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WO2018233264A1 - 一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物及其应用 - Google Patents

一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物及其应用 Download PDF

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Publication number
WO2018233264A1
WO2018233264A1 PCT/CN2017/118696 CN2017118696W WO2018233264A1 WO 2018233264 A1 WO2018233264 A1 WO 2018233264A1 CN 2017118696 W CN2017118696 W CN 2017118696W WO 2018233264 A1 WO2018233264 A1 WO 2018233264A1
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WIPO (PCT)
Prior art keywords
porcine circovirus
circovirus type
immunogenic composition
type
cap protein
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PCT/CN2017/118696
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English (en)
French (fr)
Inventor
田克恭
袁于人
李向东
殷波
肖燕
陆洲
胡冬学
孙进忠
莫小兵
张许科
Original Assignee
普莱柯生物工程股份有限公司
斯澳生物科技（苏州）有限公司
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Priority to JP2020519165A priority Critical patent/JP6882602B2/ja
Publication of WO2018233264A1 publication Critical patent/WO2018233264A1/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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A61K2039/5258—Virus-like particles
    • 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
    • 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 belongs to the field of veterinary medicine, and particularly relates to an immunogenic composition containing porcine circovirus type 3 and porcine circovirus type 2 antigen and application thereof.
  • 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 multiple systemic exhaustion syndrome, pneumonia, porcine dermatitis and nephrotic syndrome in weaned piglets.
  • reproductive disorders mainly manifested by respiratory, urinary, intestinal, lymphatic, cardiovascular, neurological, reproductive systems, and dysfunction of the skin, causing significant economic losses to pig farming worldwide.
  • PCV2 Clinically, with the widespread use of PCV2 vaccine, the variability of PCV2 is accelerated under immunological stress, and a new type of strain between PCV2b and PCV2d is becoming popular.
  • the virus is characterized by the ORF2 gene.
  • the PCV2 strain is fully protected.
  • the present invention provides an immunogenic composition for preventing and/or treating mixed infection of porcine circovirus, which is capable of mixing porcine circovirus type 2 and type 3 Infection provides effective protection and shows significant immune characteristics.
  • the present invention adopts the new porcine circovirus type 3 immunogenic protein antigen and the porcine circovirus type 2 new gene subtype strain immunogenic protein antigen for the first time to prepare for prevention and/or treatment of porcine circovirus infection.
  • the immunogenic composition not only protects against infection or mixed infection of porcine circovirus type 2 and porcine circovirus type 3, but also protects against different subtypes of porcine circovirus type 2 strain.
  • the immunogenic composition of the invention can provide complete protection against mixed infection of porcine circovirus type 3 strain and porcine circovirus type 2 strain of different geographical origins, and has broad-spectrum protection ability.
  • porcine circovirus type 3 is a genomic 2.0 kb circovirus that is less than 50% homologous to known circoviruses, whether nucleotide or amino acid sequences, and is a new porcine ring.
  • the virus which can be mixed with a variety of pathogens, causes porcine dermatitis and nephrotic syndrome, proliferative necrotizing pneumonia, reproductive disorders, and heart and multi-system inflammatory responses.
  • “porcine circovirus type 2 new gene subtype” refers to a novel subtype of PCV2 gene, which has a mutation in the ORF2 gene or is recombined between ORF2 genes of different gene subtypes, and has a tag sequence of PCV2b. However, in the genetic evolution analysis, an independent branch is formed.
  • the clinical manifestations of pigs infected with the subtype of the gene include: persistent high temperature, loss of appetite, depression, rough hair, weight loss and slowing of growth. There were different degrees of lung consolidation, lymphadenopathy, and necrosis of the kidney.
  • the present invention relates to an immunogenic composition
  • an immunogenic composition comprising porcine circovirus type 3 and porcine circovirus type 2 antigen
  • the immunogenic composition comprises an immunological amount of porcine circovirus type 3 Cap protein antigen, An immunological amount of porcine circovirus type 2 new gene subtype Cap protein antigen and a pharmaceutically acceptable carrier; wherein the porcine circovirus type 3 Cap protein antigen is porcine circovirus type 3 Cap protein or recombinant a live vector of the porcine circovirus type 3 Cap protein gene, the porcine circovirus type 2 new gene subtype Cap protein antigen is a porcine circovirus type 2 new gene subtype Cap protein or recombinantly has the pig ring A live vector of the viral type 2 Cap protein gene.
  • the immunogenic composition of the present invention is immunized once, and the antigenic component thereof provides complete protection against the respective pathogens, and protects the pig from prevention and resistance against mixed infection of porcine circovirus type 3 and porcine circovirus type 2.
  • the immunogenic compositions of the present invention provide complete protection against mixed infection of porcine circovirus type 3 and porcine circovirus type 2 of different gene subtypes.
  • the immunogenic composition of the present invention protects pigs from complete infection of mixed infections of porcine circovirus type 3 virus and porcine circovirus type 2 virus from different regions.
  • immunogenic composition refers to a pharmaceutical composition comprising porcine circovirus type 3, porcine circovirus type 2 immunogenicity, which induces, stimulates or enhances pigs against porcine circovirus type 3 , the immune response of porcine circovirus type 2 virus.
  • immuno amount is understood to mean “immunologically effective amount”, also known as an immunoprotective amount or an effective amount for generating an immune response, which is an amount of an antigen effective to induce an immune response in a recipient, which amount is sufficient to prevent or ameliorate the signs of the disease. Or symptoms, including adverse health effects or complications.
  • the immune response may be sufficient for diagnostic or other testing, or may be suitable for preventing signs or symptoms of the disease, including adverse health outcomes or complications resulting from infection by the pathogen. Humoral immunity or cell-mediated immunity or both can be induced.
  • the immune response of an animal to an immunogenic composition can be assessed indirectly by, for example, measuring antibody titer, lymphocyte proliferation assay, or directly by monitoring the signs or symptoms after challenge with a wild-type strain, which is immunogenic.
  • the protective immunity provided by the composition can be assessed by measuring, for example, clinical signs of the test animal, such as a reduction in healthy litter size, an increase in the number of stillbirths, overall physiological condition of the test subject, and overall health and performance.
  • the immune response can include, but is not limited to, induction of cellular and/or humoral immunity.
  • the porcine circovirus type 3 Cap protein antigen and the porcine circovirus type 2 Cap protein antigen of the present invention may be recombinantly expressed Cap protein subunit antigen, and the expression system thereof may be a prokaryotic expression system or a eukaryotic expression system. It may be a synthetic peptide antigen synthesized by artificial synthesis; or may be a live vector recombinant with the porcine circovirus Cap protein gene.
  • 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, i.e., an artificially synthesized method in which a protective short peptide is synthesized in the amino acid sequence of a natural protein, and an antigen is prepared by attaching an adjuvant to a carrier.
  • 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.
  • the porcine circovirus type 3 Cap protein is a sequence SEQ The protein encoded by .ID NO.1, the porcine circovirus type 2 new gene subtype Cap protein is the protein encoded by SEQ.ID NO.
  • the immunogenic proteins of the invention are immunogenic and provide complete protection with one immunization.
