WO2018176887A1 - 一种猪圆环病毒3型毒株、及其疫苗组合物、制备方法和应用 - Google Patents

一种猪圆环病毒3型毒株、及其疫苗组合物、制备方法和应用 Download PDF

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WO2018176887A1
WO2018176887A1 PCT/CN2017/113945 CN2017113945W WO2018176887A1 WO 2018176887 A1 WO2018176887 A1 WO 2018176887A1 CN 2017113945 W CN2017113945 W CN 2017113945W WO 2018176887 A1 WO2018176887 A1 WO 2018176887A1
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strain
porcine circovirus
circovirus type
culture
vaccine composition
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PCT/CN2017/113945
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English (en)
French (fr)
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田克恭
李向东
肖燕
孙进忠
张许科
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普莱柯生物工程股份有限公司
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Priority to JP2019537797A priority Critical patent/JP6821818B2/ja
Priority to US16/483,941 priority patent/US10869919B2/en
Publication of WO2018176887A1 publication Critical patent/WO2018176887A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/55—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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C12N2750/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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

Definitions

  • the invention relates to a strain, a prepared vaccine composition, a preparation method and an application thereof, and belongs to the field of animal virology.
  • PCV Porcine circoviruses
  • PCV1 porcine circovirus type 1
  • PCV2 porcine circovirus type 2
  • PCV1 was first identified in PK cell culture as a contaminant in 1974 and was not pathogenic to pigs.
  • PCV2 was first reported in 1998, which can cause Porcine circovirus associated diseases (PCVAD) in pigs under clinical conditions, mainly causing piglet multisystemic wasting syndrome, pneumonia, porcine dermatitis and nephrotic syndrome.
  • PCVAD Porcine circovirus associated diseases
  • Reproductive disorders mainly manifested by respiratory, urinary, intestinal, lymphatic, cardiovascular, neurological, reproductive systems, and dysfunction of the skin, have caused significant economic losses to pig farming worldwide.
  • the pathogenic pathogen was isolated and confirmed to be a 2.0 kb circovirus with a viral genome, which was confirmed by subsequent experiments, whether it is a nucleotide or an amino acid with a known circovirus.
  • the homology of the sequences is less than 50%. According to the International Committee of Virology, the same virus in the genus Circovirus should have >75% homology to the genomic nucleotide sequence, and Cap protein >70%.
  • the amino acid sequence is homologous, so it is certain that this is a new porcine circovirus. It can cause dermatitis and nephrotic syndrome in pigs, reproductive disorders and heart and multi-system inflammatory reactions, so the preparation of new vaccines for this new virus is very important for disease control in pig farms.
  • the present invention provides a vaccine composition for preventing and/or treating a novel porcine circovirus infection, which provides effective protection against a novel porcine circovirus and exhibits significant immunity. characteristic.
  • porcine circovirus type 3 strain SG strain which is capable of effectively protecting an epidemic strain and providing complete porcine circovirus type 3 from different sources. protection.
  • Another object of the present invention is to provide a vaccine composition for preventing and/or treating a novel porcine circovirus infection comprising an immunological amount of porcine circovirus type 3 SG strain and inactivation of the culture thereof Antigen and carrier.
  • Another object of the present invention is to provide a method for preparing a porcine circovirus type 3 inactivated vaccine, comprising: step (1) proliferating porcine circovirus type 3 SG strain; step (2) inactivating said step (1)
  • the proliferating cultured porcine circovirus type 3 SG strain is emulsified by adding an adjuvant.
  • Another object of the present invention is to provide a use of the above vaccine composition for the preparation of a medicament for preventing and/or treating porcine circovirus type 3 related diseases.
  • the SG strain of the present invention has good immunogenicity, can stimulate the body to rapidly produce immunity, and effectively protects the attack of the epidemic strain, and has a good protective effect;
  • the present invention adopts the vaccine composition prepared by the current novel porcine circovirus type 3 strain for the first time, which can achieve better immunoprotection with lower antigen content and further reduce production cost;
  • the vaccine of the present invention can provide complete protection against porcine circovirus type 3 strains of different geographical origins, and has broad-spectrum protection ability;
  • the vaccine of the present invention can provide a therapeutic effect on pigs that have been infected with porcine circovirus type 3 virus and exhibit initial clinical symptoms.
  • Porcine circovirus type 3 is a genomic 2.0 kb circovirus with known circovirus Whether the nucleotide or amino acid sequence homology is less than 50%, it is a new porcine circovirus that causes porcine dermatitis and nephrotic syndrome, reproductive disorders, and heart and multi-system inflammatory responses.
  • An object of the present invention is to provide a Porcine Circovirus type 3 (strain SG) deposited in the China Center for Type Culture Collection under the accession number CCTCC NO.V201712, and the deposit date is March 23, 2017. Day, deposit address: Wuhan University, Wuhan, China.
  • the vaccine composition prepared for porcine circovirus type 3 may comprise an immunizing antigen, an attenuated whole virus antigen, a subunit antigen or a synthetic peptide antigen of an immunological amount of porcine circovirus type 3 or a culture thereof, and a coding subunit.
  • 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 to porcine circovirus type 3.
  • Inactivated antigen also known as an inactivated vaccine, refers to a suspension of inactivated virus that is used as an antigen to produce immunity.
  • inactivated vaccines include whole virus vaccines and lytic vaccines.
  • Inactivated vaccines can be readily produced using known methods. For example, a whole virus inactivated vaccine can be obtained by treating the virus with a formaldehyde solution. The lytic vaccine can be prepared from the viral envelope after treatment with ether.
  • Attenuated whole virus antigen refers to a virus that has been virulence weakened but still replicates in the host or on the cell.
  • the term "attenuated” as used in the present invention is used to artificially reduce pathogen toxicity by mutating a gene in a manner that causes the pathogen to lose pathogenicity but remains immunogenic. Typically, attenuation is achieved by UV radiation, chemical treatment, or continuous high-order subculture in vitro. Or an artificial genetic alteration, such as the deletion of a specific nucleotide in a known sequence to attenuate virulence.
  • Subunit antigen refers to an antigen prepared by genetically engineering a protective antigen gene of a pathogen into a prokaryotic or eukaryotic expression system for efficient expression. It is less likely to cause side effects than whole virus antigens.
  • Synthetic peptide antigen refers to a small peptide containing only immunological determinant components, that is, an antigen prepared by artificially synthesizing a protective short peptide in the amino acid sequence of the natural protein, and connecting the carrier with an adjuvant.
