WO2018196836A1 - 一种猪圆环病毒3型免疫原性组合物、制备方法和应用 - Google Patents

一种猪圆环病毒3型免疫原性组合物、制备方法和应用 Download PDF

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WO2018196836A1
WO2018196836A1 PCT/CN2018/084754 CN2018084754W WO2018196836A1 WO 2018196836 A1 WO2018196836 A1 WO 2018196836A1 CN 2018084754 W CN2018084754 W CN 2018084754W WO 2018196836 A1 WO2018196836 A1 WO 2018196836A1
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Prior art keywords
porcine circovirus
circovirus type
immunogenic composition
cap protein
recombinant
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PCT/CN2018/084754
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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
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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 JP2020509149A priority Critical patent/JP7005747B2/ja
Publication of WO2018196836A1 publication Critical patent/WO2018196836A1/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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of veterinary medicine, in particular to a porcine circovirus type 3 protein, an immunogenic composition containing the same, a preparation method and application.
  • 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.
  • a virus with a genome of 2.0 kb was isolated and confirmed by subsequent experiments, whether it is homologous to the known circovirus in nucleotide or amino acid sequence. Both are less than 50%.
  • the same virus in the genus Circovirus should have >75% homology to the genomic nucleotide sequence, and the Cap protein should have an amino acid sequence of >70%. Homology, 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 an immunogenic composition for preventing and/or treating a porcine circovirus type 3-related disease, which is capable of providing an effective porcine circovirus type 3 Protection shows significant immunological properties.
  • an object of the present invention is to provide a porcine circovirus type 3 immunogenic protein Cap protein whose sequence is encoded by the nucleotide sequence shown in SEQ ID No. 1, and an immunogenic composition prepared therefrom It is effective against the attacks of epidemic strains and provides complete protection against porcine circovirus type 3 from different sources.
  • Another object of the present invention is to provide an immunogenic composition for preventing and/or treating a porcine circovirus type 3 related disease, wherein the immunogenic composition comprises an immunological amount of porcine circovirus type 3 Cap
  • the recombinant protein or immunogen amount is a live vector of the porcine circovirus type 3 Cap protein gene, and a veterinary acceptable carrier.
  • Another object of the present invention is to provide a method of preparing the immunogenic composition, the method comprising: step (1) cloning the porcine circovirus type 3 Cap protein gene, and the porcine ring Recombining the viral type 3 Cap protein gene into an expression vector to obtain a recombinant expression vector containing the porcine circovirus type 3 Cap protein gene; and (2) recombining the porcine circovirus type 3 Cap protein gene
  • the expression vector and the molecular chaperone expression vector are co-transformed into E. coli to express the porcine circovirus type 3 Cap protein; and the step (3) is to isolate the expressed porcine circovirus type 3 Cap protein, which is treated with a nonionic surfactant.
  • the endotoxin is removed; and the endotoxin-removed porcine circovirus type 3 Cap protein is mixed and emulsified with an adjuvant to obtain the immunogenic composition.
  • the invention also relates to the use of the immunogenic composition described above for the manufacture of a medicament for the prevention and/or treatment of a porcine circovirus type 3 related disease.
  • the invention adopts the high-efficiency expression of the porcine circovirus type 3 immunogenic Cap protein gene for the first time, and prepares the immunogenic composition, which can prevent or treat the epidemic caused by the porcine circovirus type 3 related disease.
  • the immunogenic composition prepared by the porcine circovirus type 3 Cap protein of the invention has good immunogenicity, can completely protect the pigs from attacking, and can effectively prevent wild strains of various geographical origins, that is, It can prevent, treat and control the infection and spread of porcine circovirus type 3 in clinical application.
  • Porcine circovirus type 3 is a genomic 2.0 kb circovirus that is less than 50% homologous to known circoviruses in nucleotide or amino acid sequences and is a new porcine ring. The virus is now known to cause dermatitis and nephrotic syndrome in pigs, reproductive disorders, and inflammatory responses to the heart and multiple systems.
  • the present invention relates to a porcine circovirus type 3 immunogenic composition, wherein the immunogenic composition comprises an immunological amount of porcine circovirus type 3 Cap protein antigen or an immunological amount of recombinant porcine circovirus A live vector of the Type 3 Cap protein gene, and a veterinary acceptable carrier.