  • the porcine circovirus type 3 Cap protein encodes the gene. Having the nucleotide sequence of SEQ. ID NO. 1 or the degenerate sequence thereof, the porcine circovirus type 2 Cap protein has the nucleotide sequence of SEQ. ID NO. 2 or Degenerate sequence.
  • the porcine circovirus type 3 Cap protein content is ⁇ 20 ⁇ g. /ml; the porcine circovirus type 2 new gene subtype Cap protein content is ⁇ 20 ⁇ g / ml.
  • the immune system when the antigen content is as low as 20 ⁇ g/ml, the immune system can be stimulated to generate an immune response, and the pig is completely protected.
  • the porcine circovirus type 3 Cap protein content is 20 ⁇ g. /ml ⁇ 100 ⁇ g / ml; the porcine circovirus type 2 new gene subtype Cap protein content is 20 ⁇ g / ml ⁇ 100 ⁇ g / ml.
  • the porcine circovirus type 3 Cap protein antigen content It is 20 ⁇ g/ml to 50 ⁇ g/ml; the porcine circovirus type 2 new gene subtype Cap protein content is 20 ⁇ g/ml to 50 ⁇ g/ml.
  • the porcine circovirus type 3 Cap protein antigen content It is 30 ⁇ g/ml to 50 ⁇ g/ml; the porcine circovirus type 2 new gene subtype Cap protein content is 30 ⁇ g/ml to 50 ⁇ g/ml.
  • the porcine circovirus type 3 Cap protein antigen content may also be selected from 20 ⁇ g/ml to 30 ⁇ g/ml, 30 ⁇ g/ml to 100 ⁇ g/ml, or 50 ⁇ g/ml to 100 ⁇ g/ Ml content range.
  • the porcine circovirus type 2 new gene subtype Cap protein antigen content may also be selected from 20 ⁇ g/ml to 30 ⁇ g/ml, 30 ⁇ g/ml to 100 ⁇ g/ml, or 50 ⁇ g/ Ml ⁇ 100 ⁇ g / ml content range.
  • the porcine circovirus type 3 Cap protein antigen content and the porcine circovirus type 2 new gene subtype Cap protein antigen content may be independently selected from any of the above contents. range.
  • the recombinant porcine circovirus type 3 Cap protein gene is recombined.
  • the living vector is a recombinant attenuated Salmonella, a recombinant Newcastle disease virus, a recombinant poxvirus, or a recombinant adenovirus;
  • the living vector recombinantly having the porcine circovirus type 2 new gene subtype Cap protein gene is a recombinant attenuated Salmonella, Recombinant Newcastle disease virus, recombinant poxvirus, or recombinant adenovirus.
  • the porcine circovirus type 3 Cap protein gene and the The porcine circovirus type 2 new gene subtype Cap protein gene is recombined in the same living vector, which is recombinant attenuated Salmonella, recombinant Newcastle disease virus, recombinant poxvirus, or recombinant adenovirus.
  • the live vector immunogenic composition of the present invention has the advantages of both an inactivated vaccine and a live vaccine, and is capable of ensuring the protection of pigs in terms of immunological efficacy, and is more immunologically effective without the addition of an adjuvant.
  • the pharmaceutically acceptable carrier comprises an adjuvant, and the adjuvant Including white oil, drake oil, and animal oil, vegetable oil or mineral oil; or aluminum hydroxide, aluminum phosphate and metal salts; or Montanide TM Gel, carbomer, squalane or squalene, ISA206 adjuvant, saponin , water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
  • the immunogenic composition of porcine circovirus type 2 antigen of the present invention is 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 of adjuvant necessary or sufficient to exert their immunological effects in the host when administered in combination with an antigen of the present invention without causing excessive side effects.
  • the precise amount of adjuvant to be administered will vary depending on various 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 adjuvant content is 5 to 20 V/V%.
  • the adjuvant content is 10 V/V%.
  • the immunogenic compositions of the invention may be formulated using conventional techniques, preferably together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, 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; oils produced by oligomerization of olefins, especially 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.
  • 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.
  • compositions of the invention may further incorporate additional agents into the compositions of the 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, propellants,
  • the immunogenic composition according to the present invention can be prepared into an oral dosage form or a non-oral dosage form.
  • Non-oral dosage forms are preferably administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.
  • the present invention also relates to the use of the above immunogenic composition for the preparation of a medicament for preventing and/or treating a porcine circovirus-related disease, wherein the porcine circovirus-associated disease is caused by PCV2 and porcine circovirus type 3 alone Infected or related diseases caused by their mixed infection.
  • the preparation of the medicament for preventing and/or treating porcine circovirus-related diseases is for preparing and preventing or treating the weaned piglet multisystemic wasting syndrome, porcine dermatitis and nephrotic syndrome.
  • porcine circovirus-associated disease as used in the present invention is used to mean a disease caused by a mixed infection of porcine circovirus types 3 and 2.
  • Non-exhaustive includes, but not limited to, weaned piglet multisystemic wasting syndrome, porcine dermatitis and nephrotic syndrome, reproductive disorders, and cardiac and multi-system inflammatory responses.
  • porcine circovirus type 3 and type 2 mixed infections means inhibition of porcine circovirus type 3 and type 2 replication, inhibition of porcine circovirus type 3 and type 2 transmission or Prevents porcine circovirus types 3 and 2 from colonizing in their hosts, as well as alleviating the symptoms of diseases or conditions of porcine circovirus type 3 and type 2 infection. If the viral load is reduced, the condition is alleviated, and/or the food intake and/or growth is increased, then the prevention and/or treatment can be considered to have achieved an effect.
  • the present invention also relates to the use of the above immunogenic composition for the preparation of a medicament for preventing and/or treating a mixed infection of porcine circovirus.
  • the PCV2 gene subtype is a PCV2a, PCV2b, PCV2d, PCV2new gene subtype.
  • the immunogenic composition of the invention can provide effective protection against different PCV2 gene subtypes and PCV3, expand the application range of the vaccine, and can prevent and/or treat individual infection and mixed infection of different PCV2 gene subtypes and PCV3.
  • the immunogenic composition of the present invention has good immunogenicity, which not only prevents porcine circovirus-related diseases, but also alleviates the clinical characterization of pigs that have been infected with porcine circovirus, that is, has a therapeutic effect.
  • the chemical reagents used in the examples of the present invention are of analytical grade and are purchased from Sinopharm Group.
  • 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. After repeated three cycles of freezing and thawing, the tissue suspension was centrifuged at 12000 r/min for 15 min, 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 the culture solution was freeze-thawed twice, 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.
  • the whole genome sequence and amino acid sequence of the virus strain were less than 50% homologous to other circoviruses already reported, and according to the standards of the International Virological Classification Committee, the same virus in the genus Cyclone should be With >75% homology of the genomic nucleotide sequence, the Cap protein has >70% amino acid sequence homology, which is affirmative, it is a new porcine circovirus, also on the current pig body The third ring of viruses found.
  • the plasmid extraction kit was purchased from Tiangen Bio; the T4 DNA Ligase was purchased from BioLab; the pET28a plasmid was purchased from Novagen; the agarose gel gel recovery kit was purchased from Tianze Bio, and the other reagents were of analytical grade.
