CN109010817B - Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof - Google Patents

Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof Download PDF

Info

Publication number
CN109010817B
CN109010817B CN201710433747.XA CN201710433747A CN109010817B CN 109010817 B CN109010817 B CN 109010817B CN 201710433747 A CN201710433747 A CN 201710433747A CN 109010817 B CN109010817 B CN 109010817B
Authority
CN
China
Prior art keywords
antigen
porcine
virus
strain
inactivated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201710433747.XA
Other languages
Chinese (zh)
Other versions
CN109010817A (en
Inventor
田克恭
李向东
肖燕
孙进忠
张许科
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pulaike Biological Engineering Co Ltd
Original Assignee
Pulaike Biological Engineering Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pulaike Biological Engineering Co Ltd filed Critical Pulaike Biological Engineering Co Ltd
Priority to CN201710433747.XA priority Critical patent/CN109010817B/en
Publication of CN109010817A publication Critical patent/CN109010817A/en
Application granted granted Critical
Publication of CN109010817B publication Critical patent/CN109010817B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/0241—Mollicutes, e.g. Mycoplasma, Erysipelothrix
    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • 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)
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/10011—Arteriviridae
    • C12N2770/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The invention relates to a vaccine composition for preventing and/or treating porcine circovirus mixed infection, wherein the vaccine composition comprises an immunizing amount of porcine circovirus type 3 antigen and a carrier. The vaccine composition has good and broad-spectrum immunogenicity, and can protect the mixed infection of porcine circovirus type 3 strains from different regional sources.

Description

Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof
Technical Field
The invention relates to a vaccine composition for preventing and/or treating porcine circovirus type 3 infection, a preparation method and application, and belongs to the field of animal virology.
Background
Porcine Circovirus (PCV) is a single-stranded circular DNA virus with a genome length of approximately 1.7kb that is one of the smallest animal DNA viruses. Two types of PCV have been identified, porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV 2). PCV1 was first identified in 1974 as a contaminant in PK cell cultures, which was not pathogenic to pigs. PCV2 was first reported in 1998 to cause Porcine circovirus-associated diseases (PCVAD) in pigs under clinical conditions, mainly causing postweaning multisystemic wasting syndrome, pneumonia, Porcine dermatitis and nephropathy syndrome and reproductive disorders, mainly manifested as respiratory, urinary, intestinal, lymphatic, cardiovascular, neurological, reproductive and cutaneous dysfunction, causing significant economic losses to live pig breeding worldwide.
However, in the case of reproductive disorders in pigs, a circovirus with a 2.0kb viral genome was isolated, and it was confirmed by subsequent experiments that it has less than 50% homology with known circovirus in both nucleotide and amino acid sequences, and that viruses of the same species in the genus circovirus should have > 75% homology in nucleotide sequence of the genome and Cap protein > 70% homology in amino acid sequence according to the criteria of the international committee for taxonomic classification of virology, thereby confirming that it is a novel porcine circovirus. It can be mixed with various pathogens to cause dermatitis nephrotic syndrome, proliferative necrotizing pneumonia, reproductive disorders and inflammatory reaction of heart and multiple systems in pigs.
The systemic disease caused by PCV2 is sporadically broken out in 1985, and the disease is massively broken out in the end of the nineties due to the fact that the disease is not paid attention to, and the new porcine circovirus PCV3 has similar etiological characteristics to PCV2 in terms of PNDS and reproductive disorders, and the existing vaccine-free product can be used for preventing or treating PCV3 infection, so that the demand for developing a vaccine capable of effectively preventing PCV3 infection or massively mixed infection with other pathogens is urgent, and the preparation of a new vaccine composition aiming at the new clinical epidemic situation is very important for controlling the disease of a pig farm.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a vaccine composition for preventing and/or treating the novel porcine circovirus mixed infection, the vaccine composition can effectively protect the novel porcine circovirus mixed infection and shows remarkable immunological properties.
Therefore, the invention aims to provide a vaccine composition for preventing and/or treating novel porcine circovirus type 3 mixed infection, which contains porcine circovirus type 3 strain antigens, can effectively protect the mixed infection of the porcine circovirus type 3 epidemic strain and one or more other pathogens, and provides complete protection against infection of porcine circovirus type 3 from different sources.
The invention also aims to provide application of the vaccine composition in preparing a medicament for preventing and/or treating porcine circovirus type 3 mixed infection diseases.
The invention has the advantages that:
(1) the strain has good immunogenicity, can stimulate an organism to quickly generate immune efficacy after immunization once, effectively protects the attack of epidemic strains, has good protection effect, can achieve good immune protection effect by using lower antigen content, and further reduces the production cost;
(2) the vaccine composition for preventing and/or treating porcine circovirus type 3 mixed infection is prepared by adopting the current new porcine circovirus type 3 epidemic strain for the first time, and all antigen components in the vaccine composition are not interfered and can be applied together;
(3) the vaccine can provide complete protection against infection of porcine circovirus type 3 strains from different regional sources, and has broad-spectrum protection capability.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
The porcine circovirus type 3 is a circovirus with a genome of 2.0kb, has homology of less than 50 percent with known circovirus in nucleotide or amino acid sequence, is a novel porcine circovirus, and can cause dermatitis nephrotic syndrome, proliferative necrotizing pneumonia, reproductive failure and inflammatory response of heart and multiple systems of pigs by mixed infection with various pathogens.
The Porcine Circovirus type 3SG (Portone Circovirus type 3, Strain SG) is preserved in China center for type culture Collection with the preservation number of CCTCC NO. V201712, the preservation date of 2017, 3 and 23 months, the preservation address: wuhan, Wuhan university in China.
The invention relates to a vaccine composition for preventing and/or treating porcine circovirus type 3 mixed infection, wherein the vaccine composition comprises an immunizing dose of porcine circovirus type 3 antigen and a pharmaceutically acceptable carrier; wherein the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: classical swine fever virus antigen, porcine pseudorabies virus antigen, porcine influenza virus antigen, porcine reproductive and respiratory syndrome virus antigen, porcine parvovirus antigen, porcine encephalitis B virus antigen, haemophilus parasuis antigen, streptococcus suis antigen, bordetella suis antigen, porcine infectious pleuropneumonia antigen, porcine pasteurella multocida antigen, salmonella choleraesuis antigen, mycoplasma hyopneumoniae antigen, mycoplasma hyorhinis antigen.
The vaccine composition has good immunogenicity, can completely protect pigs by immunizing only once, and can achieve good immune protection effect even if the antigen content is low.
The vaccine composition of the present invention may comprise an immunizing amount of inactivated antigen of porcine circovirus type 3 or a culture thereof, attenuated whole virus antigen and a pharmaceutically acceptable carrier.
"cultures" are subcultures of different generations of the virus, and those skilled in the art know that only minor variations in the gene sequence are possible between generations.
"vaccine composition" refers to a pharmaceutical composition comprising porcine circovirus type 3 immunogenicity. The pharmaceutical composition can induce, stimulate or enhance the immune response of pigs against porcine circovirus type 3.
"inactivated antigen", also known as inactivated vaccine, refers to a suspension of inactivated virus used as an antigen to generate immunity. Examples of inactivated vaccines include whole virus vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, whole virus inactivated vaccines can be obtained by treating the virus with formaldehyde solution. Split vaccines can be prepared from the viral envelope after treatment with ether.
An "attenuated whole virus antigen" refers to a virus that has been attenuated for virulence but is still able to replicate in or on a host. The term "attenuated" as used herein is intended to mean that the virulence of a pathogen is artificially reduced by mutating the gene in such a way that the pathogen loses pathogenicity, but remains immunogenic. Attenuation is typically achieved by UV irradiation, chemical treatment or in vitro sequential high-order subculture. Or artificial genetic alteration, such as deletion of specific nucleotides in known sequences to attenuate virulence.
As an embodiment of the invention, the porcine circovirus type 3 antigen is porcine circovirus type 3SG strain or culture inactivated whole virus antigen thereof, and the preservation number of the SG strain is CCTCC number V201712.
In one embodiment of the present invention, in the vaccine composition of the present invention, the porcine circovirus type 3 antigen is porcine circovirus type 3 virus strain SG or a culture lytic antigen thereof.
The gene sequence of different generations of subcultures of the virus only has slight variation, the same immunogenicity can be ensured, and the inactivated vaccine prepared by the method has similar immune efficacy.
As an embodiment of the invention, in the vaccine composition of the invention, the culture of the porcine circovirus type 3SG strain is a culture of more than or equal to 1 generation.