  • Autonomously replicable vector containing a gene encoding a subunit antigen protein means a non-pathogenic microorganism which is genetically engineered to carry and express an antigen or antigenic determinant gene to produce immunogenicity;
  • the foreign gene encoding the foreign protein of an antigenic protein is directly introduced into the animal, and the antigenic protein is synthesized by the expression system of the host cell, thereby inducing the host to generate an immune response to the antigenic protein for the purpose of prevention and treatment.
  • the independently replicable vector containing the gene encoding the subunit antigen protein may be a live vector or a plasmid.
  • Live carrier refers to a gene that non-pathogenic microorganisms carry to express and express an antigen or antigenic determinant by genetic engineering, and the non-pathogenic microorganisms may be bacteria and viruses.
  • the carrier viruses include vaccinia virus, fowlpox virus, turkey herpes virus, adenovirus, pseudorabies virus, retrovirus, lentivirus; the live carrier of bacteria may be attenuated Salmonella, BCG, attenuated monocyte Li Bacillus, attenuated Vibrio cholerae, attenuated Shigella, lactobacillus, lactobacillus bud, and Streptococcus mutans.
  • “Plasmid” refers to a eukaryotic expression vector containing a gene encoding a foreign antigen, which has a specific promoter to initiate the exogenous gene to be expressed at high levels in animals, and has various types, but mostly based on PUC or pBR322. These vectors all have an enhancer, a promoter, a vector selection marker, a translation initiation sequence, a transcription termination sequence, and a PolyA element, and the foreign antigen gene thereon can induce the body to produce protective immunity.
  • the present invention relates to a vaccine composition, wherein the vaccine composition comprises an immunized amount of the porcine circovirus type 3 SG strain or an inactivated antigen thereof, an attenuated whole virus antigen, a subunit antigen or A synthetic peptide antigen, and an independently replicable vector comprising a gene encoding a subunit antigen protein, and a pharmaceutically acceptable carrier.
  • the present invention relates to a vaccine composition, wherein the vaccine composition comprises an immunized amount of the inactivated antigen of the porcine circovirus type 3 SG strain or a culture thereof and a pharmaceutically acceptable carrier.
  • the pig ring The inactivated antigen of the virus type 3 SG strain or the culture thereof is an inactivated whole virus antigen of the porcine circovirus type 3 virus SG strain.
  • the amount of the component or component of the composition of the 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 excessive side effects.
  • the precise amount of ingredients used and compositions to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal to be treated, the mode of administration, and other ingredients in the composition.
  • the inactivated whole virus antigen content of the porcine circovirus type 3 SG strain or the culture thereof is ⁇ 10 5.0 TCID 50 /ml before inactivation.
  • porcine circovirus type 3 When porcine circovirus type 3 is used in an amount less than 10 5.0 TCID 50 , the vaccine does not effectively stimulate antibody production. On the other hand, the excess may be uneconomical.
  • the inactivated whole virus antigen content of the porcine circovirus type 3 SG strain or the culture thereof is 10 5.0 to 10 7.0 TCID 50 /ml before inactivation.
  • the inactivated whole virus antigen content of the porcine circovirus type 3 SG strain or the culture thereof is 10 6.0 TCID 50 /ml before inactivation.
  • the inactivated whole virus antigen content of the porcine circovirus type 3 SG strain or the culture thereof may also be selected from the range of 10 5.0 to 10 6.0 TCID 50 /ml, or 10 6.0 to 10 7.0 TCID. 50 / ml.
  • the culture of the porcine circovirus type 3 SG strain is a culture of ⁇ 1 generation.
  • the culture of the porcine circovirus type 3 SG strain is a culture of ⁇ 5 passages.
  • the culture of the porcine circovirus type 3 SG strain is a culture of 5 to 55 passages.
  • the pharmaceutically acceptable carrier includes an adjuvant
  • the adjuvant includes (1) aluminum hydroxide, saponin, affine, DDA, (2) a polymer of acrylic acid or methacrylic acid, a polymer of maleic anhydride and an alkenyl derivative, (3) an oil-in-water emulsion, a water-in-oil emulsion or a water-in-oil-in-water emulsion, or (4) ) Montanide TM Gel.
  • the adjuvant includes (1) saponin QuilA; (2) a polyalkenyl group of an acrylic acid or methacrylic acid polymer and a sugar or a polyhydric alcohol.
  • Ether cross-linking product carbomer (3) the adjuvant comprises an emulsion based on light liquid paraffin oil, isoprenoid oil, such as squalane or squalene; olefin, especially isobutylene or decene oligomerization
  • the adjuvant is Montanide TM Gel, the volume ratio of the adjuvant in an amount of 5 to 20%; more preferably, the adjuvant in an amount of 10% by volume.
  • the adjuvants include white oil, Drake oils, and animal oils, vegetable or mineral oil; or aluminum hydroxide, aluminum phosphate and metal salts thereof; or Montanide TM Gel, carbomer, squalane or squalene, ISA206 Adjuvants, saponins, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions.
  • the adjuvant is Montanide TM Gel, the volume ratio of the adjuvant in an amount of 5 to 20%; more preferably, the adjuvant in an amount of 10% by volume.
  • the carrier in the vaccine composition of the present invention is an adjuvant comprising white oil, Drakeoil, and other animal oils, vegetable oils or mineral oils; or aluminum hydroxide, aluminum phosphate and others. metal salt; or Montanide TM Gel, carbomer, squalane or squalene, ISA206 adjuvant, saponin, oil emulsion, oil in water emulsions, water-in-oil emulsion.
  • the vaccine compositions of the invention may be formulated using available techniques, preferably together with a veterinary acceptable carrier.
  • oil can help stabilize the formulation and additionally act as a vaccine adjuvant.
  • Oil adjuvants can be either natural or synthetically obtained.
  • adjuvant refers to a substance that is added to the compositions of the present invention to increase the immunogenicity of the composition.
  • Known adjuvants include, but are not limited to, (1) aluminum hydroxide, saponine (eg QuilA), affine, DDA, (2) polymer of acrylic acid or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (3) the vaccine may be an oil in water, water-oil or water-in-oil emulsion made in the form, or (4) Montanide TM Gel.
  • the emulsion may be based on light liquid paraffinic oils, isoprenoid oils such as squalane or squalene; olefins, especially those produced by oligomerization of isobutylene or decene, linear alkyl acid or Esters formed by alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di(octanoate/caprate), triglyceride (caprylate/caprate), propylene glycol dioleate; branched fatty acid esters Or an ester of an alcohol, especially an isostearate.
