antibody titers (detection of HI antibody titers according to：Yin Zhen, Liu Jing China animal virology second edition Beijing：Science Publishing house, 1997:204-437).21 days after immune, canine parvovirus CPV- is attacked by oral route respectively to all dogs 825/98 plant (street strain of current trend) is (referring to Siedek EM, Schmidt H, Sture GH et al.Vaccination with canine parvovirus type 2(CPV-2)protects against challenge with virulent CPV-2b and CPV-2c.Berl Munch Tierarztl Wochenschr.2011,124(1-2):58-64) virus liquid 1ml (viral levels 10^3.5TCID₅₀), observe 14.It the results are shown in Table 2.

The animal test results of the Canine Parvovirus antigen of the vaccine combination of table 2

Note：# represents there is 1 dog anorexia, body temperature rise, passage of loose stools in existing vaccine group, has 2 bodies in remaining 4 Temperature slightly rise, 2 appetite slightly decline, * represent each dog of control group occur anorexia, spirit it is depressed, vomiting, diarrhoea, It is viscous just, the classical symptom of the canine parvovirus disease such as bloody stool.

As shown in Table 2：(1) caused CPV HI antibody titer phases after dog are immunized in the antibody titer after being immunized, vaccine 2,3 When, caused antibody titer after immune higher than vaccine 1, and the CPV HI antibody titer phases with existing attenuated live vaccines after immune When even slightly higher, when showing that canine parvovirus inactivation antigen and CDV, canine adenovirus attenuated antigen are used in mixed way, not shadow Ring CPV HI antibody titers；(2) malicious protection is attacked, with the popular velogen strains of CPV attacking poison after vaccine 1-3 is immune, not occur dog thin The classical symptom of minor illness viral disease, and protective rate is 100%, and existing vaccine 1 it is immune after to attack toxic effect fruit poor.

The immune duration experiment of the Canine Parvovirus antigen of 3.3 vaccine combinations

Using 28 ages in days or so healthy susceptible canine (hemagglutination suppress (HI) antibody≤1: 4) 20, be randomly divided into 4 Group, 5/group, wherein 1-3 groups, respectively through 1ml/ vaccine of subcutaneous vaccination 1,2,3, the 4th group is only existing through subcutaneous vaccination 1ml/ Vaccine 1.Raised under the same terms, head exempt from after 7 days, 14 days, 21 days, antibody titer is surveyed in blood sampling, booster immunization one again at the 21st day Secondary, head is taken a blood sample at quarterly intervals after exempting from, using hemagglutination-inhibition test (with reference to Chinese patent CN103387996A) detection antibody Potency；Continuous Observation detects 24 months, the results are shown in Table 3.

The immune duration testing result of the Canine Parvovirus antigen of the vaccine combination of table 3

As shown in Table 3：Inactivated vaccine 1,2,3 distinguish secondary immunity dog after up to anti-CPV antibody average in 18 months all Higher level is maintained, >=1:256, in its potent antibodies maintenance period (HI can be protected completely to injection dog>1∶ 128)；And start within the 7th day after first immunisation to produce specific antibody, the specificity produced for the 14th day compared with high titre resists Body.Show that inactivated vaccine 1,2,3 can make body produce the specific antibody compared with high titre, and energy within the immune rear short time Enough epidemic situations up to prevention canine parvovirus disease in 18 months even longer time.