In a preferred embodiment of the present invention, the culture of the SG type-3 strain of porcine circovirus in the vaccine composition of the present invention is a culture of 5 generations or more.
In a more preferred embodiment of the present invention, the culture of the porcine circovirus type 3SG strain in the vaccine composition of the present invention is a culture of 5 to 55 generations.
The amount of an ingredient or component of the composition of the present invention is preferably a therapeutically effective amount. The therapeutically effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The precise amounts of the ingredients used and the composition to be administered will vary depending on a variety of factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
As an embodiment of the invention, the inactivated whole virus antigen content of the porcine circovirus type 3SG strain or the culture thereof is more than or equal to 10 before inactivation5.0TCID50/ml。
The inactivated whole virus antigen of the porcine circovirus type 3SG strain or the culture thereof in the vaccine composition has good immunogenicity, can stimulate the organism to quickly generate immunity, and is 10 percent before inactivation5.0TCID50At the content of/ml, good immune protection effect can be achieved, and 100% protection rate is realized.
In a preferred embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 3SG strain or the culture thereof is 10 before inactivation5.0～107.0TCID50/ml。
As a more preferred embodiment of the present invention, the inactivated whole virus antigen content of the porcine circovirus type 3SG strain or the culture thereof is 10 before inactivation6.0TCID50/ml。
In the vaccine composition of the present invention, the inactivated whole virus antigen content range of the porcine circovirus type 3SG strain or the culture thereof can be further selected from 105.0～106.0TCID50Per ml, or 106.0～ 107.0TCID50/ml。
The vaccine compositions of the present invention further comprise other pathogen or antigen compositions for use in the preparation of combination or composite vaccines against a variety of diseases including porcine circovirus type 3 infection.
The term "combination vaccine" refers to a vaccine prepared from a mixture of viral antigens of porcine circovirus type 3 and at least one different virus according to the invention. The term "composite vaccine" refers to a vaccine prepared from the porcine circovirus type 3 and bacterial antigens of the invention. For example, porcine circovirus type 3 of the invention can be combined with antigens of: hog cholera virus, porcine pseudorabies virus, swine influenza virus, porcine reproductive and respiratory syndrome virus, porcine parvovirus, porcine encephalitis B virus and/or haemophilus parasuis, Streptococcus suis, Bordetella suis, porcine contagious pleuropneumonia, Pasteurella multocida, Salmonella choleraesuis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, and a mixture or combination thereof.
The porcine circovirus type 3 antigen can be used together with a vaccine composition consisting of a plurality of antigen components, the antigens are not interfered with each other, and the antigen components can generate complete protection against respective challenge strains.
As a preferred embodiment of the invention, the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: the antigen comprises a classical swine fever virus attenuated antigen, a porcine pseudorabies virus attenuated antigen, a porcine reproductive and respiratory syndrome virus attenuated antigen, a porcine parvovirus inactivated antigen, a haemophilus parasuis inactivated antigen and a mycoplasma hyopneumoniae inactivated antigen.
As a preferred embodiment of the invention, the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: the antigen comprises a classical swine fever virus attenuated antigen, a porcine pseudorabies virus inactivated antigen, a porcine reproductive and respiratory syndrome virus attenuated antigen, a porcine parvovirus inactivated antigen, a haemophilus parasuis inactivated antigen and a mycoplasma hyopneumoniae inactivated antigen.
As a more preferred embodiment of the present invention, the attenuated antigen of the classical swine fever virus is a lapinized attenuated strain of classical swine fever virus, the attenuated antigen of the pseudorabies virus is an attenuated strain of HN1201-R strain, the inactivated antigen of the pseudorabies virus is an inactivated whole virus antigen of HN1201 strain, the attenuated antigen of the porcine reproductive and respiratory syndrome virus is an attenuated strain of JXA1-R strain, the inactivated antigen of the porcine parvovirus is an inactivated whole virus antigen of HN-2011 strain, the inactivated antigen of the haemophilus parasuis is an inactivated whole virus antigen of JS strain type 4 or ZJ strain type 5 of haemophilus parasuis, and the inactivated antigen of the mycoplasma hyopneumoniae is an inactivated whole virus antigen of HN0613 strain.
As one embodiment of the invention, in the vaccine composition of the invention, the porcine circovirus inactivated antigen content is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml4.0～106.0TCID50The porcine pseudorabies virus attenuated antigen content is 106.0～107.0TCID50The inactivated antigen content of the porcine pseudorabies virus is 10 before inactivation6.0～107.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 105.0～107.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation6.0～ 108.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation8.0～ 1010.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation8.0～ 1010.0CCU/ml。
As a preferred embodiment of the invention, in the vaccine composition of the invention, the inactivated antigen content of porcine circovirus is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml5.0TCID50The porcine pseudorabies virus attenuated antigen content is 106.0TCID50The inactivated antigen content of the porcine pseudorabies virus is 10 before inactivation6.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 106.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation7.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation9.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation9.0CCU/ml。
In one embodiment of the present invention, the pharmaceutical composition is a vaccine composition of the present inventionAcceptable carriers include adjuvants including white oil, derek oil, and animal, vegetable or mineral oils; or aluminum hydroxide, aluminum phosphate and metal salts; or MontanideTMGel, carbomer, squalane or squalene, ISA206 adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
In a preferred embodiment of the present invention, the adjuvant is Montanide in the vaccine composition of the present inventionTM Gel。
In one embodiment of the present invention, the adjuvant is present in an amount of 5 to 20V/V% in the vaccine composition of the present invention.
As a preferred embodiment of the present invention, in the vaccine composition of the present invention, the adjuvant content is 10V/V%.
The vaccine compositions of the present invention may be formulated using available techniques, preferably together with a veterinarily acceptable carrier. For example, the oil may help stabilize the formulation and additionally serve as a vaccine adjuvant. The oil adjuvant can be natural source or obtained by artificial synthesis. The term "adjuvant" refers to a substance added to the composition of the present invention to increase the immunogenicity of the composition. Known adjuvants include, but are not limited to: (1) aluminium hydroxide, saponin (saponin) (e.g. QuilA), alfuzidine, DDA, (2) polymers of acrylic or methacrylic acid, maleic anhydride and alkenyl derivatives, (3) vaccines can be made in the form of oil-in-water, water-in-oil or water-in-oil-in-water emulsions, or (4) MontanideTM Gel。
In particular, the emulsion may be based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; oils resulting from the oligomerization of olefins, in particular isobutene or decene, esters of acids or alcohols with linear alkyl groups, more in particular vegetable oils, ethyl oleate, propylene glycol di (caprylate/caprate), glycerol tri (caprylate/caprate), propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used with an emulsifier to form an emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of polyoxyethylated fatty acids (e.g.oleic acid), sorbitan, mannitol (e.g.anhydromannitol oleate), glycerol, polyglycerol, propylene glycol and optionally ethoxylated oleic acid, isostearic acid, ricinoleic acid, hydroxystearic acid, ethers of fatty alcohols and polyols (e.g.oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers, in particular Pluronic R, especially L121 (see Hunter et al, 1995, "The Theory and Practical applications of Advances" (Steward-Tull, D.E.S. eds.) John Wiley and sons, NY, 51-94; Todd et al, Vaccine, 1997, 15, 564 + 570).
In particular, the acrylic or methacrylic acid polymers are crosslinked by polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers.
Preferably, the adjuvant selected by the invention is MontanideTM Gel。
The amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount. By "effective amount" is meant the amount of adjuvant necessary or sufficient to exert their immunological effect in a host when administered in combination with the antigen of the invention without causing undue side effects. The precise amount of adjuvant to be administered will vary depending on a variety of factors such as the ingredients used and the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the composition.
The vaccine composition of the present invention may further comprise other agents added to the composition of the present invention. For example, the compositions of the present invention may also comprise agents such as: drugs, immunostimulants (e.g., alpha-interferon, beta-interferon, gamma-interferon, granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 2(IL2)), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives. To prepare such compositions, methods well known in the art may be used.
The vaccine composition according to the present invention may be prepared in oral or non-oral dosage forms.
Preferred are non-oral dosage forms that can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, or epidural routes.
The invention also relates to application of the vaccine composition in preparing a medicament for preventing and/or treating porcine circovirus type 3 related diseases.
The antigen in the vaccine composition can effectively stimulate immune response within the content range, resist the attack of respective pathogen, do not generate interference among antigen components in the vaccine composition, and can be completely protected, and the protection rate is 100 percent.
