CN107326012A - A kind of cell line and its application - Google Patents
A kind of cell line and its application Download PDFInfo
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- CN107326012A CN107326012A CN201610278368.3A CN201610278368A CN107326012A CN 107326012 A CN107326012 A CN 107326012A CN 201610278368 A CN201610278368 A CN 201610278368A CN 107326012 A CN107326012 A CN 107326012A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C12N2510/00—Genetically modified cells
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18451—Methods of production or purification of viral material
Abstract
The present invention relates to a kind of 1F5 plants of monoclonal cell system Vero/DogSLAM, and C/HN/001 plants of the CDV strain isolated with the monoclonal cell system and its application.The monoclonal cell system of the present invention may express SLAM albumen highly stablely, can efficiently separate the CDV strain in different animals source, to the CDV isolation of strains rate in many animals source up to more than 75%；Vaccine combination prepared by the C/HN/001 strains of the present invention can effectively prevent and/or treat the relevant disease of the wild poison of current canine distemper to be highly resistant to the attack of street strain, suppress spreading for canine distemper epidemic situation.
Description
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to a kind of monoclonal cell system, a kind of hundstaupe pyreticosis
Poison strain and its vaccine combination of preparation, preparation method and application.
Background technology
CDV (Canine distemper virus, CDV) category paramyxovirus section Morbillivirus, is sub-thread
RNA, virion is rounded or not shaping.In recent years, along with ecological environment change and the evolution of animal and virus, CDV
Natural infection host extended by traditional Canidae (including dog, fox, recoon dog etc.), Procyonidae and Mustelidae (mink etc.) animal
To many animals such as all 8 sections of Carnivora and Artiodactyla Suidae, Primates Macaca and clasper mesh Phocidae, and present not
The disconnected trend expanded.
It is a kind of acute, highly contagious disease as caused by CDV that hundstaupe pyreticosis, which is, to generate heat in two-phase heat type, knot
Film is scorching, the upper respiratory tract and lung and intestines and stomach catarrhal inflammation, dermatitis, neuritis and foot pad hardening are characterized, easily it is secondary other
The mixed infections such as bacterium, virus and superinfection.Catch an illness the death rate up to 30%-80% of dog, have a strong impact on dog and it is other easily
The health of infection animal, causes large quantities of dogs dead, and can cause the serious sequelae of infected animal, to pet dog, canine farming and
Fur economic animal aquaculture causes serious economic loss.
At present, the separation method of CDV street strains includes：By the grinding of the virus-positive tissue of early infection canine distemper animal
The supernatant of liquid, the dog lymphocyte of lymphocyte, the peritoneal macrophage of SPF ferrets or mitosis primary stimuli with SPF dogs,
Dog pulmonary alveolar macrophage co-cultures to separate CDV street strains, but these lymphocytes or macrophage Subculture times it is few and
Obtain relatively difficult, the requirement SPF animals having in addition, not only limited source and also add experimental cost and operation require；
Have few thin using inoculated into chick embryo CAM (Embryonated chicken eggs, ECE), MV1Lu, Vero and MDCK
Born of the same parents system is separately cultured CDV street strains, but this method is used for the separation and passage of vaccine strain, and there is the separation of CDV street strains
The defect that rate is low and CDV street strains that are being passed on MV1Lu, Vero, mdck cell gene is easily undergone mutation.
The nineties in last century, Japanese professor Kai is using expressing marmoset SLAM B95a cell separations to many plants of Yanaka etc.
CDV street strains, but B95a cell secreting outside Epstein-Barr virus thus there is bio-safety hidden danger, and separation rate is only 43%；Seki
The Vero cell line (Vero.DogSLAMtag) of F et al. expression dog SLAM acceptors with two construction of eukaryotic expression vector, and profit
With cell line separation CDV street strains, separation rate is only 71% (Kai C, Ochikubof, Tanaka K, et al.Use of
B95a cells for isolation of canine distemper virus from clinical
cases.J.Vet.Med.Sci.,1993,55:1067-1070；Seki F,Onon,Yamaguchi R,et
al.Efficient isolation of wild strains of canine distemper virus in Vero
cells expressing canine SLAM(CD150)and their adaptability to marmoset B95a
cells.J.Virol.,2003,77(18):9943-9950)；The sensitive cell line for the expression dog SLAM acceptors that Liu Yuxiu is built
The expression quantity of SLAM albumen expressed by Vero/DogSLAM is only 46.5%, and the cell line CDV street strains separation with
At most used in passage assays to the 5th generation (structures of Liu Yu shows CDVs sensitive cell line and infection clones with just
Walk application study Jilin University Ph.D. Dissertations, 2014).As can be seen here, quickly, accurately and efficiently divide from infected animal
There is certain difficulty from CDV street strains, seriously govern further investigation of the China to different animals source hundstaupe pyreticosis.
On the other hand, prevent and control both at home and abroad at present the CDV strain genotype being related in vaccine used in canine distemper equal
For the popular North America I types in age in last century 30-40 North America, traditional CDV vaccine strains by reduction, such as Onderstepoort plants,
Hill plants of Snyder, Lederle plants, Convac plants, and Current Domestic Major Epidemic is Asia I type CDV street strains.This
A little traditional CDV vaccine strains have larger gap with domestic nearly 10 years popular CDV street strains in genotype, there is antigen pin
The defect not strong to property, so as to cause domestic canine distemper epidemic situation can not be effectively controlled always.Therefore, need badly and isolate the country
Popular CDV street strains, are prepared into vaccine strain and are spread with preventing and/or treating canine distemper epidemic situation, with stronger
Realistic meaning.
The content of the invention
To solve the deficiencies in the prior art, the invention provides a kind of monoclonal cell system, it can be efficiently from infection dog
CDV strain is separated in animal tissue, tissue sample or its culture of distemper virus, the monoclonal cell system is to more
Planting the CDV of Species origin can efficiently separate, and separation rate is up to more than 75%.
The present invention relates to a kind of Vero/DogSLAM-1F5 plants of monoclonal cell system Vero/DogSLAM cell lines, wherein,
Vero/DogSLAM-1F5 plants of Vero/DogSLAM cell lines (Vero/DogSLAM Cell line, the strain Vero/
DongSLAM-1F5 China typical culture collection center) is preserved in, preserving number is CCTCC NO.C201611, and preservation address is
Wuhan, China Wuhan University, preservation date is on 03 29th, 2016.