  • porcine circovirus type 3 virus solution was taken in a sterile 1.5 ml centrifuge tube, 0.4 ml of VB solution was added to the virus solution, vortexed, and allowed to stand at room temperature for 10 minutes.
  • the VB column was placed in a 2 ml collection tube, and 0.6 ml of the virus solution mixed with the reagent was added to the VB column, centrifuged at 14,000 g for 1 minute, and the remaining virus solution mixed with the reagent was added to the VB column, and centrifuged at 14,000 g for 1 minute.
  • the oligonucleotide primers were synthesized based on the conserved region sequences at the 5' and 3' ends of the Cap gene, and PCR was carried out.
  • the primer sequences are shown in Table 1.
  • PCV3-Cap-F CCA CAG AAG GCG CTA TGT C PCV3-Cap-R CCG CAT AAG GGT CGT CTT G
  • the PCR product was sent to Invitrogen for sequencing, and the Cap gene was codon optimized according to the sequencing result.
  • the optimized Cap gene sequence is shown in SEQ ID NO.
  • the Cap gene was sent to Suzhou Yuxun Biotechnology Co., Ltd. for full sequence synthesis and ligated to the pET28a plasmid.
  • the ligated plasmid and chaperone plasmid pG-Tf2 were co-transformed into E. coli BL21 (DE3), and the single clone was cultured overnight in LB medium containing 100 ⁇ g/ml kanamycin and 20 ⁇ g/ml chloramphenicol to extract the plasmid.
  • Isopropyl- ⁇ -D-thiogalactopyranoside IPTG was added to a final concentration of 0.1 to 1.0 mM, and cultured at 28 ° C for 24 hours with shaking. After the completion of the culture, the cells were collected, and PBS (PBS component: sodium chloride 8 g, potassium chloride 0.2 g, disodium hydrogen phosphate 1.44 g, potassium dihydrogen phosphate 0.24 g, pH adjusted to 7.4, constant volume 1 L) was used. The suspension was sonicated, the cells were sonicated, and the supernatant was removed by centrifugation of the lysate after sonication. The content of soluble protein in the expressed product was high, and the expression amount was 25% of the total amount of the bacterial protein, and the endotoxin content was 0.28 ⁇ 10 5 EU/ml.
  • PBS PBS component: sodium chloride 8 g, potassium chloride 0.2 g, disodium hydrogen phosphate 1.44 g, potassium dihydrogen phosphat
  • Example 4 Escherichia coli expresses porcine circovirus type 3 Cap protein endotoxin clearance
  • the target protein will remain in the upper layer, and the endotoxin-containing nonionic surfactant will remain in the shape of oil droplets at the bottom of the centrifuge tube, separating the two phases.
  • the entire endotoxin-removing operation cycle described above was repeated 3 times.
  • the final purified product was determined, the protein purity was not reduced, the endotoxin content was decreased to 0.008 ⁇ 10 5 EU/ml; at the same time, the magnification was 60,000 times by 200KV transmission electron microscope, and the negative staining with 5% phosphotungstic acid was observed and fixed in the spray.
  • the PCV3 virus-like particles on the carbon copper network showed a large number of virus-like particles, and the size was uniform, showing a state of hollow particles.
  • Triton X-114 can eliminate the endotoxin remaining in the recombinant protein, and has no effect on the purity of the protein; at the same time, it does not affect the formation and stable morphology of PCV3 virus-like particles.
  • 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. After repeated three cycles of freezing and thawing, the tissue suspension was centrifuged at 12000 r/min for 15 min, 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 harvested in the above procedure 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 PCV2-specific primer, and the result showed that a 1.7 kb 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. The results showed that the whole genome sequence of the virus strain was less than 96% homologous to other PCV2s already reported, and the amino acid sequence was less than 94%. Further genome-wide sequence analysis indicated that the strain was between PCV2b and PCV2d. Mutations in the ORF2 gene or recombination between ORF2 genes of different gene subtypes, which are determined by genetic evolution analysis, belong to a new subtype of PCV2 gene.
  • the plasmid extraction kit was purchased from Tiangen Bio; the T4 DNA Ligase was purchased from BioLab; the pET28a plasmid was purchased from Novagen; the agarose gel gel recovery kit was purchased from Tianze Bio, and the other reagents were of analytical grade.
  • 0.2 ml of a new porcine circovirus type 2 virus solution was used to extract the DNA genome according to the method of Example 2.
  • the oligonucleotide primers were synthesized based on the conserved region sequences at the 5' and 3' ends of the Cap gene, and PCR was carried out.
  • the primer sequences are shown in Table 2.
  • PCV2-Cap-F CCCATGCCCTGAATTTCCA
  • PCV2-Cap-R CAGCGCACTTCTTTCGTTTTCAG
  • Cap gene was genetically altered at positions 52-64, 106-108, 131-137 compared with the Cap protein amino acid sequence of PCV2 in the prior art. Recombination, the Cap gene was codon optimized according to the sequencing result, and the optimized Cap gene sequence is shown in SEQ ID NO. 2 of the Sequence Listing.
  • the Cap gene was sent to Suzhou Yuxun Biotechnology Co., Ltd. for full sequence synthesis and ligated to the pET28a plasmid.
  • the ligated plasmid and chaperone plasmid pG-Tf2 were co-transformed into E. coli BL21 (DE3), and the single clone was cultured overnight in LB medium containing 100 ⁇ g/ml kanamycin and 20 ⁇ g/ml chloramphenicol to extract the plasmid.
  • the pET28a-PCV2-Cap/pG-Tf2/E.Coli BL21 (DE3) strain prepared in Example 6 was subjected to protein-induced expression with reference to Example 3.
  • the content of soluble target protein in the expressed product is high, and the expression amount can reach 50% of the total amount of the bacterial protein, and the endotoxin content is 0.26 ⁇ 10 5 EU/ml.
  • the soluble Cap protein-containing supernatant solution expressed in Example 7 was subjected to endotoxin clearance according to Example 4. It was determined that the purity of the protein was not reduced, and the endotoxin content was decreased to 0.008 ⁇ 10 5 EU/ml. At the same time, the magnification was 60,000 times by 200KV transmission electron microscope, and the copper was negatively dyed by 5% phosphotungstic acid and fixed on the carbon-sprayed copper network. The PCV2 virus-like particles showed a large amount of virus-like particles and were uniform in size, showing a state of hollow particles.
  • Triton X-114 could eliminate the endotoxin remaining in the recombinant protein and had no effect on the purity of the protein. At the same time, it did not affect the formation and stable morphology of PCV2 virus-like particles.
  • the porcine circovirus type 3 Cap protein purified in Example 4 and the porcine circovirus type 2 Cap protein purified in Example 8 were mixed in proportion and slowly added to the water-soluble adjuvant Gel adjuvant (Ferby, France) In the process of adding, the mixture is continuously stirred for 12 minutes with an emulsifier of 800 rpm and mixed.
  • the specific formulation of the immunogenic composition is shown in Table 3.