As an embodiment of the present invention, the vaccine composition of the present invention is used. The porcine circovirus type 3 related diseases comprise weaned piglet multisystemic wasting syndrome, dermatitis nephrotic syndrome, proliferative necrotizing pneumonia, reproductive disorders and cardiac and multisystemic inflammatory responses of pigs.
The invention also relates to application of the vaccine composition in preparing a medicament for preventing and/or treating diseases caused by porcine circovirus type 3 mixed infection.
The term "porcine circovirus type 3 mixed infection disease" as used herein is intended to mean a disease caused by a co-infection of porcine circovirus type 3 with one or more pathogens. Non-exhaustive examples include but are not limited to postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, proliferative necrotizing pneumonia, reproductive disorders and cardiac and multisystemic inflammatory responses.
The term "preventing and/or treating" when referring to a porcine circovirus type 3 mixed infection means inhibiting replication, including porcine circovirus type 3, inhibiting transmission, or preventing colonization of its host by, including porcine circovirus type 3, and alleviating the symptoms of a disease or disorder, including porcine circovirus type 3 infection. Treatment is considered to be therapeutically effective if the viral load is reduced, the condition is reduced and/or the food intake and/or growth is increased.
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group.
In order that the invention may be more readily understood, reference will now be made to the following examples. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.
Example 1 isolation and identification of porcine circovirus type 3
1. Origin of disease material
Compared with the historical average value, the mortality of the sows is increased by 9.4%, the conception rate is reduced by 1.2%, and the mummy fetus is increased by 8.2% in a commercial pig farm in China. Clinically, affected sows show anorexia, and symptoms of multifocal papules, blotches, and surface dermatitis. Mummified fetuses of different gestational ages were contained in the litters of the miscarriage, consistent with symptoms of PCV 2-associated miscarriage. Although the overall clinical manifestations and abortion symptoms observed in sows were consistent with the reproductive disorders caused by porcine circovirus type 2, different tissues of all sows, including kidney, lymph nodes, lung, skin and stillborn fetuses, were negative for PCV2, PRRSV, PPV, CSFV, mycoplasma hyopneumoniae detection by immunohistochemistry and quantitative PCR. To further check the cause, various tissue disease materials are selected for pathogen separation.
2. Isolation and culture of viral strains
The disease material is mixed according to the proportion of 1: 10 (volume ratio) adding DMEM culture solution, grinding and preparing tissue suspension; repeatedly freezing and thawing the tissue suspension for 3 times, centrifuging at 12000r/min for 15min, and collecting supernatant; filtering the supernatant with 0.22 μm filter membrane filter; the filtrate was passaged on PK15 cells, cultured at 37 ℃ for 1 hour, and DMEM culture solution containing 2% calf serum was added instead, and cultured at 37 ℃ for 5 days. And (4) harvesting a culture solution containing the virus, and after the culture solution is frozen and thawed for 2 times, harvesting the virus.
3. Identification of viral species by PCR and sequencing analysis
And (3) taking the virus culture harvested in the step (1), extracting nucleic acid of a virus sample by using a nucleic acid extraction kit, and performing PCR amplification identification by using a circovirus species specific primer, wherein the result shows that a 2000bp target band is amplified by PCR. And (3) sending the PCR product to a sequencing company for nucleotide sequence determination, and carrying out genetic evolution analysis on a sequence determination result. The results show that the genome sequence and amino acid sequence of the virus strain are less than 50% homologous with other reported circovirus, and according to the standard of the international committee for virology classification, the virus of the same species in the circovirus genus should have > 75% homology of genome nucleotide sequence and the Cap protein has > 70% homology of amino acid sequence, so that the virus strain is a new porcine circovirus and is also the third circovirus found on the pig body at present.
Example 2 screening of porcine circovirus type 3 vaccine strains
According to the separated porcine circovirus type 3 specific primers, 235 suspected PCV3 positive samples collected from all over the country are analyzed through quantitative PCR, 121 PCV3 viruses are screened and separated, and in the 121 viruses, the homology of the genome nucleotide sequence among the strains is as high as 98.9-99.6%, and the homology of the amino acid sequence of Cap protein is as high as 97.7-99.5%. Through animal pathogenicity test and immunogenicity test, 1 PCV3 virus strain with strong pathogenicity, good immunogenicity and broad spectrum is finally screened out. The porcine circovirus type 3 virus is named as porcine circovirus type 3SG strain and submitted for preservation.
Example 3 pathogenicity test of porcine circovirus type 3SG strain
10 healthy piglets which are detected by ELISA at the age of 28-30 days and have PCV2 and PCV3 antigens and negative antibodies are randomly divided into two groups, 5 piglets/group, and the 1 st group uses PCV3SG strain (containing 10)5.0TCID50First), toxin counteracting, intramuscular injection, inoculating DMEM culture medium to a blank control group, and separately feeding each group of piglets. After the challenge, each group of piglets is continuously observed and judged according to clinical symptoms, pathological changes and virus detection, and specific results are shown in table 1.
TABLE 1 porcine circovirus type 3SG strain pathogenicity test results for piglets
Figure BDA0001318068550000101
Figure BDA0001318068550000111
The results show that all pigs in the virus attacking group have high temperature of more than 40.5 ℃ with 3-5 days of persistence, anorexia, mental depression, rough hair, emaciation and slow growth speed, and the autopsy has lung excess change, lymphadenectasis and kidney dead points with different degrees, and the porcine circovirus type 3 virus can be separated again by PCR detection of each organ tissue, while the blank control group has no abnormal condition, which indicates that the piglet is infected by the porcine circovirus type 3SG strain inoculation of the invention, and the clinical exterior symptoms are typical porcine circovirus infection symptoms.
Example 4 preparation of porcine circovirus type 3SG strain antigen
The cultures of the porcine circovirus type 3SG strain selected in example 2 at different generations were inoculated into monolayer-forming PK15 passaged cells at 1% (V/V) of the virus culture medium volume, adsorbed at 37 ℃ for 30 minutes, added with a cell maintenance medium, and cultured at 37 ℃. Observing for 1-2 times every day, wherein the cell growth is good, harvesting cell culture after culturing for 4-7 days at 36-37 ℃, freezing and thawing the harvested cell culture for 2-3 times, harvesting virus liquid, and determining virus titer. Filtering the virus liquid by using a hollow fiber (0.5-2 mu m) filter column to remove cell fragments, adding 0.1-0.2% of formaldehyde solution to inactivate at 37 ℃ for 24h, and preparing the vaccine by using the virus antigen after complete inactivation.
Example 5 preparation of classical swine fever virus antigens
The ST cells growing into a good single layer and highly sensitive to the hog cholera lapinized virus strain are digested and dispersed by a digestive juice containing 0.125 percent pancreatin and 0.03 percent EDTA, the cells are counted and inoculated into a cell culture bottle, a DMEM cell culture solution containing 3 percent calf serum is added, meanwhile, hog cholera virus seed viruses (purchased from Central institute under the control of China, the accession number of AVCC NO. AV1412) are added according to the quantity of M.O.I. ═ 0.1 inoculation agents, and the mixture is placed into a 37 ℃ incubator for culture. The first detoxification is carried out after three days of culture, cell maintenance liquid containing 1.5 percent of calf serum is supplemented after detoxification, and the detoxification is carried out once every 2 days and can be continuously carried out for 5 times. Finally, mixing the detoxified antigens in batches and storing at-20 ℃.
Example 6 preparation of porcine pseudorabies virus attenuated antigen
Inoculating 1% (V/V) of porcine pseudorabies virus HN1201-R strain (disclosed in Chinese patent application CN105087506A) culture into ST cell culture forming a single layer according to the virus culture fluid amount, culturing at 37 ℃, harvesting cell culture fluid containing virus when the lesion reaches 80%, freezing and thawing the cell culture fluid for 2 times, collecting the virus, measuring the virus price, and storing at low temperature.
Example 7 preparation of inactivated antigen of porcine pseudorabies Virus
Inoculating the gE gene deleted strain (disclosed in Chinese patent application CN103923884A) culture of porcine pseudorabies virus HN1201 strain into an ST cell culture forming a single layer according to 1% (V/V) of the virus culture solution, placing the ST cell culture in a rotary culture at 37 ℃, harvesting a virus-containing cell culture solution when the lesion reaches 80%, performing freeze thawing for 2 times, harvesting a virus solution, and determining the virus price. Adding 10% (V/V) formaldehyde solution to the virus solution to make the final concentration of formaldehyde be 0.2% (V/V), inactivating at 37 deg.C for 18 hr, stirring every 4 hr for 1 time, stirring for 10min each time, and inactivating completely.