  • the oil is used with an emulsifier to form an emulsion.
  • the emulsifier is preferably a nonionic surfactant, in particular a polyoxyethylated fatty acid (for example oleic acid), sorbitan, mannitol (for example dehydrated mannitol oleate), glycerol, polyglycerol, propylene glycol and optionally B.
  • a nonionic surfactant in particular a polyoxyethylated fatty acid (for example oleic acid), sorbitan, mannitol (for example dehydrated mannitol oleate), glycerol, polyglycerol, propylene glycol and optionally B.
  • the acrylic or methacrylic polymer is crosslinked by a polyalkenyl ether of a sugar or a polyol. These compounds are called carbomers.
  • the present invention is selected adjuvant Montanide TM Gel.
  • the amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount.
  • the "effective amount” refers to the amount necessary for the adjuvant to be necessary or sufficient to exert its immunological effects in the host when administered in combination with the antigen of the present invention without causing excessive side effects.
  • the precise amount of adjuvant to be administered will vary depending on factors such as the ingredients employed and the type of disease being treated, the type and age of the animal to be treated, the mode of administration, and other ingredients in the composition.
  • the present invention also relates to a method of preparing the vaccine composition, wherein the method comprises: step (1) proliferating and culturing the porcine circovirus type 3 SG strain or a culture thereof; and step (2) inactivating In the step (1), the cultured porcine circovirus type 3 SG strain or a culture thereof is cultured, and an adjuvant is added to the inactivated porcine circovirus type 3 SG strain or a culture thereof. emulsification.
  • compositions of the present invention may further incorporate additional agents into the compositions of the present invention.
  • the composition of the present invention may further comprise the following agents, such as: drugs, immunostimulants (eg, ⁇ -interferon, ⁇ -interferon, ⁇ -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.
  • agents such as: drugs, immunostimulants (eg, ⁇ -interferon, ⁇ -interferon, ⁇ -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, propellant
  • the vaccine composition according to the invention may be prepared as an oral dosage form or a parenteral dosage form.
  • parenteral dosage forms which can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.
  • the present invention also relates to the use of the above vaccine composition for the preparation of a medicament for preventing and/or treating a porcine circovirus type 3 related disease.
  • porcine circovirus type 3 related disease as used in the present invention is used to mean a disease caused by porcine circovirus type 3 infection.
  • Non-exhaustive includes, but is not limited to, porcine dermatitis and nephrotic syndrome, reproductive disorders, and inflammatory responses to the heart and multiple systems.
  • prevention refers to all actions which inhibit the porcine circovirus type 3 infection or delay the onset of the disease by administering a vaccine composition according to the present invention.
  • treatment refers to all behaviors that alleviate or ameliorate the symptoms caused by porcine circovirus type 3 infection by administering a vaccine composition according to the present invention.
  • the disease material was added to the DMEM culture solution at a ratio of 1:10 (volume ratio), and ground to prepare a tissue suspension.
  • the tissue suspension was centrifuged at 12000 r/min for 15 min after repeated freeze-thaw cycles, and the supernatant was collected, and the supernatant was collected. After filtration through a 0.22 ⁇ m filter, the filtrate was passaged on PK15 cells, cultured at 37 ° C for 1 h, replaced with DMEM medium containing 2% yak serum, and cultured at 37 ° C for 5 days.
  • the virus-containing culture solution was harvested, and after two freeze-thaw cycles, the virus was harvested.
  • the virus culture of the above step was taken, and the nucleic acid of the virus sample was extracted with a nucleic acid extraction kit, and PCR amplification was performed using a circovirus-specific primer. The result showed that a 2000 bp target band was amplified by PCR.
  • the PCR product was sent to a sequencing company for nucleotide sequence determination, and the sequence determination results were subjected to genetic evolution analysis.
  • porcine circovirus type 3 design-specific primers 235 suspected PCV3 positive samples collected from all over the country were analyzed by quantitative PCR, and 121 PCV3 viruses were isolated and screened.
  • the genomic nucleotides of the 121 strains were isolated.
  • the sequence homology is as high as 98.9 ⁇ 99.6%, and the amino acid sequence homology of Cap protein is as high as 97.7 ⁇ 99.5%.
  • a strain of PCV3 strain with strong pathogenicity, good immunogenicity and broad spectrum was screened out.
  • the porcine circovirus type 3 virus was named porcine circovirus type 3 SG strain and submitted for preservation.
  • the cultures of the porcine circovirus type 3 SG strain screened in Example 2 were inserted into the monolayer-derived PK15 passage cells at 1% (V/V) of the amount of the virus culture solution, and adsorbed at 37 ° C. Minutes, then add the cell maintenance solution and incubate at 37 °C. The cells were observed 1 to 2 times a day, and the cells grew well. After the cells were cultured at 36 to 37 ° C for 4 to 7 days, the cell cultures were harvested, and the harvested cell cultures were frozen and thawed 2-3 times, and the virus solution was harvested to determine the virus virulence.
  • the virus solution was filtered through a hollow fiber (0.5 ⁇ m to 2 ⁇ m) filter column to remove cell debris, and then inactivated by adding 0.1% to 0.2% formaldehyde solution at 37 ° C for 24 hours to inactivate the complete virus antigen to prepare a vaccine.
  • the inactivated antigen of the porcine circovirus type 3 SG strain prepared in Example 4 was slowly added to the water-soluble adjuvant gel adjuvant (Sybex, France), and the mixture was continuously stirred for 12 minutes with an emulsifier at 800 rpm. , mix.
  • the specific formulation of the vaccine is shown in Table 2.
  • Example 5 Twenty-five healthy piglets with PCV2, PCV3 antigen and antibody negative by ELISA at 28 to 30 days of age were randomly divided into 5 groups, 5 groups/group, and the porcine circovirus type 3 SG strain inactivated vaccine prepared in Example 5 was immunized. Groups 3 to 6 were immunized with vaccines 1 to 4, respectively, and group 7 was not immunized as a control group. Each immunization group was injected with 2 ml/head of vaccine, and the control group was inoculated with 2 ml/head of DMEM medium. On the 28th day after immunization, the challenge was SG strain porcine circovirus 10 5.0 TCID 50 / head. After the challenge, each piglet was observed continuously, and the results were determined according to the clinical symptoms, pathological changes and virus detection results of each piglet. See Table 3.
  • porcine circovirus type 3 SG strain inactivated vaccine can be used for piglets after immunization of piglets. 100% (5/5) protection was provided, while all control piglets developed symptoms after challenge. It shows that the inactivated vaccine of porcine circovirus type 3 SG strain provided by the invention has good protection.