The animal experiment of the CDV antigen of 3.4 vaccine combinations

CDV antigens, health in the 2~March age beasle dog 25 of negative antibody are chosen, 5 groups is randomly divided into, 5/group, distinguishes Immune vaccine 1-3,1 part/only.4th group is prior art control group, and it is tiny that the canine distemper purchased from Intervet company, dog is immunized (as existing vaccine 2, it contains CDV Onderstepoort strains at least 10 to viral bigeminal live vaccine^5.0TCID₅₀, dog 154 plants at least 10 of parvovirus^7.0TCID₅₀/ head part), 1 part/only.Separately setting the 5th group is used as blank control group, is not immunized, and isolates Raising.Before immune and immune latter 14 days, blood sampling in 21 days is resisted with detecting the i.e. CDV SN of the neutralizing antibody of CDV Body is (referring to Yin Zhen, Liu Jing China animal virology second edition Beijing：Science Press, 1997:204-437).After immune 21 days, To all dogs respectively by collunarium and intraperitoneal injection approach attack the strain virus liquid of CDV CDV 93039 (with reference to meeting the elegant tinkling of pieces of jade, Tian Kegong dog experimental infections CDV pathological research China livestock and poultry infectious disease, 1998,20 (2):76-78), observe 14, knot Fruit is shown in Table 4.

The CDV antigen animal test results of table 4

Note：There are 1 body temperature rise, anorexia in the existing vaccine group of # expressions, just and dead, * expression control groups of having loose bowels There is the classical symptom of the CDV such as body temperature rise, One's spirits are drooping, anorexia, loose stools disease in each dog.

As shown in Table 4：(1) caused CDV SN antibody titer phases after dog are immunized in the antibody titer after being immunized, vaccine 2,3 When caused antibody titer, the antibody titer after being immunized also above existing vaccine 2, shows hundstaupe pyreticosis after immune higher than vaccine 1 Immune effect caused by poison attenuation antigen is better than the immune effect of the existing vaccine 2 of CDV；And make in vaccine 1 of the present invention CDV antigen is identical with the antigen of existing vaccine 2, when content is suitable be vaccine 1 of the present invention caused by CDV SN resist The antibody titer of the more existing vaccine 2 of body potency is higher, illustrates to be acted synergistically in the vaccine combination of the present invention；(2) attack Poison protection, the malicious symptom for not occurring CDV disease, and protective rate are attacked with the popular velogen strains of CDV after vaccine 1-3 is immune It is 100%；And being attacked after existing vaccine immunity with the popular velogen strains of CDV after poison has 3 symptoms for CDV disease occur, Protective rate is 80%, less than vaccine 1-3 protective rate.

The animal experiment of the hepatitis infectiosa canis virus antigen of 3.5 vaccine combinations

Choose CAV antigen negatives, antibody≤1:The healthy age beasle dog 15 at 2 2~3 monthly ages, is randomly divided into 3 groups, and 5 Only/group, 3,1 part of the 1st group of immune vaccine/only.2nd group is used as prior art control group, is immunized purchased from Pfizer Inc. Wei Jia -5 (as existing vaccine 3).Separately setting the 3rd group is used as blank control group, is not immunized, isolated rearing.Before immune and 14 days after immune, take a blood sample within 21 days, detection CAV neutralizing antibodies (i.e. CAV SN antibody tests according to：Yin Zhen, Liu Jing China animal virus Learn second edition Beijing：Science Press, 1997:204-437).Two exempt from 21 days afterwards, distinguish nasal attack dog gland to all dogs The viral type A8301 strains of CAV- I are (referring to the reality of the naturally weak malicious YCA18 strains of the type hepatitis infectiosa canis viruses of the such as Xia Xianzhu, model spring, Hu Rongliang II Test immune Research China veterinary drug magazine, 2001,35 (2):1-4) virus liquid 1ml (viral levels 10^5.5TCID₅₀), observation 14 Day, it the results are shown in Table 5.

The animal test results of the hepatitis infectiosa canis virus antigen of the vaccine combination of table 5

Note：* there are the dogs such as body temperature rise, poor appetite, thirsty drink increase, depressed, bloody stool, " blue eye ", cough in control dog The classical symptom of adenovirus infection.