Example 8 preparation of attenuated antigens of porcine reproductive and respiratory syndrome Virus
Selecting a required number of Marc-145 cells (cell spinner flasks which have grown into a monolayer), discarding growth liquid, inoculating a production virus strain JXA1-R strain (disclosed in Chinese patent application CN101307305A) according to 1% (V/V), adding DMEM cell culture liquid containing 2% fetal calf serum, rotating (9-12R/h) for culturing at 37 ℃, observing cytopathic effect (CPE) caused by viruses under a microscope, harvesting cell cultures when the CPE reaches more than 70%, freeze-thawing the cell cultures for 1 time, removing cell fragments by centrifugation or filtration, and freezing and storing at the temperature of below-40 ℃.
Example 9 preparation of porcine parvovirus inactivated antigen
Inoculating a porcine parvovirus HN-2011 strain (disclosed in Chinese patent application CN102886043A) culture into an ST cell culture forming a monolayer according to the inoculation dose of M.O.I. ═ 0.01, placing the ST cell culture at 37 ℃ for rotary culture, harvesting a cell culture solution containing toxin when lesion reaches 80%, freezing and thawing the cell culture solution for 2 times, harvesting virus, and measuring the toxin value. Adding 10% (V/V) formaldehyde solution to the virus solution to make the final concentration of formaldehyde be 0.2% (V/V), inactivating at 37 deg.C for 18 hr, stirring every 4 hr for 1 time, stirring for 10min each time, and inactivating completely.
Example 10 preparation of inactivated antigen of Haemophilus parasuis
Propagation of first-order seeds: haemophilus parasuis serum 4 type JS strain and 5 type ZJ strain (disclosed in Chinese patent application CN102908615A) freeze-dried strains are respectively streaked and inoculated on a TSA/NAD (TSA is produced by BD company, NAD is produced by Roche company) flat plate, the flat plate is placed at 37 ℃ for culturing for 18-24 hours, colonies meeting the requirement are selected and inoculated in a TSB/NAD (TSB is produced by BD company, NAD is produced by Roche company) liquid culture medium, and the liquid culture medium is cultured for 12-16 hours at 37 ℃ to serve as a first-grade seed;
and (3) propagation of secondary seeds: adding the prepared primary seed cultures of the JS strain 4 and ZJ strain 5 of the haemophilus parasuis serum into a TSB/NAD liquid culture medium according to the amount of 1%, culturing for 12-16 hours at 37 ℃, and taking the primary seed cultures as secondary seeds after pure examination;
adding 0.01-0.05% NAD, 5-10% calf serum (Hangzhou Biotechnology limited company in Zhejiang and 0.1-5% glucose into a TSB culture medium, adding a secondary seed bacterial solution of a JS strain of haemophilus parasuis serum type 4 and a ZJ strain type 5 into the culture medium according to the amount of 1% respectively for culture, uniformly mixing, placing at 37 ℃ for culture for 16-18 hours, and stopping culture when the concentration OD600 of the bacterial solution reaches more than 2.5, the DO value begins to rise and the pH value is reduced to below 6.5.
After counting the viable bacteria of the culture, adding a formaldehyde solution (double chemical industries, Ltd.) with the concentration of 37% according to the total amount of 0.2% (V/V), inactivating the mixture at the temperature of 37 ℃ for 24 hours, stirring the mixture for 3-5 times during the inactivation, and keeping the mixture for later use.
Example 11 preparation of inactivated antigen of Mycoplasma hyopneumoniae
Propagation of first-order seeds: unsealing a freeze-dried strain HN0613 (disclosed in Chinese patent application CN103031258A), inoculating a liquid culture medium according to the inoculation amount of 10%, placing the inoculated liquid culture medium at 37 ℃ for shaking culture for 3-7 d, harvesting a culture when the pH value is reduced from 7.5 to 6.8, and taking the culture as a primary seed after pure inspection;
and (3) propagation of secondary seeds: inoculating the prepared primary seeds into a liquid culture medium according to the inoculation amount of 5%, carrying out shaking culture at 37 ℃ for 3-7 d, harvesting a culture when the pH value is reduced from 7.5 to 6.8, and carrying out pure inspection to obtain secondary seeds;
formulation of liquid medium (1065 ml): 300ml of bovine heart extract, 360ml of ddH2O (redistilled water), pH adjusted to 7.4, and sterilized at 121 ℃ for 15 minutes. The following filter sterilized components were added: 40ml of Hank's balanced salt solution (10X) (10 times concentrated), 10ml of 0.25% phenol red, 200ml of horse serum, 100ml of 5% hydrolyzed milk protein, 20ml of 25% yeast extract, 10ml of 10000IU/ml penicillin, and 25m1 of 1% thallium acetate solution;
inoculating the secondary seed liquid into a liquid culture medium at 5% (v/v), culturing at 37 ℃ for 3-7 days, and harvesting the bacterial liquid when the pH value is reduced from 7.5 to 6.8.
And (3) slowly adding the qualified mycoplasma hyopneumoniae bacterial liquid prepared by the inspection into a formaldehyde solution (v/v) with the final concentration of 0.2% according to the total volume of the bacterial liquid, placing the mixture at 37 ℃ for inactivation, stirring the mixture once every 3-4 h, taking the mixture out after 24h, and completely inactivating the mixture for later use.
Example 12 preparation of vaccine composition containing porcine circovirus type 3SG strain
The classical swine fever virus attenuated antigen prepared in example 5, the porcine pseudorabies virus attenuated antigen prepared in example 6, and the porcine reproductive and respiratory syndrome virus attenuated antigen prepared in example 8 were mixed with a heat-resistant protective agent (2 wt% gelatin aqueous solution and 15 wt% lactose aqueous solution at a ratio of 1:1 (v/v)) at a ratio of 1:1(v/v), and then sufficiently mixed, quantitatively packaged, and rapidly subjected to freeze vacuum drying, thus obtaining a classical swine fever virus live vaccine portion, a porcine pseudorabies virus live vaccine portion, and a porcine reproductive and respiratory syndrome virus live vaccine portion.
Slowly adding the porcine circovirus type 3SG strain inactivated antigen prepared in the example 4 into a water-soluble adjuvant Gel adjuvant (French Saybolt Corp.), continuously stirring for 12min by an emulsifying machine with the rotating speed of 800rpm in the adding process, and uniformly mixing to obtain the porcine circovirus type 3 inactivated vaccine part. In use, the live vaccine portion is diluted with the inactivated vaccine portion, and the ratio of the two antigens is shown in table 2, for example.
TABLE 2 composition ratio of porcine circovirus type 3SG strain-containing vaccine composition (live vaccine part)
Figure BDA0001318068550000151
The inactivated antigen of porcine circovirus type 3SG strain prepared in example 4, the inactivated antigen of porcine pseudorabies virus prepared in example 7, the inactivated antigen of porcine parvovirus prepared in example 9, the inactivated antigen of haemophilus parasuis prepared in example 10 and the inactivated antigen of mycoplasma hyopneumoniae prepared in example 11 are respectively taken, five antigens are prepared according to the final antigen content of the composition, added into a water-soluble adjuvant Gel adjuvant (Saibek corporation, France) and continuously stirred by an emulsifying machine with the rotating speed of 800rpm for 12min in the adding process and evenly mixed. The specific formulation and content of the vaccine composition are shown in table 3.
TABLE 3 composition ratio of porcine circovirus type 3SG strain-containing vaccine composition (inactivated vaccine part)
Figure BDA0001318068550000152
Figure BDA0001318068550000161
Example 13 immunogenicity test of vaccine compositions containing porcine circovirus type 3SG strain
1. Partial immunogenicity assay for porcine circovirus type 3
45 healthy piglets which are detected by ELISA for PCV2 and PCV3 antigens and negative antibodies at the age of 28-30 days are randomly divided into 9 groups, 5 groups and one group, and the vaccine group prepared in the immunization example 12A compound (I) is provided. The 3 rd to 10 th groups are respectively immune vaccines 1 to 8, and the 11 th group is used as a challenge control group without immunization. Each immunization group was injected with 2 ml/head of vaccine, and the challenge control group was inoculated with 2 ml/head of DMEM medium. Performing virus attack 28 days after immunization, wherein the virus attack dose is SG strain porcine circovirus 105.0TCID50And/or continuously observing each piglet after the virus attack, and judging according to the clinical symptoms, pathological changes and virus detection results of each piglet, wherein the specific results are shown in table 4.