  • Example 7 Broad-spectrum protection test of inactivated vaccine against porcine circovirus type 3 SG strain
  • the challenge was carried out on the 28th, and the 8th and 13th groups were challenged with the virulent strain of porcine circovirus type 3 HN12 strain newly isolated from Henan province of China; the 9th and 14th groups were newly used from Jiangsu province, China.
  • the isolated porcine circovirus type 3 JS08 strain virulent strain was challenged; the 10th and 15th groups were challenged with the porcine circovirus type 3 JL11 strain virulent strain isolated from Jilinzhou, China; the 11th group, the first group Group 16 was challenged with a virulent strain of porcine circovirus type 3 CQ04 strain isolated from Chongqing, China; Group 12 and group 17 were newly virulent strains of porcine circovirus type 3 GD05 strain isolated from Guangdong province, China.
  • the attacking dose was 10 5.0 TCID 50 /head. After the challenge, each piglet was continuously observed, and the clinical symptoms, pathological changes and virus detection of each piglet were determined. The specific results are shown in Table 4.
  • the porcine circovirus type 3 virus was isolated by PCR detection of each organ tissue; and the 8th to 12th groups were immunized. There were no abnormal clinical symptoms after the attack, and no abnormalities were observed in all tissues and organs. PCR detection was performed on each organ, and PCV3 was negative.
  • the PCV3 strain that was attacked could not be detected from each organ tissue.
  • the vaccine compositions of the present invention have a broad spectrum of immunogenicity and provide complete protection against porcine circovirus type 3 from a variety of geographic sources.
  • the immunized group was injected with 2 ml/head of vaccine, and the blank control group was inoculated with 2 ml/head of DMEM medium.
  • the results of the two groups of sows were counted. The results are shown in Table 5.
  • the immunized group had no abnormalities in the production of sows, producing healthy piglets with an average of 11.85 heads/well, and the health rate was as high as 99.6%.
  • the control group showed obvious mummies and weak piglets.
  • the average number of healthy piglets was 7.7 per litter, and the health rate was 66.1%.
  • the difference between the immunized group and the control group was significant.
  • the piglets from the control group B were isolated and nested separately, and 18 litters were divided into two groups: group (including B-1 litter to B-17 litter, except for B-5 litter and B-13 litter. Except for a total of 15 litters of piglets, B2 (including B-18 to B-20 litter, a total of 3 litters), the B1 group was immunized with the porcine circovirus type 3 SG inactivated vaccine prepared in Example 5 before breastfeeding. 1, group B2 is a blank control group. The immunized group was injected with 2 ml/head of vaccine, and the blank control group was inoculated with 2 ml/head of DMEM medium. Each piglet was continuously observed and judged according to the clinical symptoms, pathological changes and virus detection of each piglet. The specific results are shown in Table 6.