As shown in Table 5：(1) caused CAV SN antibody titers are slightly above existing vaccine after dog is immunized in vaccine 3, show When the type antigen of hepatitis infectiosa canis virus II is used in mixed way with CDV, Canine Parvovirus antigen, the anti-of the type of hepatitis infectiosa canis virus II is not influenceed Body is horizontal, and the more existing vaccine 3 of CAV SN antibody titers reaches higher level earlier caused by invention vaccine combination；(2) Malicious protection is attacked, attacking poison with the popular velogen strain of hepatitis infectiosa canis virus after vaccine 3 is immune does not occur the sick symptom of hepatitis infectiosa canis virus, and protects Rate is 100%, with after existing vaccine immunity to attack toxic effect fruit suitable.

In summary：Not only without eating after vaccine combination containing Canine Parvovirus antigen prepared by the present invention is immune Be intended to decline, spirit is depressed, vomiting, diarrhoea, it is viscous just, the typical clinical symptoms of the canine parvovirus disease such as bloody stool, and immune effect Fruit is better than existing commercial vaccine, especially, does not have immune interference effect to other contained antigens in vaccine combination.

Described above is only the preferred embodiments of the present invention, not does any formal limitation 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 fall within the scope of technical solution of the present invention It is interior.

Claims

1. a kind of vaccine combination, wherein, Canine Parvovirus antigen of the vaccine combination including immune amount, its that amount is immunized His antigen and veterinarily acceptable carrier；Wherein, the Canine Parvovirus antigen is canine parvovirus S0425 strain antigens, Canine parvovirus S0425 strains preserving number is CCTCC NO.V201634.

2. vaccine combination according to claim 1, wherein, the canine parvovirus S0425 strains antigen is the full disease of inactivation Malicious antigen, the content of the totivirus antigen of the inactivation is before inactivation 10⁵-10⁹TCID₅₀/ ml, 10 before preferably inactivating⁶- 10⁸TCID₅₀/ ml, 10 before more preferably inactivating⁷TCID₅₀/ml。

3. vaccine combination according to claim 1, wherein, other described antigens include CDV antigen, dog adenopathy Malicious I types antigen, hepatitis infectiosa canis virus II types antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, Rabies Virus Antigen, canine influenza virus antigen, dog reovirus antigen, dog Pseudorabies virus antigen, dog wheel virus antigen, Canine alphaherpesvirus antigen, canine viral papilloma virus antigens, dog parvovirus antigen, dog Mumps virus antigens, dog lymph One or more in cellularity choriomeningitis virus antigen, bordetella branchiseptica antigen；Preferably, it is described its His antigen is CDV antigen, canine coronavirus antigen, canine parainfluenza virus antigen, hepatitis infectiosa canis virus I types antigen, dog adenopathy At least one of malicious II types antigen, leptospira canicola antigen, Rabies Virus Antigen.

4. vaccine combination according to claim 3, wherein, the CDV antigen resists including CDV R-20/8 strains Original, 11 plants of antigens of CDV, CDV XN112 strains antigen, CDV Snyder Hill strains antigen, CDV Onderstepoort strains resist Original, 150 plants of antigens of CDV BA5-Vero, CDV Lederle strain antigens, CDV YBR strains antigen, CDV CC12-30 strains antigen, 93039 plants of CDV YB strains antigen, CDV NJ01 strains antigen, CDV TM-CC strains antigen, CDV LIU strains antigen, CDV antigens, H eggs In vain, the subunit antigen of F protein, M albumen or its domain, and the mixing of these antigen forms components；

The hepatitis infectiosa canis virus II types antigen includes CAV-II YCA18 strains antigen, CAV-II XN3 strains antigen, CAV-II NL-CPI Strain antigen, CAV-II Manhattan LPV3 strain antigens, CAV-II CGF2004/75 strains antigen, CAV-II Manhattan strains The subunit antigen of antigen, CAV YCA18 strain antigens, E3 albumen or its domain, and the mixing of these antigen forms components；