TABLE 4 partial immunogenicity test results for vaccine composition PCV3 containing porcine circovirus type 3SG strain antigen
Figure BDA0001318068550000162
Figure BDA0001318068550000171
The result shows that the vaccine composition containing the porcine circovirus type 3SG strain antigen can provide 100 percent (5/5) protection for piglets aiming at porcine circovirus type 3 infection after the piglets are immunized once, and the piglets of an attacking control group are all attacked after the attack. The result shows that the porcine circovirus type 3 antigen part in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect, can provide 100% protection for pigs, and the virus detection is negative.
The porcine circovirus type 3SG strain antigen does not interfere with a plurality of viral antigens and bacterial antigens, and can be jointly used after forming a vaccine composition.
2. Partial immunogenicity test for hog cholera
The 20-day-old healthy piglets tested for PCV2, PCV3, CSFV antigen, antibody negative by ELISA were randomly divided into 2 groups of 8, 4/group. Group 12 immunization vaccine 1 prepared in example 12, and group 13 immunization as a challenge control group. Each 2ml vaccine 1 is diluted 3000 times, intramuscular injection is carried out, each 1ml, and a virus challenge control group is inoculated with 1ml of DMEM culture medium per head. After 10-14 days, 4 control groups with the same conditions were injected with 1.0 ml/head (10) of HCG-GIT5MLD), observed for 16 days. The results are shown in Table 5.
TABLE 5 partial immunogenicity test results for vaccine compositions CSFV containing porcine circovirus type 3SG strain antigen
Group of Immune head Rate of protection Clinical symptoms
12 1/3000 100％(4/4) No body temperature reaction
13 DMEM Medium 1ml 0％(0/4) All 4 deaths
According to the standard of Chinese veterinary drug, the immune efficacy of the classical swine fever virus vaccine is detected, the vaccine needs to be diluted by more than 300 times and then is detected, and the result of the embodiment shows that 1/3000 heads (diluted by 3000 times) of the vaccine composition containing the porcine circovirus type 3SG strain antigen can provide 100% (4/4) protection for piglets aiming at swine fever virus infection after the piglets are immunized by 1/3000 heads (diluted by 3000 times), and the piglets do not have body temperature reaction, but all the piglets in a virus attack control group are attacked and die. The swine fever virus antigen part in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect and safety.
3. Partial immunogenicity test for porcine pseudorabies virus
15 healthy piglets which were 9 days old and were tested for PCV2, PCV3, PRV antigen, and antibody negativity by ELISA were randomly divided into 3 groups, 5 groups, and 14 groups for immunization of vaccine 2 prepared in example 12, 15 groups for immunization of vaccine 4 prepared in example 12, and 16 groups for immunization as challenge control groups. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. The virus is attacked 21 days after the immunization, and the attacking dose is 10 of porcine pseudorabies virus HN1201 strain7.0TCID50The body temperature of piglets is measured every day after the challenge, and the clinical symptoms and death condition are observed, and the specific results are shown in table 6.
TABLE 6 partial immunogenicity test results for vaccine compositions containing porcine circovirus type 3SG strain antigen
Figure BDA0001318068550000181
The result shows that the vaccine composition containing the porcine circovirus type 3SG strain antigen can block virus infection (clinical symptoms) after immunizing piglets, can provide 100 percent (5/5) protection for the piglets aiming at the porcine pseudorabies virus infection, and all die 4 days after the piglets of an attacking control group are attacked by virus. The inactivated antigen or the live virus antigen part of the porcine pseudorabies virus in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect.
4. Partial immunogenicity test for porcine reproductive and respiratory syndrome virus
10 healthy piglets which are 4-6 weeks old and are detected to be PCV2, PCV3, PRRSV antigen and antibody negative by ELISA are randomly divided into 2 groups, 5 groups/group, 17 group is used for immunizing the vaccine 3 prepared in the example 12, and 18 group is used as a challenge control group. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. The virus is attacked 28 days after the immunization, and the attacking dose is 10 of porcine reproductive and respiratory syndrome virus NVDC-JXA1 strain5.0TCID50Head, daily thermometry and observation of clinical symptoms and mortality. The specific results are shown in Table 7.
TABLE 7 partial immunogenicity test results for vaccine compositions containing porcine circovirus type 3SG strain antigen PRRSV
Figure BDA0001318068550000191
The result shows that the vaccine composition containing the porcine circovirus type 3SG strain antigen can provide 100 percent (5/5) protection for piglets against porcine reproductive and respiratory syndrome virus infection after immunizing the piglets, and the piglets of the challenge control group are all attacked and die for 3 times after challenge. The porcine reproductive and respiratory syndrome virus antigen part in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect.
5. Partial immunogenicity assay for porcine parvovirus
10-20 kg of healthy piglets which are detected to be PCV2, PCV3, PPV antigen and antibody negative by ELISA are randomly divided into 2 groups, 5 groups/group, 19 group is used for immunizing the vaccine 5 prepared in the example 12, and 20 group is not immunized and used as a blank control group. The immunization group was injected with 2 ml/head of vaccine, and the blank control group was inoculated with 2 ml/head of DMEM medium. And (3) collecting blood together with a control group 28 days after immunization, and measuring the antibody, wherein the control group is negative (the HI antibody titer is less than or equal to 1:8), at least 4 heads of the immune group have antibody reaction, and the HI antibody titer is more than or equal to 1: 64. The specific results are shown in Table 8.
TABLE 8 partial immunogenicity test results for vaccine compositions containing porcine circovirus type 3SG strain antigen PPV
Figure BDA0001318068550000192
Figure BDA0001318068550000201
The results show that after the vaccine composition containing the porcine circovirus type 3SG strain antigen is used for immunizing piglets, the HI antibodies of the PPV are all more than 1:64, and 100% (5/5) protection can be provided for the piglets against porcine parvovirus infection. The porcine parvovirus antigen part in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect.
6. Partial immunogenicity assay for haemophilus parasuis
30 healthy piglets which are 14-21 days old and have negative antigens and antibodies of PCV2, PCV3 and PCV HPs detected by ELISA are randomly divided into 6 groups, 5 groups/group, 21 st and 22 nd groups for immunizing the vaccine 6 prepared in the example 12, 23 nd and 24 th groups for immunizing the vaccine 8 prepared in the example 12, and 25 th and 26 th groups for immunizing not to serve as challenge control groups. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. 35 days after immunization, 21 st group, 23 th group and 25 th group of the challenge control group were selected and challenged with 4 type JS strain, 3ml bacterial solution was intraperitoneally injected, and the dose of the challenge was 9.0 × 109CFU/head; collecting 22 nd and 24 th groups of immune group and 26 th group of challenge control group, performing challenge with 5 type ZJ strain, and performing intraperitoneal injection of 3ml of bacterial solution with dose of 6.0 × 109CFU/head; the clinical manifestations of the pigs were observed after the challenge, and the pigs were killed 14 days later for pathological observation. The specific results are shown in Table 9.
TABLE 9 partial immunogenicity test results for vaccine composition HPs containing porcine circovirus type 3SG strain antigen
Group of Number of piglets Virus attacking protection rate of type 4 JS strain Type 5 ZJ strain toxicity attacking protection rate
21 5 5/5 —
22 5 — 5/5
23 5 5/5 —
24 5 — 5/5
25 5 0/5 —
26 5 — 0/5
Note: the pathogenic standard of haemophilus parasuis disease: the sick pigs have clinical symptoms of fever (body temperature of more than 40.5 ℃ and lasting for 1-5 days), listlessness, cough, dyspnea, emaciation, lameness, rough fur and the like. The autopsy of the dying pig shows the pathological changes of multiple serositis (pleuritis, pericarditis and peritonitis), arthritis, meningitis and the like, and serous or cellulosic exudates appear on each serosal surface (joint capsule, pericardium, pleura and peritoneum).
The result shows that the vaccine composition containing the porcine circovirus type 3SG strain antigen can provide 100 percent (5/5) protection for piglets infected by haemophilus parasuis type 4 and type 5 after immunizing the piglets, and the piglets in the challenge control group are all attacked by the virus. The vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention is proved to have good protective effect on the antigen parts of the haemophilus parasuis type 4 and type 5.
7. Partial immunogenicity assay for Mycoplasma hyopneumoniae
15 healthy piglets which are 14-21 days old and have PCV2, PCV3, Mhp antigen and negative antibody detected by ELISA are randomly divided into 3 groups, 5 groups/group, 27 group is used for immunizing the vaccine 7 prepared in the example 12, 28 group is used for immunizing the vaccine 8 prepared in the example 12, and 29 group is used for immunizing and is used as a challenge control group. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. 35 days after immunization, 27 th group and 28 th group of the immune group and 29 th group of the virus challenge control group are taken, 5 ml/head (100MID) of CVCC354 strain (purchased from China veterinary medicine institute, the strain is a strain for swine mycoplasma pneumonia vaccine efficacy test stored in China veterinary medicine institute) is injected into a trachea, 28 days after virus challenge are observed, lungs are cut and taken, the asthma pneumonia lesion of the test pig is scored according to 28 minutes method, and the pneumonia lesion reduction rate is calculated according to the following formula. The specific results are shown in Table 10.