  • PCR detection was performed by necropsy of the organs of the pigs, all of which showed PCV3 negative, while the control piglets showed different degrees of body temperature rise. 40.5 °C or more, lasting 3 to 5 days, clinical symptoms such as loss of appetite, depression, rough hair, weight loss and slowing of growth, some pigs died, and different degrees of lung consolidation, lymphadenopathy, kidney Pathological changes of necrotic spots, PCR detection of each organ tissue, can be isolated again to porcine circovirus type 3.

Abstract

一种猪圆环病毒3型病毒毒株，以及该毒株的免疫原性物质制备的疫苗组合物，该猪圆环病毒3型病毒株免疫原性好，其制备的疫苗组合物能针对不同来源的猪圆环病毒3型病毒提供完全的保护。

Description

一种猪圆环病毒3型毒株、及其疫苗组合物、制备方法和应用 技术领域
本发明涉及一种毒株、制备的疫苗组合物、制备方法和应用，属于动物病毒学领域。
背景技术
猪圆环病毒(Porcine circoviruses，PCV)是单股环状的DNA病毒，基因组长度约为1.7kb，是最小的动物DNA病毒之一。已经确定有两种类型的PCV，即猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2)。PCV1于1974年首次在PK细胞培养物中作为一种污染物鉴定而发现，其对猪只没有致病性。PCV2于1998年首次报道，其在临床条件下能引起猪只的猪圆环病毒相关疾病(Porcine circovirus associated diseases，PCVAD)，主要引起仔猪多系统衰竭综合征，肺炎，猪皮炎和肾病综合征和繁殖障碍，主要表现为呼吸、泌尿、肠道、淋巴、心血管、神经、繁殖系统以及皮肤的功能紊乱，对全世界的生猪养殖造成了重大的经济损失。
然而，在一起猪的繁殖障碍病例中，分离并确认致病病原为一株病毒基因组为2.0kb的圆环病毒，经后续试验证实，其与已知的圆环病毒无论在核苷酸还是氨基酸序列的同源性均小于50％，根据国际病毒学分类委员会的标准，圆环病毒属中同一种的病毒应该具有＞75％的基因组核苷酸序列的同源性，Cap蛋白＞70％的氨基酸序列的同源性，因此可以肯定这是一种新的猪圆环病毒。其能引起猪的皮炎肾病综合征、繁殖障碍及心脏和多系统的炎症反应，因此针对该新病毒制备新的疫苗，对猪场疾病控制十分重要。
发明内容
为解决现有技术的不足，本发明提供一种预防和/或治疗新型猪圆环病毒感染的疫苗组合物，该疫苗组合物能对新型猪圆环病毒提供有效的保护，显示出显著的免疫特性。
为此，本发明的一个目的在于提供一种猪圆环病毒3型毒株SG株，其制备的疫苗组合物能有效保护流行株的攻击，且针对不同来源的猪圆环病毒3型提供完全的保护。
本发明的另一个目的在于提供一种预防和/或治疗新型猪圆环病毒感染的疫苗组合物，所述疫苗组合物包含免疫量的猪圆环病毒3型SG株及其培养物的灭活抗原以及载体。
本发明的另一个目的是提供一种猪圆环病毒3型灭活疫苗的制备方法，包括：步骤(1)增殖猪圆环病毒3型SG株；步骤(2)灭活所述步骤(1)的所述增殖培养的猪圆环病毒3型SG株，加入佐剂，乳化。
本发明的另一个目的在于提供上述疫苗组合物在制备预防和/或治疗猪圆环病毒3型相关疾病的药物中的应用。
发明优点：
(1)本发明毒株SG株具有良好的免疫原性，能够刺激机体快速地产生免疫力，有效保护流行株的攻击，具有很好的保护作用；
(2)本发明首次采用目前新的猪圆环病毒3型流行的毒株制备的疫苗组合物，能以较低的抗原含量达到较好的免疫保护作用，进一步降低生产成本；
(3)本发明的疫苗，能针对不同地域来源的猪圆环病毒3型毒株提供完全保护，具有广谱的保护能力；
(4)本发明的疫苗，能针对已经感染了猪圆环病毒3型病毒并表现初期临床症状的猪提供治疗作用。
具体实施方式
以下，对本发明的实施方式进行说明。
猪圆环病毒3型为基因组2.0kb的圆环病毒，其与已知的圆环病毒 无论在核苷酸还是氨基酸序列的同源性均小于50％，是一种新的猪圆环病毒，其能引起猪的皮炎肾病综合征、繁殖障碍及心脏和多系统的炎症反应。
本发明的一个目的在于提供猪圆环病毒3型SG株(Porcine Circovirus type 3，strain SG)，保藏于中国典型培养物保藏中心，保藏号为CCTCC NO.V201712，保藏日期为2017年3月23日，保藏地址：中国武汉·武汉大学。
针对猪圆环病毒3型制备的疫苗组合物，可以包含免疫量的猪圆环病毒3型或其培养物的灭活抗原、减毒全病毒抗原、亚单位抗原或合成肽抗原、含有编码亚单位抗原蛋白的基因的可独立复制的载体以及药学上可以接受的载体。
“培养物”是病毒的不同代次传代培养物，本领域技术人员知晓不同代次之间其基因序列仅可能会发生微小的变异。
“疫苗组合物”指含有猪圆环病毒3型免疫原性的药物组合物。该药物组合物可诱发、刺激或增强猪只针对猪圆环病毒3型的免疫反应。
“灭活抗原”，也称作失活疫苗，指的是用作抗原以产生免疫力的灭活病毒的混悬液。灭活疫苗的例子包括全病毒疫苗和裂解型疫苗。使用已知方法可以很容易地产生灭活疫苗。例如，通过用甲醛溶液处理病毒可获得全病毒灭活疫苗。裂解型疫苗可在用醚处理后由病毒包膜制备得到。
“减毒全病毒抗原”指的是以毒力已经减弱但仍可在宿主体内或细胞上复制的病毒。本发明所用的术语“减毒”用于指以使病原丧失致病性、但保持免疫原性的方式对基因进行突变来人工降低病原体毒性。通常，通过UV辐射、化学处理或体外连续高阶继代培养实现减毒。或者是人工的基因改变，例如将已知序列中的特定核苷酸缺失以使毒力减弱。
“亚单位抗原”指的是利用基因工程方法将病原体的保护性抗原基因克隆到原核或真核表达系统中，使其高效表达而制成的抗原。它比全病毒抗原引起副反应的可能性小。
“合成肽抗原”指的是一种仅含免疫决定簇组分的小肽，即用人工方法按天然蛋白质的氨基酸顺序合成保护性短肽，与载体连接后加佐剂所制成的抗原。
“含有编码亚单位抗原蛋白的基因的可独立复制的载体”指的是非致病微生物通过基因工程的方法使之携带并表达某种抗原或抗原决定簇的基因，产生免疫原性；也可以是编码某种抗原蛋白的外源蛋白的外源基因直接导入动物体内，并通过宿主细胞的表达系统合成抗原蛋白，诱导宿主产生对该抗原蛋白的免疫应答，以达到预防和治疗的目的。
含有编码亚单位抗原蛋白的基因的可独立复制的载体可以为活载体或质粒。
“活载体”指的是非致病微生物通过基因工程的方法使之携带并表达某种抗原或抗原决定簇的基因，产生免疫原性，非致病微生物可以是细菌和病毒，病毒活载体常作为载体的病毒有痘苗病毒、禽痘病毒、火鸡疱疹病毒、腺病毒、伪狂犬病毒、反转录病毒、慢病毒；细菌活载体可以是减毒沙门菌、卡介苗、减毒单核细胞李氏杆菌、减毒霍乱弧菌、减毒志贺氏菌、乳酸球菌、芽胚乳酸杆菌、高氏链球菌。