Described monoclonal cell system Vero/DogSLAM-1F5 plants separation CDVs are used the invention further relates to one kind
The method of strain, wherein, methods described includes：The animal sources source for infecting CDV is ground or handled, then will
Different lapping liquids or treatment fluid are inoculated in the monoclonal cell system Vero/DogSLAM-1F5 plants of progress virus purification, and passage is steady
Surely occur after syncytium formation, poison is received in freeze thawing.
Using method of the invention, it is possible to efficiently separating CDV strain from many animals tissue and sample.
The invention further relates to according to a kind of isolated CDV strain of methods described, wherein, the canine distemper
Contain F eggs shown in the gene of H protein shown in coding SEQ ID NO.2 and/or coding SEQ ID NO.4 in virus stain genome
White gene.
The CDV strain genotype of the present invention is consistent with the genotype of current trend strain, is prepared using its antigen
Vaccine combination be highly resistant to the infection of existing popular street strain.
The invention further relates to a kind of vaccine combination, wherein, the vaccine combination includes the canine distemper of immune amount
The antigen of virus stain and veterinarily acceptable carrier, the antigen of the CDV strain include the totivirus of inactivation
Antigen and/or at least one subunit antigen.
The invention further relates to the vaccine combination in the medicine for preparing prevention and/or treatment canine distemper relevant disease
Using.
The invention further relates to CDV strain H protein, F protein, the antigen protein has good immunogenicity, system
Animal can be effectively immunized in standby subunit vaccine.
Monoclonal cell system energy highly stable ground high efficient expression SLAM albumen constructed by the present invention, can be efficiently separated
The CDV strain of separate sources；The separated CDV strain of the present invention is the wild poison of popular CDV
Strain, vaccine combination is prepared into it can prevent and/or treat canine distemper epidemic situation to spread.
Brief description of the drawings
Fig. 1 is the empty carrier of Vero/DogSLAM-1F5 plants of indirect immunofluorescene assay monoclonal cell system and Vero cells
The qualification result of blank control, wherein, Figure 1A is Vero/DogSLAM-1F5 plants of expression SLAM of monoclonal cell system Vero/
DogSLAM-1F5 plants of qualification result, Figure 1B is the blank control qualification result of Vero cells after transfection empty carrier.
Embodiment
Hereinafter, embodiments of the present invention are illustrated.
The present invention relates to a kind of Vero/DogSLAM-1F5 plants of monoclonal cell system Vero/DogSLAM cell lines, wherein,
Vero/DogSLAM-1F5 plants of Vero/DogSLAM cell lines (Vero/DogSLAM Cell line, the strain Vero/
DogSLAM-1F5 China typical culture collection center) is preserved in, preserving number is CCTCC NO.C201611, and preservation address is
Wuhan, China Wuhan University, preservation date is on 03 29th, 2016.
The invention further relates to described monoclonal cell system Vero/DogSLAM-1F5 plants of construction method, wherein, the structure
Construction method includes：A, with molecular biology method build recombinant vector pCAGGS-Neo-IgK-HA；B, collection dog periphery anti-freezing
Blood, separates PBLC with dog lymphocyte separation medium and extracts total mRNA, dog SLAM bases are obtained by PCR method
Cause；C, structure plasmid pCAGGS-Neo/SLAM；D, the plasmid pCAGGS-Neo/SLAM of structure transfected to Vero cells, used
The stable expression SLAM of G418 monoclonals screening cell line, obtains described monoclonal cell system Vero/DogSLAM-1F5 plants.
Described monoclonal cell system Vero/DogSLAM-1F5 plants separation CDVs are used the invention further relates to one kind
The method of strain, wherein, methods described includes：The animal sources source for infecting CDV is ground or handled, then will
Different lapping liquids or treatment fluid are inoculated in the monoclonal cell system Vero/DogSLAM-1F5 plants of progress virus purification, and passage is steady
Surely occur after syncytium formation, poison is received in freeze thawing
As one embodiment of the present invention, the animal sources of CDV strain are infected described in methods described to be included
Canidae source, cat family source, Viverridae source, Ursidae source, Phocidae source, panda section source, skunk section source, Procyonidae source, Mustelidae source, monkey section
Source is He Xi Tuan sections source；The animal sources source of the infection CDV strain includes lungs, liver, spleen, intestines, brain, small
Brain, kidney, excrement, urine, eye nose swab or PBLC.
As one embodiment of the present invention, wherein, the animal of CDV strain is infected described in methods described
Source includes but is not limited to Canidae (Canidae) source, cat family (Felidae) source, Viverridae (Vivierridae) source, Ursidae
(Ursidae) source, Phocidae (Phocidae) source, panda section (Ailuridae) source, skunk section (Mephitidae) source, racoon
Section (Procyonidae) source, Mustelidae (Mustelidae) source, monkey section (Cercopithecidae) source are He Xi Tuan sections
(Tayassuidae) source.
As one embodiment of the present invention, wherein, the animal of CDV strain is infected described in methods described
It is thin that source source includes lungs, liver, spleen, intestines, brain, cerebellum, kidney, excrement, urine, eye nose swab or periphery hemolymph
Born of the same parents.
It is stabilization that stable generation syncytium formation is passed on as one embodiment of the present invention, described in methods described
Passed for 3 generations, be specially：It is stable to pass 1st generation, that is, described monoclonal cell system Vero/DogSLAM-1F5 plants are seeded in after upper 24 hours
The cytopathy for the cytopathy of multinucleate giant cell fusion occur or drawing in the net, cytopathy reaches 80% after being inoculated with 60-96 hours,
Freeze thawing receives poison and is used as 1st generation virus liquid；It is stable to pass 2nd generation, i.e., malicious method is connect with monolayer adsorption and connect the virus liquid that 1st generation is obtained
Plant in the monoclonal cell system Vero/DogSLAM-1F5 strains, typical cells fusion lesion occurs in inoculation after 24 hours, connect
There is syncytium formation after kinds 60-96 hour and reach more than 80%, freeze thawing, which is received, malicious is used as 2nd generation virus liquid；It is stable to pass the 3rd
Generation, i.e., poison disease vaccination that malicious method obtained 2nd generation is connect with monolayer adsorption in the monoclonal cell system Vero/DogSLAM-
Cultivated in 1F5 strains, the cytopathy for the cytopathy of multinucleate giant cell fusion occur or drawing in the net.
Term " CDV " (Canine Distemper Virus, CDV) refers to be under the jurisdiction of Paramyxoviridae measles
The sub-thread minus strand non-segmented negative RNA virus of infection Canidae, Mustelidae and the Procyonidae of Tobamovirus etc., the clinical symptoms triggered include
Diphasic fever, fever, expiratory dyspnea, visible dermis are rubescent, eye has suppurate sexual secretion, hind leg of a large amount of secretion, nose to stand
Natural occurrence and it is dead；The necrosis of cut open inspection lungs large-area hemorrhage, spleen enlargement, mesenteric adenophyma are big.