The pneumonia lesion reduction rate is (average score of virus attacking control pig pneumonia lesions-average score of immune pig pneumonia lesions)/average score of virus attacking control pig pneumonia lesions is multiplied by 100%
TABLE 10 partial immunogenicity test results for vaccine compositions containing porcine circovirus type 3SG strain antigen
Figure BDA0001318068550000211
Figure BDA0001318068550000221
Note: in the difference statistical analysis, compared among groups, the difference is not significant when the letters are the same, and the difference is significant when the letters are different (P < 0.05)
The results show that after the vaccine composition containing the porcine circovirus type 3SG strain antigen is used for immunizing piglets, aiming at the mycoplasma hyopneumoniae infection, the average lung lesion of an immune group and an attack control group is obviously different compared with the attack control group in each immune group. The mycoplasma hyopneumoniae antigen part in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect.
In conclusion, from the results of immunogenicity tests of the different vaccine compositions against PCV3, CSFV, PRV, PRRSV, PPV, HPs type 4 bacteria, HPs 5 type 5 bacteria and Mhp, each vaccine composition can achieve a better protective effect, which indicates that different antigen components in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention have a good protective effect. The inactivated antigen of the porcine circovirus type 3SG strain can be combined with different pathogenic antigen components to form an immune composition for co-administration.
Example 14 broad-spectrum protection assay for porcine circovirus type 3SG strain-containing vaccine compositions
225 healthy piglets with 28-30 days old and negative by PCV2 and PCV3 antigen detected by ELISA are randomly divided into 45 groups and 5 groups/group, 30-34 groups immunize the vaccine 1 prepared in the example 12, 35-39 groups immunize the vaccine 2 prepared in the example 12, 40-44 groups immunize the vaccine 3 prepared in the example 12, 45-49 groups immunize the vaccine 4 prepared in the example 12, 50-54 groups immunize the vaccine 5 prepared in the example 12, 55-59 groups immunize the vaccine 6 prepared in the example 12, 60-64 groups immunize the vaccine 7 prepared in the example 12, 65-69 groups immunize the vaccine 8 prepared in the example 12, and 70-74 groups are not immunized as an attack control group. Each immunization group was injected with 2 ml/head of vaccine, and the challenge control group was inoculated with 2 ml/head of DMEM medium. Performing virus challenge 28 days after immunization, and performing virus challenge on 30 th group, 35 th group, 40 th group, 45 th group, 50 th group, 55 th group, 60 th group, 65 th group and 70 th group by using porcine circovirus type 3 HN12 strain newly separated from Henan province of China; the 31 st, 36 th, 41 th, 46 th, 51 th, 56 th, 61 th, 66 th and 71 th groups were challenged with a virulent strain of porcine circovirus type 3 JS08 newly isolated from jiangsu province, china; group 32, group 37, group 42, group 47, group 52, group 57Group 62, group 67, and group 72 were challenged with virulent strains of porcine circovirus type 3 JL11 newly isolated from gillin province, china; group 33, group 38, group 43, group 48, group 53, group 58, group 63, group 68, group 73 were challenged with a virulent strain of porcine circovirus type 3 CQ04 newly isolated from chongqing city, china; group 34, group 39, group 44, group 49, group 54, group 59, group 64, group 69, group 74 were challenged with a virulent strain of porcine circovirus type 3 GD05 newly isolated from guangdong province of china; the dose of the antidote is 105.0TCID50And/or continuously observing each piglet after the challenge, and judging according to clinical symptoms, pathological changes and virus detection of each piglet, wherein specific results are shown in tables 11-19.
TABLE 11 broad Spectrum protection test results for vaccine 1
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
30 No abnormality No abnormality 0％(0/5) 100％(5/5)
31 No abnormality No abnormality 0％(0/5) 100％(5/5)
32 No abnormality No abnormality 0％(0/5) 100％(5/5)
33 No abnormality No abnormality 0％(0/5) 100％(5/5)
34 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 12 vaccine 2 broad-spectrum protection test results
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
35 No abnormality No abnormality 0％(0/5) 100％(5/5)
36 No abnormality No abnormality 0％(0/5) 100％(5/5)
37 No abnormality No abnormality 0％(0/5) 100％(5/5)
38 No abnormality No abnormality 0％(0/5) 100％(5/5)
39 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 13 vaccine 3 broad-spectrum protection test results
Figure BDA0001318068550000231
Figure BDA0001318068550000241
TABLE 14 vaccine 4 broad-spectrum protection test results
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
45 No abnormality No abnormality 0％(0/5) 100％(5/5)
46 No abnormality No abnormality 0％(0/5) 100％(5/5)
47 No abnormality No abnormality 0％(0/5) 100％(5/5)
48 No abnormality No abnormality 0％(0/5) 100％(5/5)
49 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 15 broad Spectrum protection test results for vaccine 5
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
50 No abnormality No abnormality 0％(0/5) 100％(5/5)
51 No abnormality No abnormality 0％(0/5) 100％(5/5)
52 No abnormality No abnormality 0％(0/5) 100％(5/5)
53 No abnormality No abnormality 0％(0/5) 100％(5/5)
54 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 16 broad-spectrum protection test results for vaccine 6
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
55 No abnormality No abnormality 0％(0/5) 100％(5/5)
56 No abnormality No abnormality 0％(0/5) 100％(5/5)
57 No abnormality No abnormality 0％(0/5) 100％(5/5)
58 No abnormality No abnormality 0％(0/5) 100％(5/5)
59 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 17 broad-spectrum protection test results for vaccine 7
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
60 No abnormality No abnormality 0％(0/5) 100％(5/5)
61 No abnormality No abnormality 0％(0/5) 100％(5/5)
62 No abnormality No abnormality 0％(0/5) 100％(5/5)
63 No abnormality No abnormality 0％(0/5) 100％(5/5)
64 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 18 broad Spectrum protection test results for vaccine 8
Group of Clinical symptoms Pathological changes Virus detection (Positive rate) Rate of protection
65 No abnormality No abnormality 0％(0/5) 100％(5/5)
66 No abnormality No abnormality 0％(0/5) 100％(5/5)
67 No abnormality No abnormality 0％(0/5) 100％(5/5)
68 No abnormality No abnormality 0％(0/5) 100％(5/5)
69 No abnormality No abnormality 0％(0/5) 100％(5/5)
TABLE 19 broad Spectrum protection test challenge control test results
Figure BDA0001318068550000251
Figure BDA0001318068550000261
The results show that after the 70 th to 74 th groups of virus attacking control groups are subjected to virus attacking, the body temperature rises by more than 40.5 ℃ to different degrees, the clinical symptoms such as anorexia, depression, rough and disordered hair, emaciation, slow growth speed and the like are kept for 3 to 5 days, pathological changes such as lung consolidation, lymphadenectasis and necrotic spots in the kidney to different degrees are generated in the autopsy, PCR detection is carried out on each organ tissue, and porcine circovirus type 3 virus can be separated again; and after 30 th to 69 th immune groups are subjected to toxicity attack, no abnormal clinical symptoms exist, no abnormality exists in tissues and organs after the autopsy, and PCV3 negativity is shown by PCR detection of all organ tissues. The vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention can provide effective and complete immune protection for pigs against porcine circovirus type 3 virus attack from different regional sources, and the PCV3 strain which is attacked cannot be detected from all visceral organs and tissues. The vaccine composition has broad-spectrum immunogenicity, and can completely protect porcine circovirus type 3 from different regional sources.
In conclusion, from the above test results of the immunogenicity of different vaccine compositions containing the porcine circovirus type 3SG strain antigen against porcine circovirus type 3 from different regions, each vaccine composition can achieve a better protection effect, which indicates that the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has a very good broad-spectrum protection effect.