“质粒”指的是含有外源抗原编码基因的真核表达载体，其具有特定的启动子启动外源基因在动物体内高水平表达，其有多种种类，但多以PUC或pBR322为基本骨架，这些载体均具有增强子、启动子、载体的选择标记、翻译起始序列、转录终止序列和PolyA等元件，其上的外源抗原基因能够诱发机体产生保护性免疫。
本发明涉及一种疫苗组合物，其中，所述疫苗组合物包含免疫量的所述的猪圆环病毒3型SG株或其培养物的灭活抗原、减毒全病毒抗原、亚单位抗原或合成肽抗原、以及含有编码亚单位抗原蛋白的基因的可独立复制的载体以及药学上可以接受的载体。
本发明涉及一种疫苗组合物，其中，所述疫苗组合物包括免疫量的所述猪圆环病毒3型SG株或其培养物的灭活抗原和药学上可以接受的载体。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述猪圆环 病毒3型SG株或其培养物的灭活抗原为猪圆环病毒3型病毒SG株灭活全病毒抗原。
本发明的组合物的成分或组分的量优选地是治疗有效量。所述治疗有效量是指在组合物施用的宿主中发挥它们的免疫学作用而不导致过度副作用所必需量。所用的成分和待施用的组合物的精确的量将根据因素如治疗的疾病的类型，待治疗的动物的类型和年龄，施用的方式，以及组合物中的其它成分而变化。
作为本发明的一种实施方式，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量为灭活前≥105.0TCID50/ml。
当猪圆环病毒3型以少于105.0TCID50的量使用时，疫苗不能有效刺激抗体产生。另一方面，超过的量可能是不经济的。
作为本发明的一种优选实施方式，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量为灭活前105.0～107.0TCID50/ml。
作为本发明的一种更优选实施方式，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量为灭活前106.0TCID50/ml。
本发明疫苗组合物中，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量范围还可选自105.0～106.0TCID50/ml，或106.0～107.0TCID50/ml。
作为本发明的一种实施方式，所述的疫苗组合物中，所述猪圆环病毒3型SG株的培养物为≥1代次的培养物。
作为本发明的一种优选实施方式，所述的疫苗组合物中，所述猪圆环病毒3型SG株的培养物为≥5代次的培养物。
作为本发明的一种更优选实施方式，所述的疫苗组合物中，所述猪圆环病毒3型SG株的培养物为5～55代次的培养物。
作为本发明的一种实施方式，本发明所述疫苗组合物中，所述药学上可接受的载体包括佐剂，所述佐剂包括(1)氢氧化铝、皂苷、阿夫立定、DDA，(2)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的聚合物，(3)水包油乳剂、油包水乳剂或水包油包水乳剂，或(4)MontanideTMGel。
作为本发明的一种优选实施方式，本发明所述疫苗组合物中，所述佐剂包括(1)皂苷QuilA；(2)丙烯酸或甲基丙烯酸聚合物与糖或多元醇的聚链烯基醚交联产物卡波姆；(3)所述佐剂包括基于轻液体石蜡油、类异戊二烯油的乳剂，例如角鲨烷或角鲨烯；烯烃，特别是异丁烯或癸烯低聚化产生的油，带直链烷基的酸或醇形成的酯，更特别是植物油，油酸乙基酯，丙二醇二(辛酸酯/癸酸酯)，甘油三(辛酸酯/癸酸酯)，丙二醇二油酸酯；分支脂肪酸酯或醇的酯，特别是异硬脂酸酯、油与乳化剂一起使用形成乳剂，乳化剂优选非离子表面活性剂；特别是聚氧乙烯化脂肪酸(例如油酸)，脱水山梨糖醇、甘露醇(例如脱水甘露醇油酸酯)、甘油、聚甘油、丙二醇和可选地乙氧基化的油酸、异硬脂酸、蓖麻油酸、羟基硬脂酸形成的酯，脂肪醇和多元醇(例如油醇)的醚，聚氧丙稀-聚氧乙烯嵌段共聚物，特别是PluronicR，尤其是L121；或(4)MontanideTMGel。
优选地，所述佐剂为MontanideTMGel，所述佐剂用量为体积比5～20％；更优选地，所述佐剂用量为体积比10％。
所述佐剂包括白油、德雷克油，以及动物油、植物油或矿物油；或氢氧化铝、磷酸铝及金属盐；或MontanideTM Gel、卡波姆、角鲨烷或角鲨烯、ISA206佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂。
优选地，所述佐剂为MontanideTMGel，所述佐剂用量为体积比5～20％；更优选地，所述佐剂用量为体积比10％。
本发明所述疫苗组合物中的所述载体为佐剂，所述佐剂包括白油、德雷克油(Drakeoil)，以及其他动物油、植物油或矿物油；或氢氧化铝、磷酸铝及其它金属盐；或MontanideTM Gel、卡波姆、角鲨烷或角鲨烯、ISA206佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂。
本发明的疫苗组合物可使用可用技术来调配，优选为兽医学上可接受的载体一起调配。例如，油可有助于稳定调配物，且另外充当疫苗佐剂。油佐剂既可以是自然来源，也可以是经过人工合成获得的。术语“佐 剂”指加入到本发明的组合物中以增加组合物的免疫原性的物质。已知的佐剂包括，但不限于：(1)氢氧化铝、皂苷(Saponine)(例如QuilA)、阿夫立定、DDA，(2)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的聚合物，(3)疫苗可以以水包油、油包水或水包油包水乳剂形式制成，或(4)MontanideTMGel。
尤其是，乳剂可以基于轻液体石蜡油、类异戊二烯油，例如角鲨烷或角鲨烯；烯烃，特别是异丁烯或癸烯低聚化产生的油，带直链烷基的酸或醇形成的酯，更特别是植物油，油酸乙基酯，丙二醇二(辛酸酯/癸酸酯)，甘油三(辛酸酯/癸酸酯)，丙二醇二油酸酯；分支脂肪酸酯或醇的酯，特别是异硬脂酸酯。油与乳化剂一起使用形成乳剂。乳化剂优选非离子表面活性剂，特别是聚氧乙烯化脂肪酸(例如油酸)，脱水山梨糖醇、甘露醇(例如脱水甘露醇油酸酯)、甘油、聚甘油、丙二醇和可选地乙氧基化的油酸、异硬脂酸、蓖麻油酸、羟基硬脂酸形成的酯，脂肪醇和多元醇(例如油醇)的醚，聚氧丙稀-聚氧乙烯嵌段共聚物，特别是PluronicR，尤其是L121(参照Hunter等，1995，“The Theory and Practical Application of Adjuvants”(Steward-Tull，D.E.S主编)John Wiley and Sons，NY，51-94；Todd等，Vaccine，1997，15，564-570)。
特别地，丙烯酸或甲基丙烯酸聚合物通过糖或多元醇的聚链烯基醚交联。这些化合物被称作卡波姆。
优选地，本发明选用的佐剂为MontanideTMGel。
适用于本发明的组合物的佐剂的量优选地是有效量。所述“有效量”是指佐剂在同本发明抗原联合施用时在宿主中发挥它们的免疫学作用而言必须或足够的而不导致过度副作用所必需量。待施用的佐剂的精确的量将根据因素如所用的成分和治疗的疾病的类型，待治疗的动物的类型和年龄，施用的方式，以及组合物中的其它成分而变化。
本发明还涉及一种制备所述疫苗组合物的方法，其中，所述方法包括：步骤(1)增殖培养所述猪圆环病毒3型SG株或其培养物；以及步骤(2)灭活所述步骤(1)增殖培养的猪圆环病毒3型SG株或其培养物，在所述灭活后的猪圆环病毒3型SG株或其培养物中加入佐剂， 乳化。