  • the first group, the third group, the fifth group, the seventh group, the ninth group, and the eleventh group were challenged with Porcine Circovirus type 3 (Strain SG, Deposited in the China Center for Type Culture Collection, the deposit number is CCTCC NO: V201712, the deposit date is March 23, 2017, the deposit address: Wuhan University, Wuhan, China), the dose is 10 5.0 TCID 50 / head, the first Group 2, Group 4, Group 6, Group 8, Group 10, Group 12, porcine circovirus type 2 HH3 strain (Porcine Circovirus type 2, Strain HH3, deposited in the China Center for Type Culture Collection, The deposit number is CCTCC NO: V201726, the deposit date is June 4, 2017, the deposit address: Wuhan University, Wuhan, China), the dose is 10 5.0 TCID 50 / head, and each piglet is continuously observed after the attack, according to each The clinical symptoms, pathological changes and virus detection results of piglets were determined. The specific results are shown in Table 4.
  • the immunogenic composition containing porcine circovirus type 3 and type 2 antigen can provide 100% (5/5) protection for piglets after one immunization, while all the piglets in the challenge control group were infected. It is indicated that the porcine circovirus type 3 and type 2 antigen immunogenic compositions provided by the invention have good protective efficacy. Moreover, the virus of the porcine circovirus type 3 and type 2 antigen immunogenic composition containing different levels of the invention can not detect the virus in the pig after the immune challenge, which means that after immunizing the immunogenic composition of the invention, even Wild poisons infect immunized pigs, and do not affect pig growth, eating and fattening.
  • Example 11 broad-spectrum protection test of immunogenic composition containing porcine circovirus type 3 and porcine circovirus type 2 antigen
  • Example 9 100 healthy piglets with PCV2, PCV3 antigen and antibody negative by ELISA at 28 to 30 days of age were randomly divided into 20 groups, 5 heads/group, and the immunogenic composition 1 of group 13-22 was immunized with Example 9. 23 to 32 groups were not immunized as a challenge control group. Each immunization group was injected with 2 ml/head of the immunogenic composition, and the challenge control group was inoculated with 2 ml/head of DMEM medium.
  • the challenge was carried out on the 28th, and the 13th and 23rd groups were challenged with the virulent strain of the porcine circovirus 2a subtype HN06 strain newly isolated from Henan province of China; the 14th and 24th groups were newly used from China.
  • the virulent strain of the porcine circovirus 2b subtype JS04 strain isolated from Jiangsu province was attacked; the 15th and 25th groups were challenged with the virulent strain of the porcine circovirus 2d subtype JL13 strain newly isolated from Jilin province, China.
  • Groups 16 and 26 were challenged with the newly bred circovirus 2new subtype CQ14 strain isolated from Chongqing, China; the 17th and 27th groups were newly isolated from Guangdong province, China.
  • the virus 2new subtype GD15 strain was virulent; the 18th and 28th groups were challenged with the porcine circovirus type 3 HN12 strain, which was newly isolated from Henan province, China; the 19th and 29th groups were used.
  • the experimental results show that the immunogenic composition containing the porcine circovirus type 3 and type 2 antigens provided by the invention can be used for one-time immunization, and the pig can be provided for the porcine circovirus type 2 attack of different geographical sources and different gene subtypes. Effective, fully immunoprotective, and PCV2 that cannot be detected from various organ tissues.
  • the experimental results show that the porcine circovirus type 3 and type 2 antigen immunogenic compositions provided by the invention can provide effective and complete immune protection against pig circovirus type 3 challenge against different geographical sources.
  • the challenged PCV3 strain could not be detected in each organ tissue.
  • the immunogenic composition provided by the present invention has a broad spectrum of immunogenicity and provides complete protection against the popular porcine circovirus type 3 and type 2 wild-type viruses.
  • the immunogenic composition of the present invention is immunized, even if the wild poison infects the immunized pig, it does not affect the development and growth of the pig and the feeding and fattening.
  • Groups 34 and 35 The immunized group could not effectively prevent the mixed infection of PCV2 and PCV3, and it was still in the state of onset.