Term " CDV strain (CDV strains) " is if be in the present invention the wild poison of CDV without specified otherwise
Strain (CDV street strains), wild-type canine distemper virus strain (CDV velogen strains), wild type CDV strains, are used interchangeably.
The invention further relates to Vero/DogSLAM-1F5 plants described of monoclonal cell system in the infection of separation different animals source
CDV strain in application.
As one embodiment of the present invention, the different animals source includes Canidae (Canidae) source, cat family
(Felidae) source, Viverridae (Vivierridae) source, Ursidae (Ursidae) source, Phocidae (Phocidae) source, panda section
(Ailuridae) source, skunk section (Mephitidae) source, Procyonidae (Procyonidae) source, Mustelidae (Mustelidae) source,
Monkey section (Cercopithecidae) source is He Xi Tuan sections (Tayassuidae) source.
The invention further relates to according to a kind of isolated CDV strain of methods described, wherein, the canine distemper
Contain F eggs shown in the gene of H protein shown in coding SEQ ID NO.2 and/or coding SEQ ID NO.4 in virus stain genome
White gene.
As one embodiment of the present invention, SEQ ID NO.1 institutes are contained in the CDV strain genome
Show the gene of F protein shown in the gene and SEQ ID NO.3 of H protein.
As one embodiment of the present invention, the CDV strain is C/HN/001 plants of CDV, institute
State C/HN/001 plants of CDV (Canine Distemper Virus, Strain C/HN/001) and be preserved in Chinese Typical Representative
Culture collection, preserving number is CCTCC NO.V201604, and preservation address is Wuhan, China Wuhan University, preservation date
For on March 29th, 2016.
The invention further relates to a kind of vaccine combination, wherein, the vaccine combination includes the canine distemper of immune amount
The antigen of virus stain and veterinarily acceptable carrier, the antigen of the CDV strain include the totivirus of inactivation
Antigen and/or at least one subunit antigen.
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, inactivation, subunit or attenuation, virus living or bacterium, or polysaccharide, or its combination, it is administered
Carry out costimulatory receptor to react the humoral and cellular immune response for being present in one or more antigens in immunogenic composition.Immunity inoculation
It is to apply vaccine combination and stimulate the process to the immune or immunogenic response of antigen in host, host preferably is
Thing 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 that the amount of antigen of immune response can be effectively induced in recipient's body, 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 infects as caused by pathogen caused unfavorable healthy result
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 the protective immunity that should be provided by vaccine can be by measurement
The clinical symptom of such as 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 " CDV antigen " refers at least contain a kind of any combinations thing of CDV antigen forms, institute
Stating CDV antigen can induce, stimulate or can resist the immune response of CDV infection, and the antigen forms include
But be not limited to inactivation, attenuation or subunit antigen.Wherein, the antigen of inactivation can be included at chemistry by various methods
Reason, physical treatment (such as sonication, irradiation, heat) or any other common method for being enough tissue biological's body duplication or growing come real
Now inactivate and maintain its immunogenicity, preferably pathogen is inactivated after collection and optionally receives clarification purification, passes through chemistry
Processing is using such as formalin or formaldehyde, beta-propiolactone, aziridine, binary aziridine BEI, thimerosal；Inactivation
Method 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 " refer to vaccine combination species of the present invention except CDV antigen it
Other outer all the components, do not stimulate body 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；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 uses 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 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, 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 the propylene of crosslinking
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, and preferably more than 8, the hydrogen atom of 3 hydroxyls of wherein at least is by former with least two carbon
Unsaturated lipid alkyl (aliphatic radical) substitution of son.It is preferred that group be 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.Carbopol 974P, 934P and 971P, most preferably with carbopol 971P are can be mentioned that 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, physiological pH are preferably neutralized to, to produce
Assist agent solution, immunogenicity, immunogenicity or vaccinal composition can be mixed thereto in itself.Term " adjuvant " also 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
The polymer of acid or methacrylic acid, the copolymer of maleic anhydride and alkenyl (alkenyl) derivative, RIBI adjuvants system
System, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli are intolerant to warmheartedness poison
One or more in element, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvants.
As one embodiment of the present invention, the vaccine combination includes the CDV C/HN/001 of immune amount
Strain antigen and veterinarily acceptable carrier, the totivirus that described CDV C/HN/001 plants of antigen includes inactivating resist
Former and/or at least one subunit antigen.
As one embodiment of the present invention, the vaccine combination includes the totivirus antigen and adjuvant of inactivation；Its
In, the totivirus antigenic content of the inactivation is inactivation preceding 104-109TCID50/ml；Wherein, the adjuvant include aluminium glue adjuvant,
Saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion, polymer, the maleic two of acrylic or methacrylic acid
The copolymer, RIBI adjuvant systems, Block co-polymer, SAF-M, single phosphorus of acid anhydrides and alkenyl (alkenyl) derivative
Acyl lipid A, Avridine lipids-amine adjuvant, E.coli LT, cholera toxin, IMS 1314, muramyl dipeptide
Or the one or more in Gel adjuvants.
As a kind of preferred embodiment of the present invention, the totivirus antigenic content of the inactivation is inactivation preceding 105-
107TCID50/ml。
As one embodiment of the present invention, the vaccine combination includes H protein and adjuvant, the H protein amino
Acid sequence is the amino acid sequence that SEQ ID No.2 are encoded；The H protein content is 10-200 μ g/ml；Wherein, the adjuvant
Including aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion, acrylic or methacrylic acid it is poly-
Copolymer, RIBI adjuvant systems, the Block co- of compound, maleic anhydride and alkenyl (alkenyl) derivative
Polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, E.coli LT, cholera toxin,
One or more in IMS 1314, muramyl dipeptide or Gel adjuvants.
As a kind of preferred embodiment of the present invention, the vaccine combination also includes F protein and/or M albumen, described
F protein amino acid sequence is the amino acid sequence that SEQ ID No.4 are encoded；The F protein content is 10-160 μ g/ml, the M
Protein content is 10-180 μ g/ml.Term " H protein " refers to the hemagglutinin (Attachment of CDV
Protein or Haemagglutinin protein, H or HA), it is the sugared egg of typical II type on CDV cyst membrane surface
In vain, mediate retroviral and infection cell, infection cell and merging between non-infected cell, make virus have what is spread in host
Ability, induction of antibodies produces neutralizing antibody, is one of main immunogenic albumen of canine parvovirus prevention.