本发明疫苗组合物还可以进一步将其他的试剂加入到本发明的组合物中。例如，本发明的组合物还可以包含以下试剂，如：药物，免疫刺激剂(如：α-干扰素、β-干扰素、γ-干扰素、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)和白介素2(IL2))、抗氧化剂、表面活性剂、着色剂、挥发性油、缓冲剂、分散剂、推进剂和防腐剂。为了制备这样的组合物，可以使用本领域公知的方法。
可以根据本发明的疫苗组合物制备成口服剂型或非口服剂型。
优选的是可通过皮内、肌肉、腹膜内、静脉内、皮下、鼻内或硬脑膜外途径给予的非口服剂型。
本发明还涉及上述疫苗组合物在制备预防和/或治疗猪圆环病毒3型相关疾病的药物中的应用。
本发明所用术语“猪圆环病毒3型相关疾病”用于指由猪圆环病毒3型感染引起的疾病。非穷举性包括，猪的皮炎肾病综合征、繁殖障碍及心脏和多系统的炎症反应，但不限于此。
本发明所用的术语“预防”指通过给予根据本发明的疫苗组合物抑制猪圆环病毒3型感染或推迟疾病发作的所有行为。术语“治疗”指通过给予根据本发明的疫苗组合物使猪圆环病毒3型感染引起的症状减轻或好转的所有行为。
实施例1猪圆环病毒3型的分离鉴定
1、病料来源
在国内一商品化猪场，与历史平均值相比，出现母猪死亡率增加9.4％，受孕率降低了1.2％，木乃伊胎增加8.2％的现象。临床表现上，受影响的母猪表现出厌食，呈多灶性丘疹，斑点和表面皮炎的症状。在流产的窝中含有不同胎龄的木乃伊化胎儿，与PCV2相关流产的症状一致。虽然在母猪中观察到的总体临床表现以及流产症状与猪圆环病毒2型导致的繁殖障碍疾病一致，但所有母猪的不同组织，包括肾、淋巴结、肺、皮肤以及死胎，通过免疫组化和定量PCR对PCV2、PRRSV、PPV、 CSFV、猪肺炎支原体检测均为阴性。为进一步查清原因，选取各组织病料进行病原分离。
2、病毒株的分离与培养
将病料以1∶10(体积比)加入DMEM培养液，研磨，制备组织悬液，组织悬液经反复3次冻融后，于12000r/min离心15min，收集上清液，上清液再经0.22μm滤膜滤器过滤，滤液在PK15细胞上传代，37℃培养1h，替换加入含2％犊牛血清的DMEM培养液，于37℃培养5日。收获含病毒的培养液，经2次冻融后，收获病毒。
3、PCR及测序分析鉴定病毒种属
取以上步骤的病毒培养物，用核酸提取试剂盒提取病毒样品的核酸，使用圆环病毒种属特异性引物进行PCR扩增鉴定，结果显示，PCR扩增出2000bp目的条带。PCR产物送测序公司进行核苷酸序列测定，序列测定结果进行遗传进化分析。结果显示该病毒株全基因组序列和氨基酸序列同已经报道过的其他圆环病毒的同源性都少于50％，而根据国际病毒学分类委员会的标准，圆环病毒属中同一种的病毒应该具有＞75％的基因组核苷酸序列的同源性，Cap蛋白＞70％的氨基酸序列的同源性，因此可以肯定，它是一种新的猪圆环病毒，也是目前猪体上发现的第三种圆环病毒。
实施例2猪圆环病毒3型疫苗株的筛选
根据上述分离到的猪圆环病毒3型设计特异性引物，通过定量PCR分析从全国各地采集的235份疑似PCV3阳性样品，筛选分离出121株PCV3病毒，这121株中，其基因组核苷酸序列同源性高达98.9～99.6％，Cap蛋白氨基酸序列同源性高达97.7～99.5％。经本动物致病性试验及免疫原性测试，最终筛选出1株致病性强、免疫原性良好、广谱性强的PCV3病毒株。该猪圆环病毒3型病毒命名为猪圆环病毒3型SG株，提交保藏。
实施例3猪圆环病毒3型SG株致病性试验
用28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪10头随机分成两组，5头/组，第1组用PCV3SG株(含105.0TCID50/头)攻毒，肌肉注射，空白对照组接种DMEM培养基，各组仔猪隔离饲养。攻毒后连续观察各组仔猪，根据其临床症状、病理变化和病毒检测进行判定，具体结果见表1。
表1 猪圆环病毒3型SG株对仔猪致病性试验结果
Figure PCTCN2017113945-appb-000001
Figure PCTCN2017113945-appb-000002
结果显示，攻毒组所有猪只均出现3～5日持续性40.5℃以上高温，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒3型病毒，而空白对照组无异常，表明本发明猪圆环病毒3型SG株接种仔猪均可导致仔猪发病。
实施例4猪圆环病毒3型SG株抗原的制备
将实施例2筛选到的猪圆环病毒3型SG株不同代次的培养物按照病毒培养液量的1％(V/V)接入形成单层的PK15传代细胞中，置37℃吸附30分钟，然后加入细胞维持液，置37℃培养。每日观察1～2次，细胞生长良好，置36～37℃培养4～7日后收获细胞培养物，收获的细胞培养物冻融2-3次后，收获病毒液，测定病毒毒价。将病毒液用中空纤维(0.5μm～2μm)滤柱过滤，以除去细胞碎片，再加入0.1％～0.2％的甲醛溶液在37℃灭活24h，灭活完全后的病毒抗原用以制备疫苗。
实施例5猪圆环病毒3型SG株灭活疫苗的制备
用实施例4制备的猪圆环病毒3型SG株灭活抗原缓缓加入到水溶性佐剂gel佐剂(法国赛比克公司)中，加的过程中不断用转速为800rpm乳化机搅拌12min，混匀。疫苗具体配方见表2。
表2 猪圆环病毒3型SG株灭活疫苗配方及含量
Figure PCTCN2017113945-appb-000003
实施例6 猪圆环病毒3型SG株灭活疫苗免疫原性试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪25头随机分成5组，5头/组，免疫实施例5制备的猪圆环病毒3型SG株灭活疫苗。第3～6组分别免疫疫苗1～4，第7组不免疫作为对照组。各免疫组注射疫苗2ml/头，对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，攻毒剂量为SG株猪圆环病毒105.0TCID50/头，攻毒后连续观察各仔猪，根据各仔猪临床症状、病理变化和病毒检测结果进行判定，具体结果见表3。
表3 猪圆环病毒3型SG株灭活疫苗免疫原性试验结果
Figure PCTCN2017113945-appb-000004
结果显示，猪圆环病毒3型SG株灭活疫苗免疫仔猪后，能为仔猪 提供100％(5/5)保护，而对照仔猪攻毒后全部发病。表明本发明提供的猪圆环病毒3型SG株灭活疫苗具有很好的保护力。
实施例7猪圆环病毒3型SG株灭活疫苗广谱性保护试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪50头随机分成10组，5头/组，第8～12组免疫实施例5制备的猪圆环病毒3型SG株灭活疫苗1，第13～17组不免疫作为对照组。各免疫组注射疫苗2ml/头，对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，第8组、第13组用新近从中国河南省分离的猪圆环病毒3型HN12株强毒株攻毒；第9组、第14组用新近从中国江苏省分离的猪圆环病毒3型JS08株强毒株攻毒；第10组、第15组用新近从中国吉林省分离的猪圆环病毒3型JL11株强毒株攻毒；第11组、第16组用新近从中国重庆市分离的猪圆环病毒3型CQ04株强毒株攻毒；第12组、第17组用新近从中国广东省分离的猪圆环病毒3型GD05株强毒株攻毒；攻毒剂量为105.0TCID50/头，攻毒后连续观察各仔猪，根据各仔猪临床症状、病理变化和病毒检测进行判定，具体结果见表4。