  • the immunogenic group was injected with 2 ml/head of the immunogenic composition, 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 8.
  • the blank control group showed obvious mummies and weak piglets, and the average number of healthy piglets was 6.9 per litter.
  • the health rate was 59.0%, among which three sows had a miscarriage, and the whole nest was mummified.
  • Example 9 The prepared immunogenic composition 1 and the B2 group were blank control groups. The immunogenic group was injected with 2 ml/head of the immunogenic composition, 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 9.
  • the PCR was performed by necropsy of the organs of the pigs. PCV3 and PCV2 were negative, while the piglets in the blank control group appeared to varying degrees. The body temperature increased by 40.5 °C or more for 5 days, and the clinical symptoms such as loss of appetite, depression, rough hair, weight loss and slowing of growth rate, some pigs died, and different degrees of lung consolidation and lymphadenopathy occurred in the necropsy.
  • the pathological changes of the kidney have necrotic spots, and PCR detection of each organ tissue can further isolate porcine circovirus type 3 and porcine circovirus type 2 virus.

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Abstract

一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，该免疫原性组合物包括免疫量的猪圆环病毒3型抗原、免疫量的猪圆环病毒2型new基因亚型抗原以及药学上可接受的载体。该免疫原性组合物具有良好的免疫原性，一次免疫即能完全保护猪圆环病毒3型、猪圆环病毒2型的混合感染。

Description

一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物及其应用 技术领域
本发明属于兽药领域，具体涉及一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物及其应用。
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)，主要引起断奶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。
临床上，随着PCV2疫苗的普遍应用，在免疫压力下，PCV2的变异速度加快，一类介于PCV2b与PCV2d之间的新的基因亚型的毒株开始流行，这类病毒特征是ORF2基因中存在突变或经不同基因亚型的ORF2基因之间重组而来。由于该新的PCV2基因亚型毒株的流行，其抗原与现有的基因亚型之间存在差异，而现有的商品化疫苗均以PCV2a或PCV2b为疫苗株来制备的，不能对最新流行的PCV2毒株进行完全的保护。
在一起猪的繁殖障碍病例中，分离到一株病毒基因组为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蛋白具有＞70％的氨基酸序列的同源性，据此可以肯定这是一种新的猪圆环病毒(PCV3)。
当初早在1985年由PCV2导致的系统疾病就已经以零星状态爆发，由于未能重视，导致九十年代末大规模爆发该疾病，而新的猪圆环病毒在猪皮炎肾病综合征(Porcine dermatitis and nephropathy syndrome,PDNS)和繁殖障碍方面具有与PCV2相似病原学特性，PCV2和PCV3之间蛋白同源性很低，使用PCV2疫苗不能对PCV3进行有效预防和交叉保护。
新的猪圆环病毒与PCV2现有的和新的基因亚型之间的混合感染进一步加重临床上PCV感染的复杂情况，因此针对该新临床疫情制备新的免疫原性组合物，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/或治疗猪圆环病毒混合感染的免疫原性组合物，该免疫原性组合物能对猪圆环病毒2型和3型的混合感染提供有效的保护，显示出显著的免疫特性。
本发明的一个目的在于提供一种预防和/或治疗不同猪圆环病毒混合感染的免疫原性组合物，所述免疫原性组合物包含免疫量的猪圆环病毒3型抗原、免疫量的猪圆环病毒2型抗原以及药学上可以接受的载体。
本发明的一个目的在于提供一种预防和/或治疗不同基因亚型的猪圆环病毒2型和猪圆环病毒3型混合感染的免疫原性组合物。
本发明的一个目的在于提供一种预防和/或治疗不同地域来源的猪圆环病毒2型和猪圆环病毒3型混合感染的免疫原性组合物。
本发明的一个目的在于提供上述免疫原性组合物在制备预防和/或治疗猪圆环病毒相关疾病的药物中的应用。
本发明首次采用目前新的猪圆环病毒3型免疫原性蛋白抗原和猪圆环病毒2型新的基因亚型毒株免疫原性蛋白抗原，制备预防和/或治 疗猪圆环病毒感染的免疫原性组合物，不仅能对猪圆环病毒2型和猪圆环病毒3型的感染或混合感染进行免疫保护，还能针对不同基因亚型猪圆环病毒2型毒株进行保护。
本发明的免疫原性组合物，能针对不同地域来源的猪圆环病毒3型毒株、猪圆环病毒2型毒株混合感染提供完全保护，具有广谱的保护能力。
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是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，其能与多种病原混合感染引起猪的皮炎肾病综合征、增生性坏死性肺炎、繁殖障碍及心脏和多系统的炎症反应。
“猪圆环病毒2型new基因亚型”是指一种新的PCV2基因亚型，其ORF2基因中存在突变或经不同基因亚型的ORF2基因之间重组而来，具有PCV2b的标签序列，但在遗传进化分析中组成一个独立分支，猪只感染该基因亚型的毒株后出现的临床表征有：持续性高温，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。
本发明涉及一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述免疫原性组合物包括免疫量的猪圆环病毒3型Cap蛋白抗原、免疫量的猪圆环病毒2型new基因亚型Cap蛋白抗原以及药学上可接受的载体；其中，所述猪圆环病毒3型Cap蛋白抗原为猪圆环病毒3型Cap蛋白或重组有所述猪圆环病毒3型Cap蛋白基因的活载体，所述猪圆环病毒2型new基因亚型Cap蛋白抗原为猪圆环病毒2型new基因亚型Cap蛋白或重组有所述猪圆环病毒2型Cap蛋白基因的活载体。
本发明的免疫原性组合物经一次免疫，其中的抗原成分就能针对各自的病原产生完全保护，保护猪只预防和抵抗对于猪圆环病毒3型和猪 圆环病毒2型的混合感染。
本发明的免疫原性组合物能针对猪圆环病毒3型和不同基因亚型的猪圆环病毒2型的混合感染产生完全保护。
本发明的免疫原性组合物能保护猪只对不同地域来源的猪圆环病毒3型病毒和猪圆环病毒2型病毒的混合感染进行完全保护。
术语“免疫原性组合物”指含有猪圆环病毒3型、猪圆环病毒2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、猪圆环病毒2型病毒的免疫反应。
术语“免疫量”应当理解为“免疫有效量”，又称免疫保护量或产生免疫应答的有效量，为可在接受者体内有效诱导免疫应答的抗原量，该量足以预防或改善疾病的体征或症状，包括不利的健康影响或其并发症。所述免疫应答可能足以用于诊断目的或其它试验，或可能适合用于预防疾病的征兆或症状，包括由病原体引起的感染所造成的不利的健康结果或其并发症。体液免疫力或由细胞介导的免疫力或此二者均可被诱导。动物对免疫原性组合物的免疫应答可通过例如测量抗体效价、淋巴细胞增殖分析而间接评估，或在以野生型毒株攻击后通过监测征兆或症状来直接评估，而该由免疫原性组合物提供的保护性免疫力可通过测量例如受试动物的临床征兆如健康产仔的减少、死胎数量的增加、受试动物总体生理状况及总体健康和表现来评估。所述免疫应答可包括但不限于诱导细胞性和/或体液免疫力。
本发明所述的猪圆环病毒3型Cap蛋白抗原、猪圆环病毒2型Cap蛋白抗原可以是重组表达的Cap蛋白亚单位抗原，其表达体系可以为原核表达系统、真核表达系统，也可以是通过人工合成的合成肽抗原；或者可以是重组有所述猪圆环病毒Cap蛋白基因的活载体。
“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。
“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。
作为本发明的一种实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述的猪圆环病毒3型Cap蛋白为序列SEQ.ID NO.1编码的蛋白，所述猪圆环病毒2型new基因亚型Cap蛋白为SEQ.ID NO.2编码的蛋白。
本发明的免疫原性蛋白免疫原性好，一次免疫就可提供完全保护。