Term " F protein " refers to the fusion protein (Fusion protein, F) of CDV, is CDV capsule
I type glycoprotein on film, assists H protein infection permissive cell, is one of main immunogenic albumen of canine parvovirus prevention.
Term " M albumen " refers to the matrix membrane protein (Matrix protein, M) of CDV, and M albumen is to H protein
Played a crucial role with F protein in CDV surface distributed and positioning.
As one embodiment of the present invention, the vaccine combination also includes Canine Parvovirus antigen, hepatitis infectiosa canis virus
Antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, Rabies Virus Antigen, dog influenza disease
Malicious 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 lymphocytic choriomeningitis virus resist
The former, one or more of bordetella branchiseptica antigen.
As a kind of preferred embodiment of the present invention, the vaccine combination also includes Canine Parvovirus antigen, dog gland
Viral antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, Rabies Virus Antigen, dog stream
The one or more of Influenza Virus antigen.
The invention further relates to the preparation method of vaccine combination, the preparation method includes expanding the CDV poison
The step of strain, the step of inactivating the CDV strain of the amplification, and uniformly mix the inactivation of the CDV
The step of antigen and adjuvant.The CDV inactivation antigen of vaccine combination of the present invention can be in amplification CDV strain
Afterwards, prepared using the common inactivation mode in this area, as one embodiment of the present invention, the inactivator includes but do not limited
In beta-propiolactone BPL, binary ethylenimine BEI, formalin, formaldehyde, N- acetylethylenimines AEI, Polyhaxemethylenguanidine Hydrochloride
PHMG。
As one embodiment of the present invention, preparation method of the present invention includes CDV described in clonal expression
Antigen protein the step of, and be well mixed the CDV antigen protein and adjuvant the step of；Of the present invention
In the preparation method of vaccine combination, CDV antigen protein can pass through molecular biology method described in the clonal expression
Realize, including with eukaryotic expression, prokaryotic expression, or viral vector is expressed, such as Chinese patent CN102816741A is by Newcastle Disease
Poison is prepared as live vector, or such as Chinese patent CN101358202A Recombinant Canine Adenovirus Type-2s are prepared as live vector, Li YW
Deng (Li YW, Sun CL, Han NJ, et al.Construction of recombinant pseudorabies virus
expressing canine distemper virus H gene and analysis on its biological
characters.Agricultural science and technology,2011,12(6):897-900) with rabies disease
Poison is prepared for carrier.
The invention further relates to the vaccine combination in the medicine for preparing prevention and/or treatment canine distemper relevant disease
Using.
As a kind of preferred embodiment of the present invention, the canine distemper relevant disease infection host includes Canidae, racoon
Section and mustelid.
As a kind of more preferably embodiment of the present invention, the canine distemper relevant disease infection host include dog, fox,
Recoon dog, mink.
Term " prevention and/or treatment " refers to suppress the duplication of CDV, suppression when being related to CDV infection
The propagation of CDV processed prevents CDV from being settled down in its host, and mitigates the disease of CDV infection
The symptom of disease or illness.If viral loads are reduced, illness mitigates and/or food ration and/or growth increase, then can just be recognized
Therapeutic effect has been reached for the treatment.
The invention further relates to a kind of CDV strain H protein, the CDV strain H protein has SEQ ID
Amino acid sequence shown in NO.2.
The invention further relates to a kind of CDV strain F protein, the CDV strain F protein has SEQ ID
Amino acid sequence shown in NO.4.
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 constitute any limitation to the scope of the present invention.Those skilled in the art
It should be understood that can be carried out without departing from the spirit and scope of the invention to the details and form of technical solution of the present invention
Modifications or substitutions, but these are changed and replacement is 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, if being conventional method without specified otherwise；Described biomaterial, if without specified otherwise, can be from business way
Footpath is obtained.
Structure, Screening and Identification and the application of the cell line of embodiment 1
The structure of 1.1 Vero/DogSLAM-1F5 plants of monoclonal cell systems
With pDisplay carriers (being purchased from Invitrogen companies) for template, the specific primer Neo-F of design is utilized:
5'-GCCTGTGGAATGTGTGTCAGT-3' and Neo-R:5'-GCTCAGAAGAACTCGTCAAGAAG-3', passes through PCR method
The Neo genetic fragments (NCBI accession number is JQ624676) containing SV40 promoters and Ploy (A) tail are cloned respectively and containing small
Mouse globulin IgK chain V-J2-C signal peptides are (with reference to the signal peptide Murine of the pSecTag2-B carriers of Invitrogen companies
Igk-chain V-J2-C singnal peptide, its sequence GAATTCGCCACCATGGAGACAGACACACTCCTGCTATG
GGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGAC) and HA labels gene (sequence is
TATCCATATGATGTTCCAGATTATGCTGGGGCCCTCGAG) fragment, is then connected in pCAGGS carriers by digestion,
By pCAGGS vector modifications into having Neo neomycin resistances and differentiate the recombinant vector of label containing high expression signal peptide and HA
pCAGGS-Neo-IgK-HA。
Aseptic collection dog periphery anticoagulation 10ml, (is cured with dog lymphocyte separation medium purchased from Chinese Academy of Medical Sciences's biology
Learn Graduate School of Engineering) separation PBLC is operated to specifications, afterwards with 0.1mg/ml ConA10%RPMI cultures
Liquid overnight incubation.Carried according to classical total RNA extraction reagent box (being purchased from Shanghai Hua Shun bioengineering Co., Ltd) specification operation
The total mRNA of canine peripheral blood lymphocyte of culture is taken, cDNA is synthesized using Random primers (being purchased from Dalian TAKARA) reverse transcription
Afterwards, primer SLAM-F is used：GCGAATTCATGGATTCCCAGGGGCT and SLAM-R：GCAGATCTTCAGCTCTCTGGGAACGT
Dog SLAM genes are expanded, the target gene fragment of amplification is then connected to pCAGGS-Neo-IgK-HA, pCAGGS- is screened
Neo/SLAM eukaryon expression plasmids.