The rabies virus antigen includes RV Flury strains antigen, RV Rb/E3 strain antigens, RV Flury LEP strains antigen, RV SAD strains antigen, RV CVS-11 strains antigen, RV CTN-1 strains antigen, RV Pasteur RIV strains antigen, RV HCP-SAD strains resist The antigen of original, RV G52 strains antigen, RV VP12 strain antigens, RV G subunit proteins antigens or its domain, and these antigens The mixing of form component；

The canine parainfluenza virus antigen include CY-c strains antigen, A-20/8 strains antigen, Solvary strains antigen, D008 strains antigen, Manhattan strains antigen, FDL strains antigen, NL-CPI-5 strains antigen, Cornell strains antigen, A-20/8 strain antigens,Naramune^TM、 Univac 2、Univac 5、Kennel-Jec^TM、Plus 5 canine parainfluenza virus strain antigen；D008 strains Antigen, XN931 strains antigen, XN934 strains antigen, 78238 plants of antigens, yz0506 strains antigen, subunit antigen CPIV F subunits Proteantigen, HN subunit proteins antigen, M subunit proteins antigen, NP subunit proteins antigen, P subunit proteins antigen or The antigen of L subunit proteins antigen or its domain, and the mixing of these antigen forms components；

The canine coronavirus antigen include CCV YS1/V60 strains antigen, NL-18 strains antigen, CCV S subunit proteins antigens or The antigen of its domain, and the mixing of these antigen forms components；

The leptospira canicola antigen includes Lep dog type C-51 strains antigen, icteric NADL11403 strains antigen, Lep dog types CA-12-000 strains antigen, hook end type 820K strains antigen, Lep subunit proteins antigen, the antigen of outer membrane protein or its domain, And the mixing of these antigen forms components.

5. vaccine combination according to claim 1, wherein, the CDV antigen is CDV Onderstepoort strain low virulent strains, content 10⁵-10⁶TCID₅₀/ head part；The hepatitis infectiosa canis virus II types antigen is CAV-II YCA18 strain Natural Avirulent Strains, content 10⁵TCID₅₀/ head part.

6. vaccine combination according to claim 1, the veterinarily acceptable carrier includes adjuvant, the adjuvant Including：(1) aluminium glue adjuvant, saponin(e, Avridine, DDA；(2) water-in-oil emulsion, oil in water emulsion, W/O/W emulsion；Or (3) copolymer of the polymer of acrylic or methacrylic acid, maleic anhydride and alkenyl derivative；And RIBI adjuvants System, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli are intolerant to warmheartedness One or more in 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, because caused by olefin oligomerisation isoprenoid oil (such as saualane or squalene oil, particularly alkene, isobutene or the last of the ten Heavenly stems It is oily caused by alkene oligomerization), ester (more specifically vegetable oil, ethyl oleate, propane diols two-(octanoic acid containing linear alkyl of acid or alcohol 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)；Emulsifying agent is that (ester, the sorb of especially polyoxyethylated fatty acid (such as oleic acid) gather nonionic surfactant 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 the acrylic or methacrylic acid polymer of crosslinking, especially With sugar poly alkenyl ether or polyalcohols crosslinking compound carbomer, be preferably carbopol 974P, 934P and 971P；

Preferably, the copolymer of maleic anhydride and alkenyl derivative is the copolymer EMA of maleic anhydride and ethene；

Preferably, the adjuvant is GEL A adjuvants；

7. a kind of kit, wherein, the kit include the Canine Parvovirus antigen of effective dose, effective dose other antigens and Veterinarily acceptable carrier, the Canine Parvovirus antigen are canine parvovirus S0425 strain antigens, canine parvovirus S0425 strains preserving number is CCTCC NO.V201634；Wherein, Canine Parvovirus antigen is distinguished with other antigens in the kit Place or together place；