表4 猪圆环病毒3型SG株灭活疫苗广谱性保护试验结果
Figure PCTCN2017113945-appb-000005
Figure PCTCN2017113945-appb-000006
结果显示，第13～17组对照组攻毒后均不同程度出现体温升高40.5℃以上，持续3～5天，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓等临床症状，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点的病理变化，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒3型病毒；而第8～12组免疫组攻毒后无异常临床症状，剖检各组织器官也无异常，通过各脏器组织进行PCR检测，均显示PCV3阴性。
上述结果表明本发明提供的猪圆环病毒3型SG株灭活疫苗可对猪只针对不同地域来源的猪圆环病毒3型攻毒提供有效的、完全免疫保 护，且从各脏器组织中不能检出攻毒的PCV3株。本发明的疫苗组合物具有广谱的免疫原性，对于不同地域来源的猪圆环病毒3型均能提供完全保护。
实施例8猪圆环病毒3型SG株灭活疫苗应用试验
在国内一商品化猪场，与历史平均值相比，出现母猪死亡率增加9.4％，受孕率降低了1.2％，木乃伊胎增加8.2％的现象。临床表现上，受影响的母猪表现厌食，呈多灶性丘疹，斑点和表面皮炎的症状。在流产的窝中含有不同胎龄的木乃伊化胎儿，将具有临床表现症状的母猪中选取40头怀孕母猪随机分成两组：A组、B组，20头/组，A组为疫苗接种组，A组免疫实施例5制备的猪圆环病毒3型SG株灭活疫苗1，B组为空白对照组。免疫组注射疫苗2ml/头，空白对照组接种DMEM培养基2ml/头。统计两组母猪产子情况，结果见表5。
表5 母猪产子统计结果
Figure PCTCN2017113945-appb-000007
Figure PCTCN2017113945-appb-000008
结果显示，免疫组母猪产子无异常，生产健康仔猪，平均11.85头/窝，健康率高达99.6％，而对照组出现明显的木乃伊胎及弱仔猪情况， 生产健康仔猪平均7.7头/窝，健康率66.1％，其中还有两头母猪出现流产情况，全窝木乃伊胎，免疫组和对照组差异显著。
表5的结果证明了本发明猪圆环病毒3型SG株灭活疫苗对感染猪圆环病毒3型的母猪有着良好的免疫保护作用，且能针对已经感染了PCV3病毒的母猪进行保护。
同时，对B组对照组所产仔猪分别隔离按窝饲养，18窝分成两组：组(包括B-1窝至B-17窝，除B-5窝和B-13窝因全窝木乃伊胎不计外，共15窝仔猪)、B2组(包括B-18至B-20窝，共3窝仔猪)，B1组吃母乳之前免疫实施例5制备的猪圆环病毒3型SG株灭活疫苗1，B2组为空白对照组。免疫组注射疫苗2ml/头，空白对照组接种DMEM培养基2ml/头。连续观察各仔猪，根据各仔猪临床症状、病理变化和病毒检测进行判定，具体结果见表6。
表6 猪圆环病毒3型SG株灭活疫苗对仔猪的免疫保护试验
Figure PCTCN2017113945-appb-000009
Figure PCTCN2017113945-appb-000010
Figure PCTCN2017113945-appb-000011
结果显示，免疫组仔猪无异常临床症状，随机剖检各组织器官也无异常，通过剖检猪只各脏器组织进行PCR检测，均显示PCV3阴性，而对照组仔猪均不同程度出现体温升高40.5℃以上，持续3～5天，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓等临床症状，部分猪只死亡，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点的病理变化，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒3型。
由于PCV3在猪群中是可以通过垂直传播的，因此表6的结果证明了本发明猪圆环病毒3型SG株灭活疫苗对感染猪圆环病毒3型的仔猪有着良好的免疫保护作用，且能针对已经感染了PCV3病毒的仔猪进行保护，保护率为100％。
以上所述仅是本发明的优选实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以优选实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (14)

  1. 猪圆环病毒3型SG株，保藏于中国典型培养物保藏中心，保藏号为CCTCC NO.V201712，保藏日期为2017年3月23日，保藏地址：中国武汉·武汉大学。
  2. 一种疫苗组合物，其中，所述疫苗组合物包括免疫量的所述猪圆环病毒3型SG株或其培养物的灭活抗原和药学上可以接受的载体。
  3. 根据权利要求2所述的疫苗组合物，其中，所述猪圆环病毒3型SG株或其培养物的灭活抗原为猪圆环病毒3型病毒SG株或其培养物的灭活全病毒抗原。
  4. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株灭活全病毒抗原或其培养物的含量为灭活前≥105.0TCID50/ml。
  5. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环3型病毒SG株或其培养物的灭活全病毒抗原含量为灭活前105.0～107.0TCID50/ml。
  6. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量为灭活前106.0TCID50/ml。
  7. 根据权利要求2或3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株的培养物为≥1代次的培养物。
  8. 根据权利要求7所述的疫苗组合物，其中，所述猪圆环病毒3型SG株的培养物为≥5代次的培养物。
  9. 根据权利要求8所述的疫苗组合物，其中，所述猪圆环病毒3型SG株的培养物为5～55代次的培养物。
  10. 根据权利要求2或3所述的疫苗组合物，其中，所述药学上可接受的载体包括佐剂，所述佐剂包括白油、德雷克油，以及动物油、植物油或矿物油；或氢氧化铝、磷酸铝及金属盐；或MontanideTMGel、卡波姆、角鲨烷或角鲨烯、ISA206佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂。
  11. 根据权利要求10所述的疫苗组合物，其中，优选地，所述佐 剂为MontanideTMGel，所述佐剂用量为体积比5～20％。
  12. 根据权利要求11所述的疫苗组合物，其中，所述佐剂用量为体积比10％。
  13. 一种制备权利要求2或3所述的疫苗组合物的方法，其中，所述方法包括：
    步骤(1)增殖培养所述猪圆环病毒3型SG株或其培养物；以及
    步骤(2)灭活所述步骤(1)增殖培养的猪圆环病毒3型SG株或其培养物，在所述灭活后的猪圆环病毒3型SG株或其培养物中加入佐剂，乳化。
  14. 根据权利要求2～12所述的疫苗组合物在制备预防和/或治疗猪圆环病毒3型相关疾病的药物中的应用。
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