作为本发明的一种实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述的猪圆环病毒3型Cap蛋白其编码基因具有SEQ.ID NO.1所示的核苷酸序列或其简并序列，所述的猪圆环病毒2型Cap蛋白其编码基因具有SEQ.ID NO.2所示的核苷酸序列或其简并序列。
作为本发明的一种实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述猪圆环病毒3型Cap蛋白含量为≥20μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为≥20μg/ml。
本发明的免疫原性组合物中，当抗原含量低至20μg/ml也能刺激免疫系统产生免疫反应，对猪只产生完全保护。
作为本发明的一种优选实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述猪圆环病毒3型Cap蛋白含量为20μg/ml～100μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为20μg/ml～100μg/ml。
作为本发明的一种更优选实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述猪圆环病毒3型 Cap蛋白抗原含量为20μg/ml～50μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为20μg/ml～50μg/ml。
作为本发明的一种更优选实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述猪圆环病毒3型Cap蛋白抗原含量为30μg/ml～50μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为30μg/ml～50μg/ml。
在本发明的免疫原性组合物中，所述猪圆环病毒3型Cap蛋白抗原含量还可以选自20μg/ml～30μg/ml，30μg/ml～100μg/ml，或50μg/ml～100μg/ml含量范围。
在本发明的免疫原性组合物中，所述猪圆环病毒2型new基因亚型Cap蛋白抗原含量还可以选自20μg/ml～30μg/ml，30μg/ml～100μg/ml，或50μg/ml～100μg/ml含量范围。
在本发明的免疫原性组合物中，所述猪圆环病毒3型Cap蛋白抗原含量和所述猪圆环病毒2型new基因亚型Cap蛋白抗原含量可以独立地选自各自上述任一含量范围。
作为本发明的一种实施方式，在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述重组有猪圆环病毒3型Cap蛋白基因的活载体为重组减毒沙门氏菌、重组新城疫病毒、重组痘病毒、或重组腺病毒；所述重组有所述猪圆环病毒2型new基因亚型Cap蛋白基因的活载体为重组减毒沙门氏菌、重组新城疫病毒、重组痘病毒、或重组腺病毒。
作为本发明的一种实施方式,在本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述猪圆环病毒3型Cap蛋白基因和所述猪圆环病毒2型new基因亚型Cap蛋白基因重组于同一个活载体，所述活载体为重组减毒沙门氏菌、重组新城疫病毒、重组痘病毒、或重组腺病毒。
本发明的活载体免疫原性组合物因为兼具有灭活疫苗和活疫苗的优点，在免疫效力上可以保证能够对猪进行保护，且其免疫效力更强，可以不添加佐剂。
作为本发明的一种实施方式，本发明含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，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Montanide TM 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。
作为本发明的一种优选实施方式，本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述佐剂为Montanide TMGel。
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需的量。待施用的佐剂的精确的量将根据多种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。
作为本发明的一种实施方式，本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述佐剂含量为5～20V/V％。
作为本发明的一种优选实施方式，本发明的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物中，所述佐剂含量为10V/V％。
本发明的免疫原性组合物可使用常规技术来调配，优选为药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)Montanide TMGel。
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 ofAdjuvants”(Steward-Tull，D.E.S主编)John Wiley andSons，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Montanide TMGel。
本发明免疫原性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。
可以根据本发明的免疫原性组合物制备成口服剂型或非口服剂型。
优选地可通过皮内、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。
本发明还涉及上述免疫原性组合物在制备预防和/或治疗猪圆环病毒相关疾病的药物中的应用，其中，所述猪圆环病毒相关疾病为由PCV2和猪圆环病毒3型单独感染或它们混合感染导致的相关疾病。
作为本发明的一种实施方式，所述制备预防和/或治疗猪圆环病毒相关疾病的药物中的应用为，制备预防和/或治疗断奶仔猪多系统衰竭综合征、猪的皮炎肾病综合征、增生性坏死性肺炎、繁殖障碍及心脏和多系统的炎症反应的药物中的应用。
本发明所用术语“猪圆环病毒相关疾病”用于指由猪圆环病毒3型和2型混合感染引起的疾病。非穷举性包括，断奶仔猪多系统衰竭综合征、 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。
术语“预防和/或治疗”在涉及猪圆环病毒3型和2型混合感染时是指抑制猪圆环病毒3型和2型的复制、抑制猪圆环病毒3型和2型的传播或防止猪圆环病毒3型和2型在其宿主体内定居，以及减轻猪圆环病毒3型和2型感染的疾病或病症的症状。若病毒荷载量减少、病症减轻和/或摄食量和/或生长增加，那么就可以认为所述预防和/或治疗达到了效果。
本发明还涉及上述免疫原性组合物在制备预防和/或治疗猪圆环病毒混合感染的药物中的应用。
作为本发明的一种实施方式，本发明制备预防和/或治疗猪圆环病毒混合感染的药物中的应用中，所述PCV2基因亚型为PCV2a、PCV2b、PCV2d、PCV2new基因亚型。
本发明的免疫原性组合物能针对不同PCV2基因亚型以及PCV3提供有效保护，扩大了疫苗的应用范围，能对PCV2不同基因亚型和PCV3的单独感染和混合感染进行预防和/或治疗。
本发明的免疫原性组合物具有良好的免疫原性，其不仅能预防猪圆环病毒相关疾病，还能减轻已经感染了猪圆环病毒的猪只的临床表征，也即具有治疗作用。
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
本发明实施例中所用到的化学试剂均为分析纯，购自国药集团。
本发明所述的实验方法，若无特殊说明，均为常规方法；所述的生物材料，若无特殊说明，均可从商业途径获得。
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，于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，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蛋白具有＞70％的氨基酸序列的同源性，据此可以肯定，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2pET28a-PCV3-Cap表达载体的构建
1、PCV3病毒DNA的提取
质粒提取试剂盒购自天根生物；T4DNA Ligase购自BioLab；pET28a质粒购自Novagen；琼脂糖凝胶胶回收试剂盒购自天泽生物，其他试剂均为分析纯。
按照病毒DNA提取试剂盒说明书，取0.2ml猪圆环病毒3型病毒液于无菌1.5ml离心管中，加0.4ml VB溶液于病毒液中，旋涡混匀，室温静置10分钟。加0.45ml AD buffer于病毒液中，用力混匀。将VB柱放入2ml收集管中，取0.6ml上述混合了试剂的病毒液加入VB柱中，14000g离心1分钟，再将剩余的上述混合了试剂的病毒液加入VB柱中，14000g离心1分钟，弃掉2ml收集管，将VB柱放入新的2ml收集管中，加入0.4ml W1buffer，14000g离心30秒，再加0.6ml Wash buffer于VB柱，14000g离心30秒，然后不加buffer再空置离心3分钟，将该VB柱放入新的1.5ml EP管中，加入50μl RNase free water置于膜中央，静置3分钟，14000g离心1分钟，EP管中离心下来的液体即为猪圆环病毒3型病毒DNA基因组溶液。
2、Cap基因扩增
根据Cap基因5’和3’末端的保守区序列合成寡聚核苷酸引物，进行PCR。引物序列见表1。
表1 猪圆环病毒3型Cap基因扩增引物
PCV3-Cap-F CCA CAG AAG GCG CTA TGT C
PCV3-Cap-R CCG CAT AAG GGT CGT CTT G
将PCR产物送Invitrogen公司测序，根据测序结果对Cap基因进行密码子优化，优化后的Cap基因序列如序列表SEQ ID NO.1所示。
3、表达载体构建
优化后Cap基因送苏州泓迅生物科技股份有限公司进行全序列合成，并连接到pET28a质粒上。连接后的质粒和分子伴侣质粒pG-Tf2共转化大肠杆菌BL21(DE3)，挑取单克隆在含有100μg/ml卡那霉素和20μg/ml氯霉素的LB培养基中培养过夜，提取质粒后测序分析，确定合成序列正确，阳性克隆即为pET28a-PCV3-Cap/pG-Tf2表达菌株 pET28a-PCV3-Cap/pG-Tf2/E.Coli BL21(DE3)。