Preferably, the canine parvovirus S0425 strains antigen is the totivirus antigen of inactivation, the totivirus antigen of the inactivation Content is before inactivation 10⁵-10⁹TCID₅₀/ ml, 10 before preferably inactivating⁶-10⁸TCID₅₀/ ml, before more preferably inactivating 10⁷TCID₅₀/ml；

Preferably, Canine Parvovirus antigen and other antigens are inactivation antigen and/or subunit antigen form in the kit When, Canine Parvovirus antigen is together placed with other antigens；The Canine Parvovirus antigen is inactivation antigen form, it is described other When active antigen form composition is included in antigen, the active antigen form antigenic component is put respectively with inactivation antigen form antigenic component Put.

8. kit according to claim 7, wherein, other described antigens include CDV antigen, hepatitis infectiosa canis virus I types Antigen, hepatitis infectiosa canis virus II types antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, rabies Viral antigen, canine influenza virus antigen, dog reovirus antigen, dog Pseudorabies virus antigen, dog wheel virus antigen, canine herpesvirus Viral antigen, canine viral papilloma virus antigens, dog parvovirus antigen, dog Mumps virus antigens, dog lymphatic One or more in choriomeningitis virus antigen, bordetella branchiseptica antigen；Preferably, other described antigens For CDV antigen, canine coronavirus antigen, canine parainfluenza virus antigen, hepatitis infectiosa canis virus I types antigen, hepatitis infectiosa canis virus II types One or more in antigen, leptospira canicola antigen, Rabies Virus Antigen；

Preferably, the CDV antigen resists including CDV R-20/8 strains antigen, 11 plants of antigens of CDV, CDV XN112 strains Original, CDV Snyder Hill strains antigen, CDV Onderstepoort strain antigens, CDV BA5-Vero 150 plants of antigens, CDV Lederle strain antigens, CDV YBR strains antigen, CDV CC12-30 strains antigen, CDV YB strains antigen, CDV NJ01 strains antigen, CDV 93039 plants of TM-CC strains antigen, CDV LIU strains antigen, CDV antigens, H protein, F protein, the subunit of M albumen or its domain Antigen, and the mixing of these antigen forms components；

The leptospira canicola antigen includes Lep dog type C-51 strains antigen, icteric NADL11403 strains antigen, Lep dog types CA-12-000 strains antigen, hook end type 820K strains antigen, Lep subunit proteins antigen, the antigen of outer membrane protein or its domain, And the mixing of these antigen forms components；

Preferably, the CDV antigen is CDV Onderstepoort strain low virulent strains, content 10⁵-10⁶TCID₅₀/ head Part；The hepatitis infectiosa canis virus II types antigen is CAV-II YCA18 strain Natural Avirulent Strains, content 10⁵TCID₅₀/ head part.

9. kit according to claim 7, wherein, active antigen part also includes freeze drying protectant, institute in the kit Stating inactivation antigen and/or subunit antigen part also includes adjuvant；The freeze drying protectant includes skim milk, gelatin, marine alga Sugar, bovine serum albumin(BSA), polyvinylpyrrolidone, sorbierite, sodium glutamate, Vc, sucrose, glycine and/or arginine；

The adjuvant includes：(1) aluminium glue adjuvant, saponin(e, Avridine, DDA；(2) water-in-oil emulsion, oil in water emulsion, Shui Bao Water-in-oil emulsion；Or the copolymerization of the polymer, maleic anhydride and alkenyl derivative of (3) acrylic or methacrylic acid Thing；It is and RIBI adjuvant systems, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, big One or more in enterobacteria heat-labile toxin, cholera toxin, IMS 1314, muramyl dipeptide, Gel adjuvants；

Preferably, saponin(e is Quil A, QS-21, GPI-0100；

Preferably, the adjuvant is GEL A adjuvants；

10. any one of any one of claim 1~6 vaccine combination and claim 7~9 kit is being prepared in advance Application in anti-and/or treatment Canine parvovirus infection relevant disease medicine.