The screening and identification of 1.2 Vero/DogSLAM-1F5 plants of monoclonal cell systems
6 porocyte plates are inoculated in the 1 × 10 of non-resistant 5%DMEM cultures6/ hole Vero cells, are 70%-80% to cell
Supernatant is abandoned during individual layer, is cleaned with serum-free DMEM three times；With the transfection reagents of Lipofectamine 2000 by recombinant vector
PCAGGS-Neo/SLAM by specification step transfected Vero cells, if repeating transfection hole and normal cell controls, put 37 DEG C, 5%
CO2In culture 14-16 hours, cell liquid is changed to the 2%DMEM nutrient solutions containing 800 μ g/ml G418 and screened, and routinely exists
Digest, pass on when cell is covered with, it is all dead etc. control cell, carried out by Vero/DogSLAM-1F5 plants to experimental group per hole
The dilution factor of 1 cell carries out three monoclonals and screens to obtain SLAM-1F5 plants of monoclonal cell system Vero/Dog, using exempting from indirectly
Epidemic disease fluorescent method and FCM analysis method are (referring to Liu Yu shows CDVs sensitive cell line and the structure of infection clones
Build and Preliminary Applications research Jilin University Ph.D. Dissertations, 2014) to monoclonal cell system Vero/DogSLAM-1F5 plants enter
The qualitative and quantitative detection of row.
Detected during indirect immunofluorescene assay using Vero cells as control, as a result (see Figure 1A)：It is thin in monoclonal
The expression quantity that the SLAM albumen of anti-HA labels is detected in born of the same parents system is 100%, and green fluorescence is to surround the circle of cell surface one
It is shinny, be conducive to the ligand binding with corresponding cause of disease；And the Vero cells for transfecting empty carrier do not occur green fluorescence (see Figure 1B),
Show Vero/DogSLAM-1F5 plants of cell expression SLAM albumen of monoclonal cell system.
FCM analysis qualification result is shown in Table 1：Monoclonal cell system Vero/DogSLAM-1F5, which involves in a criminal case, resumed for 25 generations still
Can be with highly stable ground high efficient expression SLAM cells.
SLAM cell results are expressed in the monoclonal cell system continuous passage of table 1
The application of 1.3 Vero/DogSLAM-1F5 plants of monoclonal cell systems
With with the wild poison of the 15th generation cell (i.e. P15) of Vero/DogSLAM-1F5 plants of monoclonal cell system separation infection CDV
The animal of strain, illustrates that it is applied by taking lungs lapping liquid or PBLC separation CDV street strains as an example.From CDV street strains
The following clinical symptoms of infection animal appearance, which are defined, carries out the separation of CDV street strains, and clinical symptoms are：Body temperature diphasic fever, have a fever, exhale
Inhale that difficult, visible dermis is rubescent, eye there are a large amount of secretion, nose to suppurate natural occurrence that sexual secretion, hind leg can not stand and it is dead；
The necrosis of cut open inspection lungs large-area hemorrhage, spleen enlargement, mesenteric adenophyma are big, as the standard of selected animal.
By the dog of 10 infection canine distempers from Henan pet clinic, and from Luoyang City periphery plant
The minks of 8 CDV infection canine distempers, 6 CDV infect the fox of canine distemper, weigh 5g at respective lung lesion, ground device
Lungs mucous membrane liquid is inoculated in 1F5 plants of cells of individual layer to cultivate and separate CDV velogen strains after milled processed；Simultaneously by 1F5
The strain respective lymphocyte of cell culture the results are shown in Table 2 to be separated to CDV velogen strains.
The different cell line separation CDV street strains results of table 2
As shown in Table 2：Can be from infection dog, mink and fox lungs using Vero/DogSLAM-1F5 plants of P15 cells
It is respectively 100%, 75% and 83.3% to separate CDV street strains virus isolated rate, from infection dog, mink and fox periphery hemolymph
It is respectively 80%, 75% and 83.3% that CDV street strains virus isolated rate is separated in cell.
The further identification of the CDV strain of embodiment 2 and assay
The further identification of 2.1 CDV strains
All different animals source CDV velogen strains that embodiment 1 is separated further are identified, with Quan Yuan CDV street strains
One plant complete set forth below exemplified by C/HN/001 plants.
Take and be ground at dog lung lesion, after lapping liquid is inoculated in Vero/DogSLAM-1F5 prepared by embodiment 1
The P15 cells of strain carry out virus purification, are seeded in Vero/DogSLAM-1F5 plants of monoclonal cell system after upper 24 hours on cell
There is the cytopathy or the cytopathy drawn in the net of multinucleate giant cell fusion, after inoculation 96 hours cytopathy reach 80% with
On, freeze thawing receives poison and is used as 1st generation virus liquid；Poison disease vaccination that malicious method obtained 1st generation is connect with monolayer adsorption in monoclonal
In cell line Vero/DogSLAM-1F5 strains, there is typical fused cell lesion after 24 hours or draws in the net cytopathy in inoculation,
There is syncytium formation after 60-96 hour and reaches more than 80% in inoculation, and freeze thawing, which is received, malicious is used as 2nd generation virus liquid；Use individual layer
Absorption connects the poison disease vaccination that malicious method obtained 2nd generation and cultivated in monoclonal cell system Vero/DogSLAM-1F5 strains, goes out
Now typical fused cell lesion or draw in the net cytopathy, freeze thawing receives poison and is used as the 3rd generation virus liquid.After 3 generations of stable passage
Virus liquid carries out electron microscopic observation to carry out morphologic detection, as a result shows：Virion with typical paramyxovirus feature sample
Son occurs.
The specificity of virus is identified according to the indirect immunofluorescene assay method of embodiment 1, is as a result shown：Virus inoculation is formed
Syncytium formation position there is specific green fluorescence, show that specificity is good.
With CDV H-F:5'TTAGGGCTCAGGTAGTCCA3'、CDV H-R:5'CTAAGKCCAATTGARATGTGT3' is
Primer, according to EasyScript One-Step RT-PCR SuperMix (being purchased from Beijing Quanshijin Biotechnology Co., Ltd)
Kit specification carries out the H gene sequence that One step RT-PCR technology expands C/HN/001 plants, and amplification purpose fragment connection T is carried
Converted after body, select positive colony plasmid and be sequenced in Invitrogen (Shanghai) Trading Co., Ltd., as a result such as SEQ ID No.1 institutes
Show, be consistent with nucleic acid electrophoresis with 1800bp or so；And carry out MegAlign processing with CDV H proteins gene order in GenBank
To analyse and compare, as a result show：The strain is Asia I type virus, with traditional vaccine Onderstepoort plants of H protein genes of strain
The homology of sequence is 90%.
Meanwhile, with CDV F-F:5'-CCGGAATTCACAGGCAAGCCCCATGCAC-3'、CDV F-R:5'-
CCGCTCGAGAATTAGGCGGGACTTATGGA-3' is primer, according to EasyScript One-Step RT-PCR
SuperMix (being purchased from Beijing Quanshijin Biotechnology Co., Ltd) kit specification carries out One step RT-PCR technology amplification
Converted after C/HN/001 plants of F gene orders, amplification purpose fragment connection carrier T, select positive colony plasmid in Invitrogen
(Shanghai) trade Co., Ltd is sequenced, as a result as shown in SEQ ID No.3, is consistent with nucleic acid electrophoresis with 2000bp or so；And with
CDV sequences carry out MegAlign processing to analyse and compare in GenBank, as a result show：The strain is Asia I type virus, with biography
The homology for Onderstepoort plants of F protein gene orders of vaccine strain of uniting is 90%.
It is CDV street strains that result above, which further confirms C/HN/001 plants,.
The measure of 2.2 CDV strain contents
C/HN/001 plants are expanded in the monoclonal cell system Vero/DogSLAM-1F5 strains prepared by embodiment 1, will
C/HN/001 strains after amplification are according to Reed-Muench methods (referring to Yin Zhen animal virology Beijing:Science Press,
1997) viral level measure is carried out, is as a result shown：The content of the strain is 104-108TCID50/ml。
The preparation and identification of the inactivated vaccine of the canine distemper vaccine composition of embodiment 3
The preparation of 3.1 inactivated vaccines CDV strains
C/HN/001 plants are inactivated through final concentration of 0.025%V/V BPL (beta-propiolactone), and according to《Chinese beast
Pharmacopeia》(the Chinese veterinary drug committee, version three in 2010, Chinese agriculture publishing house, 2010) method is carrying out sterile inspection to CDV
Test, mycoplasma is examined, exogenous virus is examined, as a result show：After C/HN/001 plants of inactivations, the pollution of bacterium, mould is not affected by,
The infection of mycoplasma and exogenous virus is not affected by, pure property is good.
The preparation of 3.2 inactivated vaccines
It is mixed after being diluted or concentrated with sterile saline purchased from the Gel A adjuvants of Seppic companies and through C/HN/001 plants
The final concentration described in table 3 is bonded to, 1%V/V thimerosal solution is added to its final concentration of 0.01%V/V, and fully mixed,
Produce inactivated vaccine 1a, 1b, 1c.
Component details contained by the inactivated vaccine of the vaccine combination of table 3
| Title | C/HN/001 plants of whole content (TCID50/ml) | Adjuvant | Adjuvant end content (V/V) |
| Vaccine 1a | 104 | Gel A | 5% |
| Vaccine 1b | 106 | Gel A | 5% |
| Vaccine 1c | 109 | Gel A | 5% |
Inactivated vaccine 1a, 1b, 1c are subjected to safety verification, i.e., it is subcutaneous respectively respectively with 2-10 monthly ages healthy susceptible canine 5
Vaccine inoculation 4ml, Continuous Observation 2 weeks, as a result：It is all strong to live, show that inactivated vaccine 1a, 1b, 1c can use safely.
Inactivated vaccine 1a, 1b, 1c are subjected to efficacy test, i.e., respectively with 2-10 monthly ages healthy susceptible canine 5, each notch graft
Vaccine 1ml is planted, while setting blank control dog, immune rear 7d and 14d takes a blood sample respectively, separates the anti-CDV antibody titers of Virus monitory.Knot
Really：Immune dog canine parvovirus prevention antibody is >=1:80, control dog canine parvovirus prevention antibody all should≤1:20, show to inactivate epidemic disease
Seedling 1a, 1b, 1c are effective.
Press《Chinese veterinary pharmacopoeia》(2010 editions) remain to the beta-propiolactone in inactivated vaccine 1a, 1b, 1c and antiseptic mercurials
It is fixed to measure, as a result：Meet the regulation of veterinary biologicses general rule.
The safety testing of 3.3 inactivated vaccines
3.3.1 the safety testing that a single dose is inoculated with
Using 50 ages in days or so healthy susceptible mink (neutralizing antibody≤1: 4) 20, be randomly divided into 4 groups, 5/group, its
In 1-3 groups respectively through subcutaneous immunoprophylaxis 1ml/ inactivated vaccine 1a, 1b, 1c, the 4th group is used as blank control.The same terms
It is lower to raise, observe 14 days, whether observation mink appetite, spirit, body temperature, eye nose, excrement are normal daily, and injection portion and whole body are
It is no to have adverse reaction.It the results are shown in Table 4：Single dose is inoculated with after susceptible mink, and the state of mind of mink is good, diet is intended to just
Often, body temperature and excrement are normal, and anophthalmia nasal discharge is produced, and injection site and whole body have no adverse reaction, and all minks are good for and lived.
Safety testing result of the inactivated vaccine of table 4 to single dose inoculation of mink
Note："-" represents non-vaccinal injection therefore not detected.
3.3.2 the safety testing of single dose repeated inoculation
Using 50 ages in days or so healthy susceptible mink (neutralizing antibody≤1: 4) 20, be randomly divided into 4 groups, 5/group, its
In 1-3 groups respectively through subcutaneous vaccination first immunisation 1ml/ inactivated vaccine 1a, 1b, 1c, and when the 14th day after first immunisation
Only, the 4th group is used as blank control to booster immunization 1ml/.Raise, observe 14 days under the same terms, daily observation mink appetite, essence
Whether god, body temperature, eye nose, excrement are normal, and whether injection portion and whole body have adverse reaction.As a result (5 are shown in Table)：Single dose is repeated
It is inoculated with after susceptible mink, the state of mind of mink is good, diet is intended to normally, body temperature and excrement are normal, the generation of anophthalmia nasal discharge,
Injection site and whole body have no adverse reaction, and all minks are good for and lived.
Safety testing result of the inactivated vaccine of table 5 to mink single dose repeated inoculation
Note："-" represents non-vaccinal injection therefore not detected.
3.3.3 the safety testing that an overdose is inoculated with
Using 50 ages in days or so healthy susceptible mink (neutralizing antibody≤1: 4) 20, be randomly divided into 4 groups, 5/group, its
In 1-3 groups respectively through subcutaneous immunoprophylaxis 4ml/ inactivated vaccine 1a, 1b, 1c, the 4th group is used as blank control.The same terms
It is lower to raise, observe 14 days, whether observation mink appetite, spirit, body temperature, eye nose, excrement are normal daily, and injection portion and whole body are
It is no to have adverse reaction.As a result (6 are shown in Table)：Overdose is inoculated with after susceptible mink, and the state of mind of mink is good, diet is intended to just
Often, body temperature and excrement are normal, and anophthalmia nasal discharge is produced, and injection site and whole body have no adverse reaction, and all minks are all good for and lived.
Safety testing result of the inactivated vaccine of table 6 to overdose inoculation of mink
Note："-" represents non-vaccinal injection therefore not detected.
Result above shows：It is safe that inactivated vaccine 1a, 1b, 1c, which are used for immunity inoculation animal,.
The potency test of 3.4 inactivated vaccines
Using 50 ages in days or so healthy susceptible mink (neutralizing antibody≤1: 4) 20, be randomly divided into 4 groups, 5/group, its
In 1-3 groups respectively through subcutaneous immunoprophylaxis 1ml/ inactivated vaccine 1a, 1b, 1c, the 4th group is used as blank control.The same terms
It is lower to raise, observe and find within 14 days that mink appetite, spirit, body temperature, eye nose, excrement are normal, injection portion and whole body are without bad
Reaction.After immune blood sampling in latter 14th day, wild-type canine distemper virus strain (C/HN/ is carried out using collunarium 1ml and intraperitoneal inoculation 2ml
001 strain virus liquid 105.0TCID50/ ml) challenge test, attacking daily observation mink appetite, spirit, body temperature, eye nose, excrement after poison is
It is no normal, and whether injection site and whole body have adverse reaction, and calculate and attack malicious protective rate, it the results are shown in Table 7.
Meanwhile, using the healthy susceptible canine 20 of 45 ages in days or so, the healthy susceptible foxes 20 of 50 ages in days, 50 ages in days
The susceptible recoon dog 20 of health attacks malicious potency test only according to above method progress inactivated vaccine, the results are shown in Table 7.
The efficacy test results of the inactivated vaccine of table 7
As shown in Table 7：Inactivated vaccine 1a, 1b, 1c attack malicious CDV velogen strains again after mink, dog, fox, recoon dog is immunized respectively
Afterwards without any side reaction, and it is 100% to attack malicious protective efficacy, shows that inactivated vaccine 1a, 1b, 1c can prevent and treat hundstaupe
The attack of fever virus.
3.5 immune durations are detected
Using 28 ages in days or so healthy susceptible canine (neutralizing antibody≤1: 4) 20, be randomly divided into 4 groups, 5/group, wherein
1-3 groups are respectively through subcutaneous immunoprophylaxis 1ml/ inactivated vaccine 1a, 1b, 1c, and the 4th group is used as blank control.Under the same terms
Raise, observe 21 days, antibody titer is surveyed in blood sampling, then booster immunization is once, head exempts from blood sampling in latter every 3 months and surveys average in an each group
Neutralize antibody titers；Continuous Observation is detected 24 months, the results are shown in Table 8.
The inactivated vaccine of table 8 is to dog immune duration result of the test
As shown in Table 8：Inactivated vaccine 1a, 1b, 1c distinguish after secondary immunity dog all to be tieed up up to anti-CDV antibody in 18 months
Hold higher level (>1:256), antibody level is gradually reduced afterwards.Show that inactivated vaccine 1a, 1b, 1c can be in the long periods
Interior prevention canine distemper epidemic situation.
The preparation and identification of the subunit vaccine of the canine distemper vaccine composition of embodiment 4
The structure of 4.1 recombination rhabdovirus expression vectors
The H proteins of C/HN/001 strains according to measured by embodiment 2, F protein gene order (see successively SEQ ID No.1,
SEQ ID No.3), and SY plants of CDV M protein gene sequences (referring to NCBI accession number：KJ466106), separately design and draw
Thing sequence is as follows：
CDVH-F:5’-TTAGGGCTCAGGTAGTCCA-3’
CDVH-R:5’-CTAAGKCCAATTGARATGTGT-3’
CDVF-F:5’-CCGGAATTCACAGGCAAGCCCCATGCAC-3’
CDVF-R:5’-CCGCTCGAGAATTAGGCGGGACTTATGGA-3’
CDVM-F:5’-CCGCTCGAGAAAACTGCTTTCACTATCGC-3’
CDVM-R:5’-GGAAGATCTCAGGAGCAACACCGAGACAA-3’
According to (Liu FX, Zhao Y, the Li L ed al.Budding of peste des petits such as Liu FX
ruminants virus-like particles from insect cell membrane based on
intracellular co-expression of peste des petits ruminants virus M,H and N
proteins by recombinant baculoviruses.J Virol Methods.2014,207:78-85) the behaviour of document
Make method construction expression CDV H, F and M albumen successively, and purify acquisition H, F and M albumen.Finally recombinated respectively
H, F and M albumen, and respectively with BCA protein quantifications kit (being purchased from green skies biotechnology research institute) according to explanation after purification
Book determines protein content, as a result：H, F and M protein content are followed successively by 0.8,0.8,1mg/ml.
The preparation of 5.2 subunit vaccines
Obtained CDV is recombinated into H, F, M albumen and Gel A adjuvants are diluted with sterile saline, by table 8
Shown final concentration carries out mixed preparing and obtains mixed liquor as subunit vaccine.And carry out character inspection respectively to each mixed liquor (long
Upper strata is postponed for colourless clear liquid, lower floor is pale precipitation, is in homogenous suspension after shaking), steriling test (press 2010 editions
《Chinese veterinary pharmacopoeia》Test, equal asepsis growth) as vaccine 2a, 2b, 2c, details are shown in Table 9.
The subunit vaccine component situation of the different vaccine combinations of table 9
| Title | Protein ingredient | Protein content (μ g/ml) | Adjuvant end ratio V/V |
| Vaccine 2a | H | 100 | 5% |
| Vaccine 2b | H、F | It is 100 | 5% |
| Vaccine 2c | H、F、M | It is 100 | 5% |
The effect detection of 5.3 canine distemper subunit vaccines
Using the healthy susceptible canine 20 of 28 ages in days, 4 groups, every group 5 are randomly divided into.Preceding 3 groups of difference intradermal vaccinations 2a,
2b, 2c, every subcutaneous vaccination 1ml, the 4th group of blank control are not inoculated with.Under the same terms raise, observe 14 days find dog food be intended to,
Spirit, body temperature, eye nose, excrement are normal, and injection portion and whole body have no adverse reaction.After immune blood sampling in latter 14th day, adopt
Wild-type canine distemper virus strain (C/HN/001 strain virus liquid 10 is carried out with collunarium 2ml and intraperitoneal inoculation 4ml5.0TCID50/ ml) attack poison
Experiment, whether attack after poison observation dog food desire, spirit, body temperature, eye nose, excrement daily normal, and injection site and whole body whether
There is adverse reaction, and malicious protective rate is attacked in calculating.It the results are shown in Table 10：The immune dog state of mind good, diet that is rear, attacking after poison is intended to
Normal, body temperature and excrement are normal, and equal anophthalmia nasal discharge is produced, and injection site and whole body are showed no adverse reaction, attack poison
All dogs are all good for and lived afterwards.
The effect testing result of the subunit vaccine of table 10
Result above shows that the subunit vaccine prepared by CDV H, F, M albumen can be carried to the poison of attacking of canine distemper velogen strain
For effective immunoprotection.By contrast, in the subunit vaccine that prepared by three kinds of different antigen components, H protein is contained, and
Also the immune effect of the subunit vaccine containing other antigenic components is preferable, and 5/5 protection is can reach to the poison of attacking of velogen strain.
Describe the preferred embodiments of the present invention comprehensively above, but various alternatives and modifications can be carried out to them.Therefore,
It reference should not be made to above description to determine the scope of the present invention, but should refer to appended claims and its whole equivalents to determine
Determine the scope of the present invention.Any feature, (being whether preferred) can be with any other feature (being whether preferred) phase
With reference to.Claims of the present invention is understood not to the limitation with method+function, unless led in a certain claim
Cross term " ... method " and clearly include such limitation.
Claims (10)
1. a kind of Vero/DogSLAM-1F5 plants of monoclonal cell system Vero/DogSLAM cell lines, wherein, the Vero/
Vero/DogSLAM-1F5 plants of DogSLAM cell lines are preserved in China typical culture collection center, and preserving number is CCTCC
NO.C201611, preservation address is Wuhan, China Wuhan University, and preservation date is on 03 29th, 2016.
2. Vero/DogSLAM-1F5 plants of separation CDV strains of monoclonal cell system described in a kind of usage right requirement 1
Method, wherein, methods described includes：The animal sources source for infecting CDV strain is ground or handled, then will not
Monoclonal cell system Vero/DogSLAM-1F5 plants of progress virus point described in claim 1 is inoculated in lapping liquid or treatment fluid
From passage is stable to be occurred after syncytium formation, and poison is received in freeze thawing；Preferably, CDV is infected described in methods described
Animal sources include Canidae source, cat family source, Viverridae source, Ursidae source, Phocidae source, panda section source, skunk section source, Procyonidae source,
Mustelidae source, monkey Ke Yuan and western Tuan sections source, the infection CDV strain animal sources source include lungs, liver, spleen,
Intestines, brain, cerebellum, kidney, excrement, urine, eye nose swab and/or PBLC.
3. the isolated a kind of CDV strain of method according to claim 2, wherein, the CDV poison
Base containing F protein shown in the gene of H protein shown in coding SEQ ID NO.2 and/or coding SEQ ID NO.4 in pnca gene group
Cause.
4. CDV strain according to claim 3, the CDV strain is CDV C/HN/
001 plant, described CDV C/HN/001 plants are preserved in China typical culture collection center, and preserving number is CCTCC
NO.V201604, preservation address is Wuhan, China Wuhan University, and preservation date is on March 29th, 2016.
5. a kind of vaccine combination, wherein, the vaccine combination includes hundstaupe pyreticosis described in the claim 3 or 4 of immune amount
The antigen of poison strain and veterinarily acceptable carrier, the totivirus that the antigen of the CDV strain includes inactivating resist
Former and/or at least one subunit antigen；Preferably, the vaccine combination includes the CDV C/HN/001 of immune amount
Strain antigen and veterinarily acceptable carrier.
6. vaccine combination according to claim 4, wherein, the vaccine combination include inactivation totivirus antigen and
Adjuvant；Wherein, the totivirus antigenic content of the inactivation is inactivation preceding 104-109TCID50/ml；Preferably, the inactivation is complete
Viral antigen content is inactivation preceding 105-107TCID50/ml；Or
The vaccine combination includes H protein and adjuvant, and the H protein amino acid sequence is the amino that SEQ ID No.2 are encoded
Acid sequence, the H protein content is 10-200 μ g/ml；Preferably, the vaccine combination also includes F protein and/or M albumen,
The F protein amino acid sequence is the amino acid sequence that SEQ ID No.4 are encoded；The F protein content is 10-160 μ g/ml,
The M protein contents are 10-180 μ g/ml；
Wherein, the adjuvant includes aluminium glue adjuvant, saponin(e, water-in-oil emulsion, oil in water emulsion, W/O/W emulsion, propylene
The polymer of acid or methacrylic acid, the copolymer of maleic anhydride and alkenyl (alkenyl) derivative, RIBI adjuvants system
System, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipids-amine adjuvant, Escherichia coli are intolerant to warmheartedness poison
One or more in element, cholera toxin, IMS 1314, muramyl dipeptide or Gel adjuvants.
7. according to any one of the claim 5-6 vaccine combinations, wherein, the vaccine combination also includes canine parvovirus
Antigen, hepatitis infectiosa canis virus antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, hydrophobin
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 lymphatic train of thought
Clump meningitis virus, the one or more of bordetella branchiseptica antigen；Preferably, the vaccine combination also includes dog
It is parvovirus antigen, hepatitis infectiosa canis virus antigen, leptospira canicola antigen, canine coronavirus antigen, canine parainfluenza virus antigen, mad
The one or more of dog disease viral antigen, canine influenza virus antigen.
8. preparing prevention according to any one of the claim 4-6 vaccine combinations and/or treating canine distemper relevant disease
Application in medicine；Preferably, the canine distemper relevant disease infection host includes Canidae, Procyonidae and mustelid；It is more excellent
Selection of land, the canine distemper relevant disease infection host includes dog, fox, recoon dog, mink.
9. a kind of CDV strain H protein, the CDV strain H protein has the ammonia shown in SEQ ID NO.2
Base acid sequence.
10. a kind of CDV strain F protein, the CDV strain F protein has the ammonia shown in SEQ ID NO.4
Base acid sequence.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750006A (en) * | 2019-01-14 | 2019-05-14 | 青岛农业大学 | A kind of canine distemper virus replication defective strain and its construction method |
| CN110894215A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Peste des petits ruminants virus antigen and colloidal gold immunochromatographic test paper card for detecting Peste des petits ruminants virus antibody |
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| CN101851607A (en) * | 2010-04-21 | 2010-10-06 | 中国人民解放军军事医学科学院军事兽医研究所 | Canine-SLAM/Vero cell line and constructing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101851607A (en) * | 2010-04-21 | 2010-10-06 | 中国人民解放军军事医学科学院军事兽医研究所 | Canine-SLAM/Vero cell line and constructing method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109750006A (en) * | 2019-01-14 | 2019-05-14 | 青岛农业大学 | A kind of canine distemper virus replication defective strain and its construction method |
| CN110894215A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Peste des petits ruminants virus antigen and colloidal gold immunochromatographic test paper card for detecting Peste des petits ruminants virus antibody |
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