  • the invention finds that the porcine circovirus type 3 Cap protein has good immunogenicity for the first time, and the prepared subunit antigen or the live vector recombined with the gene can produce good immunity after immunization, and provide 100% for pigs. protection of.
  • immunogenic composition refers to a pharmaceutical composition comprising porcine circovirus type 3 immunogenicity which induces, stimulates or potentiates the immune response of pigs to porcine circovirus type 3.
  • 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, an 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.
  • porcine circovirus type 3 antigen refers to any composition comprising at least one porcine circovirus type 3 antigenic form that induces, stimulates or potentiates porcine circovirus type 3 infection.
  • the immune response including but not limited to inactivated, attenuated or subunit antigens.
  • the porcine circovirus type 3 Cap protein antigen of the present invention may be a recombinantly expressed Cap protein subunit antigen, and the expression system may be a prokaryotic expression system, a eukaryotic expression system, or a synthetic peptide antigen synthesized by artificial synthesis; Alternatively, it may be a live vector in which the porcine circovirus Cap protein gene is recombined.
  • 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.
  • 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 protein encoded by the nucleotide sequence shown in SEQ ID NO.
  • the gene encoding the porcine circovirus type 3 Cap protein has the nucleotide sequence shown in SEQ ID NO. 1 or And sequence.
  • the porcine circovirus type 3 Cap protein content is ⁇ 20 ⁇ g/ml in the immunogenic composition of the present invention.
  • the porcine circovirus type 3 Cap protein content is from 20 ⁇ g/ml to 100 ⁇ g/ml.
  • the porcine circovirus type 3 Cap protein antigen content is from 20 ⁇ g/ml to 50 ⁇ g/ml.
  • the porcine circovirus type 3 Cap protein antigen content is from 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.
  • the live vector recombinant porcine circovirus type 3 Cap protein gene 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 porcine circovirus type 3 Cap gene of the present invention can also be applied to the development of expression vectors, nucleic acid vaccines, diagnostic reagent development, and other drugs for preventing and/or treating porcine circovirus type 3 related diseases.
  • the present invention relates to a porcine circovirus type 3 Cap protein, the sequence of which is encoded by the nucleotide sequence shown in SEQ ID No. 1.
  • the present invention relates to a recombinant vector capable of expressing the porcine circovirus type 3 Cap protein of the present invention, which is immunogenic and capable of producing an immune response.
  • the present invention relates to a transformant comprising the introduced recombinant vector expressing the porcine circovirus type 3 Cap protein of the present invention.
  • the veterinary acceptable carrier comprises an adjuvant comprising (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 comprises (1) saponin QuilA; (2) a polyether of an acrylic or methacrylic polymer and a sugar or a polyhydric alcohol. Alkenyl 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 An oil produced by oligomerization, an ester of a linear alkyl acid or an alcohol, more particularly a vegetable oil, ethyl oleate, propylene glycol di(octanoate/caprate), glycerol (octanoate/ A phthalate ester, a propylene glycol dioleate; an ester of a branched fatty acid ester or an alcohol, particularly an isostearate, an oil and an emul
  • 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 immunogenic composition of the present invention is an adjuvant, which includes white oil, Drakeoil, and other animal oils, vegetable oils or mineral oils; or aluminum hydroxide, aluminum phosphate and other metal salts; or Montanide TM Gel, carbomer, squalane or squalene, ISA206 adjuvant, saponin, oil emulsion, oil in water emulsions, water-in-oil emulsion.
  • an adjuvant which includes white oil, Drakeoil, and other animal oils, vegetable oils or mineral oils; or aluminum hydroxide, aluminum phosphate and other metal salts; or Montanide TM Gel, carbomer, squalane or squalene, ISA206 adjuvant, saponin, oil emulsion, oil in water emulsions, water-in-oil emulsion.
  • the immunogenic 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 immunogenic composition may be water-in-oil, oil-in- water 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.
  • 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 an amount necessary for an adjuvant to be necessary or sufficient to exert its immunological effects in a 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 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 Cap protein of the present invention can be produced by any method known in the art.
  • the Cap protein can be produced by recombinantly expressing the Cap gene, and the expression system can use any known expression system, for example, a eukaryotic expression system, a prokaryotic expression system. Or directly synthesize Cap protein.