Example 15 Mixed infection protection test with porcine circovirus type 3SG strain-containing vaccine composition
1. Porcine circovirus type 3 and classical swine fever virus mixed infection protection test
15 healthy piglets which are detected by ELISA at the age of 28-30 days for PCV2, PCV3 and CSFV antigen and antibody negative are randomly divided into 3 groups and 5 groups. Group 75 immunization of vaccine 1 prepared in example 12, and group 76 and group 77 immunization were used as challenge control groups. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. Performing virus attack 28 days after immunization, wherein the virus attack dose is SG strain porcine circovirus 105.0TCID501.0 ml/head of hog cholera-phylum blood poison (10)5MLD), 75 th group simultaneously attacks the porcine circovirus and the swine fever virus, 76 th group is a porcine circovirus attack control group, 77 th group is a swine fever virus attack control group,after the challenge, each piglet was continuously observed and judged according to clinical symptoms and pathological changes of each piglet, and the specific results are shown in table 20.
TABLE 20 protection test results for porcine circovirus type 3, classical swine fever virus
Figure BDA0001318068550000271
The result shows that after the piglet is immunized by the vaccine composition containing the porcine circovirus type 3SG strain antigen, 100% (5/5) protection can be provided for the piglet against the mixed infection of the porcine circovirus type 3 and the classical swine fever virus, and no body temperature response exists, while the piglets of two groups of virus challenge control groups are all attacked or die. The vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect and safety, and can effectively protect the mixed infection of the porcine circovirus type 3 and the hog cholera virus.
2. Porcine circovirus type 3 and porcine pseudorabies virus mixed infection protection test
20 healthy piglets at 9 days of age, tested by ELISA for PCV2, PCV3, PRV antigen, antibody negative were randomly divided into 4 groups, 5 groups/group. Group 78 immunization of vaccine 2 prepared in example 12, group 79 immunization of vaccine 4 prepared in example 12, and group 80 and group 81 immunization were performed as challenge control groups. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. Performing virus attack 28 days after immunization, wherein the virus attack dose is SG strain porcine circovirus 105.0TCID5010 of porcine pseudorabies virus HN12017.0TCID50And the 78 th group and the 79 th group simultaneously attack the porcine circovirus and the porcine pseudorabies virus, the 80 th group is a porcine circovirus attack control group, the 81 th group is a porcine pseudorabies virus attack control group, after attack, each piglet is continuously observed, and judgment is carried out according to the clinical symptoms, pathological changes and virus detection results of each piglet, and the specific results are shown in table 21.
TABLE 21 protection test results of porcine circovirus type 3, porcine pseudorabies virus mixed infection
Figure BDA0001318068550000281
The result shows that after the piglet is immunized by the vaccine composition containing the porcine circovirus type 3SG strain antigen, 100% (5/5) protection can be provided for the piglet aiming at the mixed infection of the porcine circovirus type 3 and the porcine pseudorabies virus, and the piglets of the two groups of challenge control groups are all attacked or die. The vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective efficacy and safety, and can effectively protect mixed infection of porcine circovirus type 3 and porcine pseudorabies virus.
3. Porcine circovirus type 3, porcine reproductive and respiratory syndrome virus mixed infection protection test
15 healthy piglets which are detected by ELISA for PCV2, PCV3, PRRSV antigen and antibody at the age of 28-30 days are randomly divided into 3 groups and 5 groups. Group 82 immunization of vaccine 3 prepared in example 12, and group 83 and 84 immunization as challenge control group. The immunization group is injected with 2 ml/head of vaccine, and the challenge control group is inoculated with 2 ml/head of DMEM medium. Performing virus attack 28 days after immunization, wherein the virus attack dose is SG strain porcine circovirus 105.0TCID50Porcine reproductive and respiratory syndrome Virus NVDC-JXA1 Strain 105.0TCID50And the 82 th group is simultaneously attacked and poisoned by porcine circovirus and porcine reproductive and respiratory syndrome virus, the 83 th group is a porcine circovirus attacking and poisoning control group, the 84 th group is a porcine reproductive and respiratory syndrome virus attacking and poisoning control group, after poisoning, each piglet is continuously observed and judged according to the clinical symptoms and pathological changes of each piglet, and the specific results are shown in a table 22.
TABLE 22 porcine circovirus type 3, porcine reproductive and respiratory syndrome virus mixed infection protection test results
Figure BDA0001318068550000291
The result shows that the vaccine composition containing the porcine circovirus type 3SG strain antigen can provide 100 percent (5/5) protection for piglets aiming at the mixed infection of the porcine circovirus type 3 and porcine reproductive and respiratory syndrome virus after the piglets are immunized by the vaccine composition, and the piglets of two groups of attacking control groups are all attacked by the virus and are all attacked by the disease or die. The vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective efficacy and safety, and can effectively protect the mixed infection of the porcine circovirus type 3 virus, the porcine reproductive and respiratory syndrome virus.
4. Porcine circovirus type 3, haemophilus parasuis and mycoplasma hyopneumoniae mixed infection protection test
25 healthy piglets at 21 days of age tested for PCV2, PCV3, HPs, Mhp antigen, antibody negative by ELISA were randomly divided into 5 groups, 5 heads/group. Group 85 immunization of vaccine 8 prepared in example 12, and group 86, 87, 88, and 89 immunization as challenge control group. The immunization group was injected with 2 ml/head of vaccine, and the control group was inoculated with 2 ml/head of DMEM medium. The virus is attacked 35 days after the immunization, and the toxin attacking dose is 10 SG strain porcine circovirus5.0TCID509.0X 10 JS strain of Haemophilus parasuis type 496.0X 10 of CFU/head, type 5 ZJ strain9CFU/head, Mycoplasma hyopneumoniae CVCC354 strain 100 MID/head, 85 th group simultaneously attacking porcine circovirus, haemophilus parasuis and Mycoplasma hyopneumoniae, 86 th group is a porcine circovirus attacking control group, 87 th group is a haemophilus parasuis type 4 attacking control group, 88 th group is a haemophilus parasuis type 5 attacking control group, 89 th group is a Mycoplasma hyopneumoniae attacking control group, after attacking, each piglet is continuously observed and judged according to clinical symptoms and pathological changes of each piglet, and specific results are shown in Table 23.
TABLE 23 protection test results of mixed infection of porcine circovirus type 3, Haemophilus parasuis, and Mycoplasma hyopneumoniae
Figure BDA0001318068550000301
Figure BDA0001318068550000311
Note: in the difference statistical analysis, compared among groups, the difference is not significant when the letters are the same, and the difference is significant when the letters are different (P < 0.05)
The result shows that the vaccine composition containing the porcine circovirus type 3SG strain antigen can provide 100 percent (5/5) protection for piglets aiming at the mixed infection of the porcine circovirus type 3, the haemophilus parasuis and the mycoplasma hyopneumoniae after immunizing the piglets, and the piglets of four groups of virus challenge control groups are all attacked after virus challenge. The vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention has good protective effect and safety, and can effectively protect mixed infection of porcine circovirus type 3, haemophilus parasuis and mycoplasma hyopneumoniae.
In conclusion, from the immunogenicity test results of different vaccine compositions containing the porcine circovirus type 3SG strain antigen aiming at the mixed infection of the porcine circovirus type 3 and other pathogens, each vaccine composition can achieve better protection effect, and the different antigen components in the vaccine composition containing the porcine circovirus type 3SG strain antigen provided by the invention have good protection effect.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (17)

1. A vaccine composition for preventing porcine circovirus type 3 mixed infection, wherein the vaccine composition comprises an immunizing amount of porcine circovirus type 3 antigen and a pharmaceutically acceptable carrier;
wherein the vaccine composition further comprises one or more of the group consisting of an immunizing amount of: classical swine fever virus antigen, porcine pseudorabies virus antigen, porcine influenza virus antigen, porcine reproductive and respiratory syndrome virus antigen, porcine parvovirus antigen, porcine encephalitis b virus antigen, haemophilus parasuis antigen, streptococcus suis antigen, bordetella suis antigen, porcine infectious pleuropneumonia antigen, porcine pasteurella multocida antigen, salmonella choleraesuis antigen, mycoplasma hyopneumoniae antigen, mycoplasma hyorhinis antigen; the porcine circovirus type 3 antigen is a porcine circovirus type 3SG strain or a culture inactivated whole virus antigen thereof, and the preservation number of the SG strain is CCTCC NO. V201712.
2. The vaccine composition according to claim 1, wherein the culture of porcine circovirus type 3SG strain is a culture of more than or equal to 1 generation.
3. The vaccine composition according to claim 1, wherein the culture of the porcine circovirus type 3SG strain is a culture of more than or equal to 5 generations.
4. The vaccine composition according to claim 1, wherein the culture of the porcine circovirus type 3SG strain is a 5-55 generation culture.