实施例3猪圆环病毒3型Cap蛋白的表达
将实施例2中制备的pET28a-PCV3-Cap/pG-Tf2/E.Coli BL21(DE3)菌株接种到含50-100μg/ml卡那霉素和20μg/ml氯霉素的LB培养基中，同时LB培养基中含有5-10ng/ml四环素用于分子伴侣蛋白的诱导表达，接种量为1％(V/V)，于37℃振荡培养。当OD 600＝0.4～0.6时，于28℃放置30分钟。加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)，使其终浓度为0.1～1.0mM，于28℃振荡培养24小时。培养结束后，收集菌体，并用PBS(PBS组分：氯化钠8g，氯化钾0.2g，磷酸氢二钠1.44g，磷酸二氢钾0.24g，调节pH至7.4，定容1L)重悬菌体，超声破碎菌体，超声破碎后的裂解液离心取上清。表达产物中可溶性目的蛋白含量较高，表达量可达菌体蛋白总量的25％，内毒素含量为0.28×10 5EU/ml。
实施例4大肠杆菌表达猪圆环病毒3型Cap蛋白内毒素清除
1.5ml离心管中加入0.5ml待处理的含可溶性Cap蛋白的上清溶液和终浓度为1％(v/v)的曲拉通X-114(Triton X-114)(加入量5μl)，涡旋振荡。涡旋振荡混匀的样品在冰上放置5分钟。样品冷却后，离心管立即放入37℃水浴中温浴5min，使得产生新的两相。然后，把样品在37℃温度条件下离心60s。离心过后，目的蛋白将留在上层，而含有内毒素的非离子表面活性剂将会以油滴的形状残留在离心管底部，分离两相。上述整个去除内毒素的操作循环3次。最终的纯化产物经测定，蛋白纯度没有降低，内毒素含量下降为0.008×10 5EU/ml；同时，通过200KV透射电镜，放大倍数60000倍，观察经5％磷钨酸负染、固定于喷碳的铜网上的PCV3病毒样颗粒，结果显示大量的病毒样颗粒，且大小均匀，呈现为空心粒子状态。
实验结果表明Triton X-114能够清除重组蛋白中残留的内毒素，且对蛋白的纯度没有影响；同时，也没有影响PCV3病毒样颗粒的形成及稳定形态。
实施例5猪圆环病毒2型的分离鉴定
1、病料来源
在国内一免疫商品化PCV2疫苗的猪场,零星出现母猪流产、木乃伊胎增加的现象。受影响的母猪表现出厌食症状，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。通过免疫组化和定量PCR检测，PCV2阳性。因其是在免疫PCV2商品化疫苗后依然从发病猪组织中检测到PCV2，原因值得深究，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，于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
取以上步骤收获的病毒培养物，用核酸提取试剂盒提取病毒样品的核酸，使用PCV2特异性引物进行PCR扩增鉴定，结果显示，PCR扩增出1.7kb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同已经报道过的其他PCV2的同源性都少于96％，氨基酸序列少于94％，进一步的全基因组序列分析表明该毒株介于PCV2b与PCV2d之间，ORF2基因中存在突变或经不同基因亚型的ORF2基因之间重组而来，经遗传进化分析确定，其属于一个新的PCV2基因亚型。
实施例6pET28a-PCV2-Cap表达载体的构建
1、PCV2病毒DNA的提取
质粒提取试剂盒购自天根生物；T4DNA Ligase购自BioLab；pET28a质粒购自Novagen；琼脂糖凝胶胶回收试剂盒购自天泽生物， 其他试剂均为分析纯。
按照病毒DNA提取试剂盒说明书，参照实施例2方法取0.2ml新的猪圆环病毒2型病毒液提取DNA基因组。
2、猪圆环病毒2型Cap基因扩增
根据Cap基因5’和3’末端的保守区序列合成寡聚核苷酸引物，进行PCR。引物序列见表2。
表2 猪圆环病毒2型Cap基因扩增引物
PCV2-Cap-F CCCATGCCCTGAATTTCCA
PCV2-Cap-R CAGCGCACTTCTTTCGTTTTCAG
将PCR产物送Invitrogen公司测序，结果显示与现有技术中的PCV2的Cap蛋白氨基酸序列相比，Cap基因编码的氨基酸序列在52～64位、106～108位、131～137位发生基因突变或重组，根据测序结果对Cap基因进行密码子优化，优化后的Cap基因序列如序列表SEQ ID NO.2所示。
3、表达载体构建
优化后Cap基因送苏州泓迅生物科技股份有限公司进行全序列合成，并连接到pET28a质粒上。连接后的质粒和分子伴侣质粒pG-Tf2共转化大肠杆菌BL21(DE3)，挑取单克隆在含有100μg/ml卡那霉素和20μg/ml氯霉素的LB培养基中培养过夜，提取质粒后测序分析，确定合成序列正确，阳性克隆即为pET28a-PCV2-Cap/pG-Tf2表达菌株pET28a-PCV2-Cap/pG-Tf2/E.Coli BL21(DE3)。
实施例7猪圆环病毒2型Cap蛋白的表达
将实施例6中制备的pET28a-PCV2-Cap/pG-Tf2/E.Coli BL21(DE3)菌株参照实施例3进行蛋白诱导表达。表达产物中可溶性目的蛋白含量较高，表达量可达菌体蛋白总量的50％，内毒素含量为0.26×10 5EU/ml。
实施例8大肠杆菌表达Cap蛋白内毒素清除
将实施例7表达的含可溶性Cap蛋白的上清溶液参照实施例4进行 内毒素清除。经测定，蛋白纯度没有降低，内毒素含量下降为0.008×10 5EU/ml；同时，通过200KV透射电镜，放大倍数60000倍，观察经5％磷钨酸负染、固定于喷碳的铜网上的PCV2病毒样颗粒，结果显示大量的病毒样颗粒，且大小均匀，呈现为空心粒子状态。
结果表明Triton X-114能够清除重组蛋白中残留的内毒素，且对蛋白的纯度没有影响；同时，也没有影响PCV2病毒样颗粒的形成及稳定形态。
实施例9猪圆环病毒3型、猪圆环病毒2型免疫原性组合物的制备
将按实施例4纯化的猪圆环病毒3型Cap蛋白、实施例8纯化的猪圆环病毒2型Cap蛋白按比例混合后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。免疫原性组合物的具体配方见表3。
表3 含猪圆环病毒3型、猪圆环病毒2型抗原免疫原性组合物配比
Figure PCTCN2017118696-appb-000001
实施例10含猪圆环病毒3型、猪圆环病毒2型抗原免疫原性组合物免疫原性试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪60头随机分成12组，5头/组，免疫实施例9制备的猪圆环病毒 3型、2型免疫原性组合物。第1～2组免疫组合物1，第3～4组免疫组合物2，第5～6组免疫组合物3，第7～8组分别免疫组合物4，第9～10组分别免疫原性组合物5、组合物6，第11～12组不免疫作为攻毒对照组。各免疫组注射免疫原性组合物2ml/头，对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，第1组、第3组、第5组、第7组、第9组、第11组攻毒猪圆环病毒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)，攻毒剂量均为10 5.0TCID 50/头，第2组、第4组、第6组、第8组、第10组、第12组攻毒猪圆环病毒2型HH3株(Porcine Circovirus type 2，Strain HH3，保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：V201726，保藏日期为2017年6月4日，保藏地址：中国武汉·武汉大学)，攻毒剂量均为10 5.0TCID 50/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4。
表4 含猪圆环病毒3型、2型抗原免疫原性组合物免疫原性试验结果
Figure PCTCN2017118696-appb-000002
Figure PCTCN2017118696-appb-000003
结果显示，含猪圆环病毒3型、2型抗原免疫原性组合物一次免疫仔猪后，能为仔猪提供100％(5/5)保护，而攻毒对照组仔猪攻毒后全部发病。表明本发明提供的含猪圆环病毒3型、2型抗原免疫原性组合物具有很好的保护效力。且本发明不同含量的含猪圆环病毒3型、2型抗原免疫原性组合物免疫攻毒后在猪体内均不能检测到病毒，这说明当免疫本发明的免疫原性组合物后，即便野毒感染了免疫的猪只，也不会对猪的发育生长、进食增肥产生影响。
实施例11含猪圆环病毒3型、猪圆环病毒2型抗原免疫原性组合物广谱性保护试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪100头随机分成20组，5头/组，第13～22组免疫实施例9制备的免疫原性组合物1，第23～32组不免疫作为攻毒对照组。各免疫组注射免疫原性组合物2ml/头，攻毒对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，第13组、第23组用新近从中国河南省分离的猪圆环病毒2a基因亚型HN06株强毒株攻毒；第14组、第24组用新近从中国江苏省分离的猪圆环病毒2b基因亚型JS04株强毒株攻毒；第15组、第25组用新近从中国吉林省分离的猪圆环病毒2d基因亚型JL13株强毒株攻毒；第16组、第26组用新近从中国重庆市分离的猪圆环病毒2new基因亚型CQ14株强毒株攻毒；第17组、第27组用新近从中国广东省分离的猪圆环病毒2new基因亚型GD15株强毒株攻毒；第18组、第28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19组、第29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20组、第30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21组、第31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22组、第32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10 5.0TCID 50/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5～6。
表5 含猪圆环病毒3型、2型抗原免疫原性组合物针对PCV2感染广谱性保护试验结果
Figure PCTCN2017118696-appb-000004
Figure PCTCN2017118696-appb-000005
结果显示，第23～27组攻毒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2型病毒；而第13～17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PCV2阴性。实验结果表明本发明提供的含猪圆环病毒3型、2型抗原免疫原性组合物一次免疫，即可使得猪只针对不同地域来源、不同基因亚型的猪圆环病毒2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PCV2。
表6 含猪圆环病毒3型、2型抗原免疫原性组合物针对PCV3感染广谱性保护试验结果
Figure PCTCN2017118696-appb-000006
Figure PCTCN2017118696-appb-000007
结果显示，第28～32组攻毒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18～22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。实验结果表明本发明提供的含猪圆环病毒3型、2型抗原免疫原性组合物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。表明，本发明提供的免疫原性组合物具有广谱的免疫原性，对于流行的猪圆环病毒3型、2型野毒均能提供完全保护。且当免疫本发明的免疫原性组合物后，即便野毒感染了免疫的猪只，也不会对猪的发育生长、进食增肥产生影响。
实施例12含猪圆环病毒3型、2型抗原免疫原性组合物混合感染保护试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪20头随机分成4组，5头/组。第33组免疫实施例9制备的免疫原性组合物1，第34组免疫实施例9制备的免疫原性组合物5，第35组免疫实施例9制备的免疫原性组合物6，第36组不免疫作为攻毒对照组。各免疫组注射免疫原性组合物2ml/头，对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，所有组攻毒猪圆环病毒3型SG株和猪圆环病毒2型HH3株混合病毒液，攻毒剂量为10 5.0TCID 50/头，攻毒后连续观察各仔猪，根据各仔猪临床症状、病理变化结果进行判定，具体结果见表7。
表7 含猪圆环病毒3型、2型抗原免疫原性组合物混合感染保护试验结果
Figure PCTCN2017118696-appb-000008
Figure PCTCN2017118696-appb-000009
结果显示，第36组攻毒对照组攻毒后均不同程度出现体温升高40.5℃以上，持续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；第33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；而第34组、第35组免疫组不能有效阻止PCV2、PCV3的混合感染，依然呈发病状态。实验结果表明本发明提供的含猪圆环病毒3型、2型抗原免疫原性组合物一次免疫就可对猪只针对PCV2、PCV3联合攻毒提供有效的、完全免疫保护。
实施例13含猪圆环病毒3型、2型抗原免疫原性组合物应用试验
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9.8％，受孕率降低了1.2％，木乃伊胎增加8.6％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32头怀孕母猪随机分成两组A组、B组，16头/组，A组为免疫原性组合物接种组，A组免疫实施例9制备的免疫原性组合物1，B组为空白对照组。免疫组注射免疫原性组合物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8。
表8 免疫组和空白对照组母猪产子统计结果
Figure PCTCN2017118696-appb-000010
Figure PCTCN2017118696-appb-000011
Figure PCTCN2017118696-appb-000012
结果显示，免疫组母猪产子无异常，生产健康仔猪，平均11.7头/窝，健康率高达99.5％，而空白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6.9头/窝，健康率59.0％，其中还有三头母猪出现流产情况，全窝木乃伊胎，免疫组和空白对照组差异显著。
表8的结果证明了本发明含猪圆环病毒3型、2型抗原免疫原性组合物对感染猪圆环病毒的母猪有着良好的免疫保护作用，且能针对已经感染了PCV的母猪进行保护，即具有治疗作用。
同时，对B组对照组所产仔猪分别隔离按窝饲养，13窝分成两组B1组(包括B-1窝至B-11窝，除B-5窝因全窝木乃伊胎不计外，共10窝仔猪)、B2组(包括B-12至B-16窝，除B-14窝和B-16窝因全窝木乃伊胎不计外，共3窝仔猪)，B1组吃母乳之前免疫实施例9制备的免疫原性组合物1，B2组为空白对照组。免疫组注射免疫原性组合物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9。
表9 含猪圆环病毒3型、2型抗原免疫原性组合物对仔猪的免疫保护试验
Figure PCTCN2017118696-appb-000013
Figure PCTCN2017118696-appb-000014
Figure PCTCN2017118696-appb-000015
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、PCV2阴性，而空白对照组仔猪均不同程度出现体温升高40.5℃以上，持续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型和猪圆环病毒2型病毒。
由于PCV在猪群中是可以通过垂直传播的，因此表9的结果证明了本发明含猪圆环病毒3型、2型抗原免疫原性组合物对混合感染猪圆环病毒3型和2型的仔猪有着良好的免疫保护作用，且能针对已经混合感染了PCV3和PCV2病毒的仔猪进行保护，即具有治疗作用，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 (13)

  1. 一种含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述免疫原性组合物包括免疫量的猪圆环病毒3型Cap蛋白抗原、免疫量的猪圆环病毒2型new基因亚型Cap蛋白抗原以及药学上可接受的载体；其中，所述猪圆环病毒3型Cap蛋白抗原为猪圆环病毒3型Cap蛋白或重组有所述猪圆环病毒3型Cap蛋白基因的活载体，所述猪圆环病毒2型new基因亚型Cap蛋白抗原为猪圆环病毒2型new基因亚型Cap蛋白或重组有所述猪圆环病毒2型Cap蛋白基因的活载体。
  2. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白为SEQ.ID NO.1编码的蛋白，所述猪圆环病毒2型new基因亚型Cap蛋白为SEQ.ID NO.2编码的蛋白。
  3. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为≥20μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为≥20μg/ml。
  4. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为20μg/ml～100μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为20μg/ml～100μg/ml。
  5. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为20μg/ml～50μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为20μg/ml～50μg/ml。
  6. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为30μg/ml～50μg/ml；所述猪圆环病毒2型new基因亚型Cap蛋白含量为30μg/ml～50μg/ml。
  7. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗 原的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白基因和所述猪圆环病毒2型new基因亚型Cap蛋白基因重组于同一个活载体，所述活载体为重组减毒沙门氏菌、重组新城疫病毒、重组痘病毒、或重组腺病毒。
  8. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，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Montanide TMGel、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；
    所述佐剂含量为5～20V/V％。
  9. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述佐剂含量为10V/V％。
  10. 根据权利要求1所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述药学上可接受的载体包括佐剂，所述佐剂为Montanide TMGel；所述佐剂含量为5～20V/V％。
  11. 根据权利要求10所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物，其中，所述佐剂含量为10V/V％。
  12. 根据权利要求1～11任一项所述的含猪圆环病毒3型、猪圆环病毒2型抗原的免疫原性组合物在制备预防和/或治疗猪圆环病毒相关疾病的药物中的应用。
  13. 根据权利要求12所述的应用，所述猪圆环病毒相关疾病包括断奶仔猪多系统衰竭综合征、猪的皮炎肾病综合征、增生性坏死性肺炎、繁殖障碍及心脏和多系统的炎症反应。
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