  • Eukaryotic expression systems can include mammalian cell expression systems, yeast expression systems, and insect expression systems.
  • the present invention also relates to a method of preparing the immunogenic composition, wherein the method comprises: step (1) cloning the porcine circovirus type 3 Cap protein gene, and porcine circovirus 3 Recombining a type Cap protein gene into an expression vector to obtain a recombinant expression vector containing the porcine circovirus type 3 Cap protein gene; and (2) the recombinant expression vector containing the porcine circovirus type 3 Cap protein gene And the molecular chaperone expression vector co-transforms Escherichia coli to express the porcine circovirus type 3 Cap protein; step (3) separates the expressed porcine circovirus type 3 Cap protein, and is treated with a nonionic surfactant to remove the inside Toxin; and step (4) mixing and emulsifying the endotoxin-depleted porcine circovirus type 3 Cap protein with an adjuvant to obtain the immunogenic composition.
  • the recombinant expression vector containing the porcine circovirus type 3 Cap protein gene in the step (1) is recombinant The pET28a plasmid, wherein the expression vector of the chaperone in the step (2) is pG-Tf2, the Escherichia coli is Escherichia coli BL21 (DE3); and the nonionic surfactant in the step (3) is Triton X- 114.
  • 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.
  • drugs eg, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony-stimulating factor (GM-CSF) ), macrophage colony-stimulating factor (M-CSF) and interleukin 2 (IL2)
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • M-CSF macrophage colony-sti
  • the immunogenic composition according to the present invention can be prepared into an oral dosage form or a non-oral dosage form.
  • parenteral dosage forms which can be administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.
  • the invention also relates to the use of the immunogenic composition for the manufacture of a medicament for the prevention and/or treatment of a porcine circovirus type 3 associated 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, dermatitis, nephrotic syndrome, reproductive disorders, and cardiac and multi-system inflammatory responses in pigs.
  • porcine circovirus type 3 related disease means inhibiting the replication of porcine circovirus type 3, inhibiting the spread of porcine circovirus type 3 or preventing porcine circovirus type 3 in its
  • the host colonizes and alleviates the symptoms of a disease or condition of porcine circovirus type 3 infection. If the viral load is reduced, the condition is alleviated, and/or the food intake and/or growth is increased, then the treatment is considered to have achieved a therapeutic effect.
  • the chemical reagents used in the examples of the present invention are of analytical grade and are purchased from Sinopharm Group.
  • sow mortality rate increased by 9.4%
  • the conception rate decreased by 1.2%
  • the mummy was increased by 8.2%.
  • the affected sows showed anorexia, symptoms of multifocal papules, spots and surface dermatitis. Mummy-bearing fetuses with different gestational ages in aborted litters are consistent with the symptoms of abortion caused by PCV2 infection.
  • the overall clinical manifestations and abortion symptoms observed in sows were consistent with reproductive dysfunction caused by porcine circovirus type 2
  • different tissues of all sows including kidney, lymph nodes, lungs, skin, and stillbirth, passed the immunization group. Quantitative and quantitative PCR were negative for PCV2, PRRSV, PPV, CSFV, and Mycoplasma hyopneumoniae. In order to further find out the reasons, the pathogens of each tissue were selected for pathogen isolation.
  • the diseased material was added to DMEM culture medium 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 was harvested, and after two freeze-thaw cycles, the virus-containing culture solution 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 Committee of Virology, the same virus in the genus Cyclone should be With >75% homology of the genomic nucleotide sequence, the Cap protein should have >70% amino acid sequence homology, so it is certain that it is a new porcine circovirus and is currently on pigs. 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.
  • 0.2 ml of porcine circovirus type 3 virus solution was taken in a sterile 1.5 ml centrifuge tube, 0.4 ml of VB was added to the virus solution, vortexed, and allowed to stand at room temperature for 10 minutes.
  • the oligonucleotide primers were synthesized based on the conserved region sequences at the 5' and 3' ends of the Cap protein gene, and PCR was carried out.
  • the primer sequences are shown in Table 1.
  • the PCR product was sent to Invitrogen for sequencing, and the Cap protein gene was codon-optimized according to the sequencing result.
  • the optimized Cap protein gene sequence is shown in SEQ ID NO.
  • the optimized Cap protein gene was sent to Suzhou Jingxun 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 positive clone was the pET28a-PCV3-Cap/pG-Tf2 expression strain.
  • 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 weighed with PBS (sodium chloride, 8 g, potassium chloride, 0.2 g, disodium hydrogen phosphate, 1.44 g, potassium dihydrogen phosphate, 0.24 g, adjusted to pH 7.4, and made up to 1 L). The suspension was sonicated, and the supernatant was centrifuged. 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 sodium chloride, 8 g, potassium chloride, 0.2 g, disodium hydrogen phosphate, 1.44 g, potassium dihydrogen phosphate, 0.24 g, adjusted to pH 7.4, and made up to 1 L.
  • the target protein After centrifugation, 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 removal procedure was cycled 3 times. 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.
  • 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 PCV3 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 PCV3 virus-like particles.
  • Cap protein purified by the method of Example 4 was slowly added to the water-soluble adjuvant Gel adjuvant Montanide TM Gel (Sybex, France), and the mixture was continuously stirred for 12 minutes with an emulsifier at 800 rpm. uniform.
  • the specific formulation of the immunogenic composition is shown in Table 2.
  • 25 healthy piglets with PCV2, PCV3 antigen and antibody negative by ELISA at 28-30 days were randomly divided into 5 groups, 5 heads/group, immunogenicity of porcine circovirus type 3 Cap protein prepared in immunization example 5. combination.
  • the first to fourth groups of piglets were immunized with immunogenic compositions 1 to 4, respectively, and the fifth group of piglets were not immunized as the challenge control group.
  • the immunized group was injected with 2 ml/head of the immunogenic composition, and the control group was given 2 ml/head of normal saline. On the 28th day after immunization, each group was challenged.
  • the dose was porcine circovirus type 3 SG strain (Porcine Circovirus type 3, strain SG, deposited in China Center for Type Culture Collection, deposit number CCTCC NO.V201712, date of preservation For March 23, 2017, the deposit address: Wuhan University, Wuhan, China, 10 5.0 TCID 50 / head, continuous observation of each group of piglets after the attack, killing all the test pigs 25 days after the attack, according to the clinical characteristics of each group of piglets Symptoms, pathological changes and virus test results were determined. The specific results are shown in Table 3.
  • porcine circovirus type 3 Cap protein immunogenic composition can provide 100% (5/5) protection for piglets after immunization of piglets, while all the piglets in the challenge control group were infected. It is indicated that the porcine circovirus type 3 Cap protein immunogenic composition provided by the invention has good protection.
  • the 6th and 11th groups of piglets were challenged with the virulent strain of porcine circovirus type 3 HN12 strain newly isolated from Henan province of China; the 7th and 12th groups of piglets were newly used from China.
  • the virulent strain of porcine circovirus type 3 JS08 strain isolated from Jiangsu province was attacked; the 8th and 13th groups of piglets were challenged with the virulent strain of porcine circovirus type 3 JL11 strain isolated from Jilin province, China; Group 14 and piglets were challenged with the newly virulent strain of porcine circovirus type 3 CQ04 strain isolated from Chongqing, China; the 10th and 15th groups of piglets were newly porcine circovirus type 3 isolated from Guangdong province, China.
  • GD05 strain virulent strains were challenged; the doses were 10 5.0 TCID 50 /head. After the challenge, each piglet was observed continuously. All pigs were killed 25 days after the challenge, according to the clinical symptoms, pathological changes and viruses of each piglet. The test is judged, and the specific results are shown in Table 4.
  • the porcine circovirus type 3 virus can be isolated again by PCR detection of each visceral and organ tissue; and 6 ⁇ 10 There were no abnormal clinical symptoms after challenge in each immunization group, and no abnormalities were observed in all tissues and organs. PCR detection was performed on each viscera and organ tissues, and all showed PCV3 negative.
  • porcine circovirus type 3 Cap protein immunogenic composition provided by the invention can provide effective and complete immune protection against porcine circovirus type 3 challenge against pigs of different geographical origins, and from various visceral organs and organ tissues.
  • the PCV3 strain that was attacked could not be detected.
  • the immunogenic 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. Since the virus is not infected in the pig body after immunization, the immunogenic composition of the present invention is important for the purification of a pig farm infected with PCV3.
  • the sow mortality rate increased by 8.9%, the conception rate decreased by 1.4%, and the mummified fetus increased by 8.5%.
  • the affected sows showed anorexia, with symptoms of multifocal papules, spots and superficial dermatitis.
  • 38 pregnant sows with clinical manifestations were randomly divided into two groups, group A, group B, group 19/group, and group A was immunogenic.
  • group A was immunized with the porcine circovirus type 3 Cap protein immunogenic composition 1 prepared in Example 5, and the group B was a blank control group.
  • the immunogenic group was injected with 2 ml/head of the immunogenic composition, and the blank control group was inoculated with physiological saline 2 ml/head.
  • the results of the two groups of sows were counted. The results are shown in Table 5.
  • the immunized group had no abnormal births, producing healthy piglets with an average of 11.79 heads/well, and the health rate was 99.6%.
  • the control group showed obvious mummies and weak piglets, and the average healthy piglets produced 7.6 heads/well.
  • the rate was 65.0%, and there were two sows with abortion, and the whole nest was mummified. The difference between the immunized group and the control group was significant.
  • the healthy piglets from the control group B were isolated and nested, and as the subject of the following experiment, 17 litters were divided into two groups B1 (including B-1 to B-15, except for B-4 The nest of mummies is not included, a total of 14 litters of piglets, and the B2 group (including B-16 to B-19 litters, except for the B-18 litter due to the whole nest of mummies, a total of 3 litters), and the B1 group is immunized before breastfeeding.
  • the immunized group piglets were injected with the immunogenic composition 2 ml/head, and the blank control group piglets were inoculated with physiological saline 2 ml/head. The piglets were continuously observed, and the dead piglets were immediately dissected. The piglets in the B1 group and the piglets in the B2 group were sacrificed on the 25th day. The clinical symptoms, pathological changes and virus detection of each piglet were determined. The specific results are shown in Table 6.

Abstract

提供一种猪圆环病毒3型免疫原性蛋白，以及该免疫原性蛋白制备的免疫原性组合物。还提供了制备所述免疫原性组合物的方法以及所述免疫原性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。

Description

一种猪圆环病毒3型免疫原性组合物、制备方法和应用 技术领域
本发明涉及兽药领域，具体涉及一种猪圆环病毒3型蛋白、含该蛋白的免疫原性组合物、制备方法和应用。
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蛋白应该具有＞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/或治疗猪圆环3型病毒相关疾病的免疫原性组合物，该免疫原性组合物能对猪圆环病毒3型提供有效的保护，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型免疫原性蛋白Cap蛋白，其序列由SEQ ID No.1所示的核苷酸序列编码，由其制备的免疫原性组合物能有效抵抗流行株的攻击，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/或治疗猪圆环病毒3型相关疾病的免疫原性组合物，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蛋白或免疫量的重组有所述猪圆环病毒3型Cap蛋白基因的活载体，以及兽医学上可接受的载体。
本发明的另一个目的是提供一种制备所述免疫原性组合物的方法，所述方法包括：步骤(1)克隆所述猪圆环病毒3型Cap蛋白基因，并将所述猪圆环病毒3型Cap蛋白基因重组到表达载体以获得含有所述猪圆环病毒3型Cap蛋白基因的重组表达载体；步骤(2)将所述含有所述猪圆环病毒3型Cap蛋白基因的重组表达载体和分子伴侣的表达载体共同转化大肠杆菌，表达所述猪圆环病毒3型Cap蛋白；步骤(3)分离所述表达的猪圆环病毒3型Cap蛋白，使用非离子表面活性剂处理除去内毒素；以及步骤(4)将所述去除内毒素的猪圆环病毒3型Cap蛋白与佐剂混匀、乳化，得到所述免疫原性组合物。
本发明还涉及所述的免疫原性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。
本发明首次采用猪圆环病毒3型免疫原性Cap蛋白基因高效表达后，制备免疫原性组合物，可预防或治疗猪圆环病毒3型相关疾病引起的疫情。
本发明猪圆环病毒3型Cap蛋白制备的免疫原性组合物，其免疫原性好，能对猪只的攻毒产生完全保护，能有效对多种地域来源的野毒株进行预防，即在临床应用上能对猪圆环病毒3型的感染、蔓延进行预防、治疗和控制。
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型为一种基因组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，现在已知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。
本发明涉及一种猪圆环病毒3型免疫原性组合物，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蛋白抗原或免疫量的重组有所述猪圆环病毒3型Cap蛋白基因的活载体，以及兽医学上可接受的载体。本发明首次发现猪圆环病毒3型Cap蛋白具有良好的免疫原性，其制备的亚单位抗原或重组有其基因的活载体均能在免疫后产生良好的免疫效力，对猪只提供100％的保护。
术语“免疫原性组合物”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。
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。
术语“猪圆环病毒3型抗原”是指至少含有一种猪圆环病毒3型抗原 形式的任何组合物，所述猪圆环病毒3型抗原可诱导、刺激或增强猪圆环病毒3型感染的免疫应答，所述抗原形式包括但不限于灭活的、减毒的或亚单位的抗原。
本发明所述的猪圆环病毒3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，在本发明的免疫原性组合物中，所述的猪圆环病毒3型Cap蛋白为SEQ ID NO.1所示的核苷酸序列编码的蛋白。
作为本发明的一种实施方式，在本发明的免疫原性组合物中，所述的猪圆环病毒3型Cap蛋白的编码基因具有SEQ ID NO.1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。
作为本发明的一种优选实施方式，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。
作为本发明的一种更优选实施方式，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。
作为本发明的一种实施方式，在本发明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。
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。
本发明的猪圆环病毒3型Cap基因还可以应用于表达载体、核酸疫苗、诊断试剂开发以及其它预防和/或治疗猪圆环病毒3型相关疾病的药物开发。
本发明涉及一种猪圆环病毒3型Cap蛋白，其序列由SEQ ID No.1所示的核苷酸序列编码。
本发明涉及一种重组载体，所述重组载体能表达本发明所述的猪圆环病毒3型Cap蛋白，其具有免疫原性并能产生免疫反应。
本发明涉及一种转化子，所述转化子包含有导入的本发明所述的表达猪圆环病毒3型Cap蛋白的重组载体。
作为本发明的一种实施方式，本发明所述免疫原性组合物中，所述兽医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)Montanide TMGel。
作为本发明的一种优选实施方式，本发明所述免疫原性组合物中，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)Montanide TMGel。
优选地，所述佐剂为Montanide TMGel，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。
优选地，所述佐剂为Montanide TMGel，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％。
本发明所述免疫原性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)，以及其它动物油、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。
本发明的免疫原性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)免疫原性组合物可以以水包油、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。
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的量。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。
本发明的Cap蛋白可以通过本领域内任何已知的方法制备，例如可以通过重组表达Cap基因制备Cap蛋白，表达系统可以使用任何已知的表达系统，例如：真核表达系统、原核表达系统。或者直接合成Cap蛋白。真核表达系统可以包括哺乳动物细胞表达系统，酵母表达系统和昆虫表达系统。
本发明还涉及一种制备所述免疫原性组合物的方法，其中，所述方 法包括：步骤(1)克隆所述猪圆环病毒3型Cap蛋白基因，并将所述猪圆环病毒3型Cap蛋白基因重组到表达载体以获得含有所述猪圆环病毒3型Cap蛋白基因的重组表达载体；步骤(2)将所述含有所述猪圆环病毒3型Cap蛋白基因的重组表达载体和分子伴侣的表达载体共同转化大肠杆菌，表达所述猪圆环病毒3型Cap蛋白；步骤(3)分离所述表达的猪圆环病毒3型Cap蛋白，使用非离子表面活性剂处理除去内毒素；以及步骤(4)将所述去除内毒素的猪圆环病毒3型Cap蛋白与佐剂混匀、乳化，得到所述免疫原性组合物。
作为本发明的一种实施方式，在所述的制备所述免疫原性组合物的方法中，所述步骤(1)中所述含有猪圆环病毒3型Cap蛋白基因的重组表达载体为重组pET28a质粒，所述步骤(2)中所述分子伴侣的表达载体为pG-Tf2，所述大肠杆菌为大肠杆菌BL21(DE3)；所述步骤(3)中非离子表面活性剂为Triton X-114。
本发明免疫原性组合物还可以进一步将其它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。
本发明还涉及所述免疫原性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，猪的皮炎肾病终合症、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型相关疾病时是指抑制 猪圆环病毒3型的复制、抑制猪圆环病毒3型的传播或防止猪圆环病毒3型在其宿主体内定居，以及减轻猪圆环病毒3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治疗达到了治疗效果。
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。
为使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。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感染引起流产的症状一致。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，置于37℃培养5日。收获病毒，经2次冻融后，收获含病毒的培养液。
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它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蛋白应该具有＞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 pET28a-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；T4 DNA 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，其它试剂均为分析纯。
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于病毒液中，涡旋混匀，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混合液加入VB柱中，14000g离心1分钟，将剩余的混合液加入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 W1 buffer，14000g离心30秒，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DNA基因组溶液。
2、Cap蛋白基因扩增
根据Cap蛋白基因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 Cap蛋白基因扩增引物
Figure PCTCN2018084754-appb-000001
将PCR产物送Invitrogen公司测序，根据测序结果对Cap蛋白基因进行密码子优化，优化后的Cap蛋白基因序列如序列表SEQ ID NO.1所示。
3、表达载体构建
优化后Cap蛋白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，阳性克隆即为pET28a-PCV3-Cap/pG-Tf2表达菌株。
实施例3 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)，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，28℃振荡培养24小时。培养结束后，收集菌体，并用PBS(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，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Cap蛋白内毒素清除
在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)，涡旋振荡混匀。将混匀的样品在冰上放置5分钟。待样品冷却后，将离心管立即放入37℃水浴中温浴5min，使得产生新的两相。然后，使样品在37℃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。整个去除内毒素的操作循环3次。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。
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3型Cap蛋白免疫原性组合物的制备
将按实施例4的方法纯化后的Cap蛋白缓缓加入到水溶性佐剂Gel佐剂Montanide TMGel(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2。
表2猪圆环病毒3型Cap蛋白免疫原性组合物配比
Figure PCTCN2018084754-appb-000002
实施例6 猪圆环病毒3型Cap蛋白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、抗体均为阴性的健康仔猪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Cap蛋白免疫原性组合物。第1～4组仔猪分别免疫免疫原性组合物1～4，第5组仔猪不免疫作为攻毒对照组。各免疫组仔猪注射免疫原性组合物2ml/头，攻毒对照组仔猪接种生理盐水2ml/头。免疫后28日各组进行攻毒，攻毒剂量为猪圆环病毒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)，10 5.0TCID 50/头，攻毒后连续观察各组仔猪，攻毒后25天剖杀所有试验猪，根据各组仔猪临床症状、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Cap蛋白免疫原性组合物免疫原性试验结果
Figure PCTCN2018084754-appb-000003
结果显示，猪圆环病毒3型Cap蛋白免疫原性组合物免疫仔猪后，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。表明本发明提供的猪圆环病毒3型Cap蛋白免疫原性组合物具有很好的保护力。
实施例7 猪圆环病毒3型Cap蛋白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、抗体均为阴性 的健康仔猪50头随机分成10组，5头/组，第6～10组免疫实施例5制备的猪圆环病毒3型Cap蛋白免疫原性组合物1，第11～15组不免疫作为攻毒对照组。各免疫组仔猪注射免疫原性组合物2ml/头，对照组仔猪接种生理盐水2ml/头。免疫后28日进行攻毒，第6组、第11组仔猪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7组、第12组仔猪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8组、第13组仔猪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9组、第14组仔猪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10组、第15组仔猪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；攻毒剂量均为10 5.0TCID 50/头，攻毒后连续观察各仔猪，于攻毒后25天剖杀所有试验猪，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Cap蛋白免疫原性组合物广谱性保护试验结果
Figure PCTCN2018084754-appb-000004
Figure PCTCN2018084754-appb-000005
结果显示，第11～15组各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，通过各内脏、器官组织进行PCR检测，可再次分离到猪圆环病毒3型病毒；而第6～10组各免疫组攻毒后无异常临床症状，剖检各组织器官也无异常，通过各内脏、器官组织进行PCR检测，均显示PCV3阴性。表明本发明提供的猪圆环病毒3型Cap蛋白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，且从各内脏、器官组织中不能检出攻毒的PCV3株。本发明的免疫原性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。由于免疫后猪体中没有感染的病毒，本发明的免疫原性组合物对于感染了PCV3的猪场的净化有重要意 义。
实施例8猪圆环病毒3型Cap蛋白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8.9％，受孕率降低了1.4％，木乃伊胎增加8.5％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38头怀孕母猪随机分成两组A组、B组，19头/组，A组为免疫原性组合物接种组，A组免疫实施例5制备的猪圆环病毒3型Cap蛋白免疫原性组合物1，B组为空白对照组。免疫组注射免疫原性组合物2ml/头，空白对照组接种生理盐水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 PCTCN2018084754-appb-000006
Figure PCTCN2018084754-appb-000007
结果显示，免疫组母猪产子无异常，生产健康仔猪，平均11.79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7.6头/窝，健康率65.0％，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Cap蛋白免疫原性组合物 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、所产仔猪进行保护。
同时，对B组对照组所产健康仔猪分别隔离、按窝饲养，作为以下实验的对象，17窝分成两组B1组(包括B-1窝至B-15窝，除B-4窝因全窝木乃伊胎不计外，共14窝仔猪)、B2组(包括B-16至B-19窝，除B-18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5制备的猪圆环病毒3型Cap蛋白免疫原性组合物1，B2组为空白对照组。免疫组仔猪注射免疫原性组合物2ml/头，空白对照组仔猪接种生理盐水2ml/头。连续观察各仔猪，死亡仔猪即时解剖，于25天剖杀B1组抽检仔猪及B2组存活仔猪，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Cap蛋白免疫原性组合物对仔猪的免疫保护试验
Figure PCTCN2018084754-appb-000008
Figure PCTCN2018084754-appb-000009
Figure PCTCN2018084754-appb-000010
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，通过剖检猪只各内脏、器官组织进行PCR检测，均显示PCV3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，通过各内脏、器官组织进行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Cap蛋白免疫原性组合物对感染猪圆环病毒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. 一种猪圆环病毒3型免疫原性组合物，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蛋白或免疫量的重组有所述猪圆环病毒3型Cap蛋白基因的活载体，以及兽医学上可接受的载体。
  2. 根据权利要求1所述的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白为SEQ ID No.1所示的核苷酸序列编码的蛋白。
  3. 根据权利要求1所述的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为≥20μg/ml。
  4. 根据权利要求1所述的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为20μg/ml～100μg/ml。
  5. 根据权利要求1所述的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为20μg/ml～50μg/ml。
  6. 根据权利要求1所述的免疫原性组合物，其中，所述猪圆环病毒3型Cap蛋白含量为30μg/ml～50μg/ml。
  7. 根据权利要求1所述的免疫原性组合物，其中，所述重组有所述猪圆环病毒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。
  8. 根据权利要求1所述的免疫原性组合物，其中，所述兽医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)Montanide TMGel。
  9. 根据权利要求8所述的免疫原性组合物，其中，所述佐剂为Montanide TMGel，所述佐剂用量为体积比5～20％。
  10. 根据权利要求9所述的免疫原性组合物，其中，所述佐剂用量为体积比10％。
  11. 一种制备权利要求1所述免疫原性组合物的方法，其中，所述方法包括：步骤(1)克隆所述猪圆环病毒3型Cap蛋白基因，并将所 述猪圆环病毒3型Cap蛋白基因重组到表达载体以获得含有所述猪圆环病毒3型Cap蛋白基因的重组表达载体；
    步骤(2)将所述含有所述猪圆环病毒3型Cap蛋白基因的重组表达载体和分子伴侣的表达载体共同转化大肠杆菌，表达所述猪圆环病毒3型Cap蛋白；
    步骤(3)分离所述表达的猪圆环病毒3型Cap蛋白，使用非离子表面活性剂处理除去内毒素；以及
    步骤(4)将所述去除内毒素的猪圆环病毒3型Cap蛋白与佐剂混匀、乳化，得到所述免疫原性组合物。
  12. 根据权利要求11所述的方法，其中，所述步骤(1)中所述含有猪圆环病毒3型Cap蛋白基因的重组表达载体为重组pET28a质粒，所述步骤(2)中所述分子伴侣的表达载体为pG-Tf2，所述大肠杆菌为大肠杆菌BL21(DE3)；所述步骤(3)中非离子表面活性剂为Triton X-114。
  13. 根据权利要求1～10所述的免疫原性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。
  14. 一种猪圆环病毒3型Cap蛋白，其序列由SEQ ID No.1所示的核苷酸序列编码。
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