5. The vaccine composition according to claim 1, wherein the inactivated whole virus antigen content of the porcine circovirus type 3SG strain or the culture thereof is not less than 10 before inactivation5.0TCID50/ml。
6. The vaccine composition according to claim 1, wherein the inactivated whole virus antigen content of the porcine circovirus type 3SG strain or the culture thereof is 10 before inactivation5.0～107.0TCID50/ml。
7. The vaccine composition according to claim 1, wherein the inactivated whole virus antigen content of the porcine circovirus type 3SG strain or the culture thereof is 10 before inactivation6.0TCID50/ml。
8. The vaccine composition according to claim 1, wherein the antigens in the group comprised in the vaccine composition are: classical swine fever virus attenuated antigen, porcine pseudorabies virus attenuated antigen, porcine reproductive and respiratory syndrome virus attenuated antigen, porcine parvovirus inactivated antigen, haemophilus parasuis inactivated antigen, and mycoplasma hyopneumoniae inactivated antigen; or, the antigens in the group comprised in the vaccine composition are: the antigen comprises a classical swine fever virus attenuated antigen, a porcine pseudorabies virus inactivated antigen, a porcine reproductive and respiratory syndrome virus attenuated antigen, a porcine parvovirus inactivated antigen, a haemophilus parasuis inactivated antigen and a mycoplasma hyopneumoniae inactivated antigen.
9. The vaccine composition according to claim 8, wherein the attenuated antigen of classical swine fever virus is a lapinized attenuated strain of classical swine fever virus, the attenuated antigen of pseudorabies virus is an attenuated strain of HN1201-R strain, the inactivated antigen of pseudorabies virus is an inactivated whole virus antigen of HN1201 strain, the attenuated antigen of porcine reproductive and respiratory syndrome virus is an attenuated strain of JXA1-R strain, the inactivated antigen of porcine parvovirus is an inactivated whole virus antigen of HN-2011 strain, the inactivated antigen of haemophilus parasuis is an inactivated whole virus antigen of JS strain 4 or ZJ strain 5 of haemophilus parasuis, and the inactivated antigen of mycoplasma hyopneumoniae is an inactivated whole virus antigen of HN0613 strain.
10. The vaccine composition according to claim 8, wherein the inactivated antigen content of porcine circovirus is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml4.0～106.0TCID50Per ml, the content of the porcine pseudorabies virus attenuated antigen is 106.0～107.0TCID50The inactivated antigen content of the porcine pseudorabies virus is 10 before inactivation6.0～107.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 105.0～107.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation6.0～108.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation8.0～1010.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation8.0～1010.0CCU/ml。
11. The vaccine composition according to claim 8, wherein the inactivated antigen content of porcine circovirus is 10 before inactivation5.0～107.0TCID50The attenuated antigen content of the hog cholera virus is 10ml5.0TCID50Per ml, the content of the porcine pseudorabies virus attenuated antigen is 106.0TCID50The inactivated antigen content of the porcine pseudorabies virus is 10 before inactivation6.0TCID50The porcine reproductive and respiratory syndrome virus attenuated antigen content is 106.0TCID50The inactivated antigen content of the porcine parvovirus is 10 before inactivation7.0TCID50The content of the inactivated antigen of the haemophilus parasuis is 10 before inactivation9.0CFU/ml, the content of the mycoplasma hyopneumoniae inactivated antigen is 10 before inactivation9.0CCU/ml。
12. The vaccine composition according to claim 1, wherein the pharmaceutically acceptable carrier comprises an adjuvant, and the adjuvant content is 5-20V/V%.
13. The vaccine composition of claim 12, wherein the adjuvant is MontanideTMGel。
14. The vaccine composition according to claim 12, wherein the adjuvant content is 10V/V%.
15. Use of a vaccine composition according to any one of claims 1 to 14 in the manufacture of a medicament for the prevention of porcine circovirus type 3 associated disease.
16. The use of claim 15, wherein the porcine circovirus type 3 associated disease comprises postweaning multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, proliferative necrotizing pneumonia, reproductive disorders and cardiac and multisystemic inflammatory responses.
17. Use of a vaccine composition according to any one of claims 1 to 14 in the manufacture of a medicament for the prevention of disease caused by porcine circovirus type 3 mixed infection.
CN201710433747.XA 2017-06-09 2017-06-09 Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof Active CN109010817B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710433747.XA CN109010817B (en) 2017-06-09 2017-06-09 Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710433747.XA CN109010817B (en) 2017-06-09 2017-06-09 Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN109010817A CN109010817A (en) 2018-12-18
CN109010817B true CN109010817B (en) 2021-12-03

Family

ID=64629844

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710433747.XA Active CN109010817B (en) 2017-06-09 2017-06-09 Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN109010817B (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488895A (en) * 2011-12-30 2012-06-13 重庆大学 Porcine circovirus, porcine parvovirus and porcine reproductive and respiratory syndrome virus triple virus-like particle vaccine and its preparation method
CN102961742A (en) * 2012-01-13 2013-03-13 普莱柯生物工程股份有限公司 Bivalent inactivated vaccine of porcine circovirus type 2 and porcine parvovirus and preparation method thereof
WO2017066772A1 (en) * 2015-10-16 2017-04-20 Kansas State University Research Foundation Porcine circovirus type 3 immunogenic compositions and methods of making and using the same

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102488895A (en) * 2011-12-30 2012-06-13 重庆大学 Porcine circovirus, porcine parvovirus and porcine reproductive and respiratory syndrome virus triple virus-like particle vaccine and its preparation method
CN102961742A (en) * 2012-01-13 2013-03-13 普莱柯生物工程股份有限公司 Bivalent inactivated vaccine of porcine circovirus type 2 and porcine parvovirus and preparation method thereof
WO2017066772A1 (en) * 2015-10-16 2017-04-20 Kansas State University Research Foundation Porcine circovirus type 3 immunogenic compositions and methods of making and using the same

Also Published As

Publication number Publication date
CN109010817A (en) 2018-12-18

Similar Documents

Publication Publication Date Title
CN109125719B (en) Immunogenic composition containing porcine circovirus type 3 and porcine circovirus type 2 antigens and application thereof
CN107513506B (en) Mycoplasma hyopneumoniae, vaccine composition and application thereof
CN108653724B (en) Vaccine composition for preventing egg drop syndrome of poultry, and preparation method and application thereof
CN108220248B (en) Porcine rotavirus strain, vaccine composition, preparation method and application thereof
CN108785667B (en) Porcine circovirus type 3 immunogenic composition, preparation method and application
CN108653725B (en) Vaccine composition for preventing egg drop syndrome of poultry, and preparation method and application thereof
CN104250636B (en) A kind of cultural method of pig circular ring virus and its application
CN109207436B (en) Group I type 4 avian adenovirus strain and application thereof
CN107523556B (en) Avian adenovirus strain, vaccine composition and application thereof
CN109010818B (en) Bivalent vaccine composition for preventing and/or treating porcine circovirus infection and preparation method and application thereof
CN109134619B (en) Porcine circovirus type 2 antigen, immunogenic composition prepared from same, preparation method and application
CN112574958B (en) H9 subtype avian influenza virus isolate and application thereof
CN106929480B (en) Porcine reproductive and respiratory syndrome virus strain and application thereof
US10869919B2 (en) Porcine circovirus type 3 strain, vaccine composition, method of making the same and use thereof
CN109022368B (en) Porcine circovirus type 2 strain, vaccine composition, preparation method and application thereof
KR101102271B1 (en) Attenuated Avian Infectious Bronchitis Virus and Vaccine for Avian Infectious Bronchitis Comprising the Same
CN109010817B (en) Vaccine composition for preventing and/or treating porcine circovirus type 3 infection and preparation method and application thereof
CN109125720B (en) Immunogenic composition containing porcine circovirus type 3 antigen and application thereof
CN108624522B (en) Acinetobacter paragallinarum strain and application thereof
IQBAL et al. Immune response of rabbits to hemorrhagic septicemia vaccine formulations adjuvanted with montanide ISA-206, paraffin oil and alum
CN110713987B (en) Recombinant gene VII type Newcastle disease virus strain and vaccine composition, preparation method and application thereof
CN107865965B (en) Vaccine composition, and preparation method and application thereof
CN110343670B (en) Recombinant porcine pseudorabies virus attenuated strain for expressing porcine circovirus Cap protein gene, and preparation method and application thereof
CN114573708A (en) Avibacterium paragallinarum HA fusion protein and tripolymer thereof, vaccine composition prepared from avibacterium paragallinarum HA fusion protein, preparation method and application of vaccine composition
CN116790510A (en) Canine parainfluenza virus HeN20 strain, and product and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant