WO2014146603A1 - 败血性巴氏杆菌毒素重组蛋白及其应用 - Google Patents

败血性巴氏杆菌毒素重组蛋白及其应用 Download PDF

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WO2014146603A1
WO2014146603A1 PCT/CN2014/073851 CN2014073851W WO2014146603A1 WO 2014146603 A1 WO2014146603 A1 WO 2014146603A1 CN 2014073851 W CN2014073851 W CN 2014073851W WO 2014146603 A1 WO2014146603 A1 WO 2014146603A1
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toxin
recombinant protein
septicum
pasteurella
group
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PCT/CN2014/073851
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English (en)
French (fr)
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杨滢臻
郭村勇
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施怀哲维克生物科技股份有限公司
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Priority to EA201591861A priority Critical patent/EA201591861A1/ru
Priority to EP14768194.4A priority patent/EP2982683B1/en
Publication of WO2014146603A1 publication Critical patent/WO2014146603A1/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
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/285—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza

Definitions

  • the present invention relates to the use of a recombinant protein of Pasteurella septicum toxin in the field of animal health, and more particularly to the use of a porcine atrophic rhinitis immunological composition containing a recombinant protein of Pasteurella multocida toxin in the field of animal health.
  • Atrophic Rhinitis is one of the three major infectious diseases in the respiratory system of pigs.
  • Atrophic rhinitis in pigs can cause facial deformities and chronic suppurative rhinitis in infected pigs, causing atrophy of the turbinate.
  • lesions can also occur in the nasal cavity, humerus, and upper jaw.
  • the lower fossa of the inferior turbinate is most often infected.
  • Upper turbinate, inferior turbinate bone, nasal sputum or ethmoid bone are all infected. Book
  • Porcine atrophic rhinitis is caused by Sortfete//a bronchiseptica and Pasteurella septicum CPaWeMre//multocida) (PmA) and D-type bacteria (PmD).
  • PmA Sortfete//a bronchiseptica and Pasteurella septicum CPaWeMre//multocida
  • PmD D-type bacteria
  • the toxin produced by Pasteurella multocida D-type bacteria (PmD) is 0 PaW £ ⁇ 4/re//a multocida toxin, PMT). Inoculation of PMT toxins in muscle, abdominal and nasal inoculation in four-week-old piglets can cause lesions of the nasal atrophy, and also affect the development of systemic bones, causing growth retardation, even at high doses. It can cause damage to the liver and cause jaundice and death in pigs.
  • Porcine atrophic rhinitis (AR) is spread throughout the world in pig raising areas, with slow growth of pigs and reduced feed utilization efficiency. Although the mortality rate is not high when infected alone, the pollution rate is very large, and it is easy to induce infection of other comorbidities or pathogens, resulting in high mortality rate and increased production cost. In pig farms with severe atrophic rhinitis (AR) infection, the economic loss is about 15-38%, and there are obvious growth disorders in heavily infected pig farms. The average daily gain is about the same as that of normal pigs. Less 5-8%. Therefore, it is imperative and extremely important to develop an effective porcine atrophic rhinitis vaccine to prevent pigs from suffering from atrophic rhinitis (AR). Summary of the invention
  • the present invention provides, in the first part, a recombinant protein of Recombinant PMT (rPMT) comprising at least one epitope (EPT) of S. septicum toxin protein (PMT) for inducing animals
  • rPMT Recombinant PMT
  • EPT epitope
  • PMT S. septicum toxin protein
  • the S. septicum toxin recombinant protein (rPMT) may further comprise a full-length or partial amino acid sequence of the complement cleavage fragment C3d to increase the specific immune response. And the antigenicity of each of the S.
  • septicum toxin proteins (PMT)
  • the full length or partial amino acid sequence of each of the complement cleavage fragments C3d and the antigenic determination of the septicemia pasteurin toxin protein (PMT)
  • the position may be further linked by a linker between the full length or a partial amino acid sequence of the complement cleavage fragment C3d.
  • the S. septicum toxin recombinant protein (rPMT) provided by the present invention can be represented by the following formula: (A) m - (C3d fragment) n;
  • Each of these A represents an epitope of an independent S. septicum toxin protein, and each A line is independently selected from SEQ ID NOs: 2, 3, 4, 20, 21, 22, 23, 24, Groups of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42;
  • Each of the C3d fragments represents an amino acid sequence of an independent complement cleavage fragment C3d, and each C3d fragment is independently selected from the group consisting of SEQ ID NOs: 6, 7, 8, and 9;
  • n is an integer representing from 1 to about 30;
  • n represents an integer from 0 to about 10.
  • the present invention provides a nucleotide sequence encoding a recombinant protein of Pasteurella multocida toxin (rPMT) in the second part.
  • the S. septicum toxin recombinant protein (rPMT) comprises at least one antigenic epitope of the S. septicum toxin protein (PMT), and a partial amino acid sequence of the complement cleavage fragment C3d of 0 to 10 units.
  • the invention provides a porcine atrophic rhinitis immune composition in the third part.
  • the porcine atrophic rhinitis immunocomplex comprises a S. septicum toxin recombinant protein (rPMT) and a pharmaceutically acceptable carrier.
  • the S. septicum toxin recombinant protein (rPMT) comprises at least one epitope (epitopes) of the S. septicum toxin protein (PMT), and a partial amino acid sequence of the complement cleavage fragment C3d of 0 to 10 units.
  • the porcine atrophic rhinitis immunological composition may further comprise B. bronchiseptica ⁇ ⁇ . wwc/ ⁇ e ⁇ 'ca), S. septicum type A bacterium (PmA), and S. septicum D type bacterium ( PmD).
  • the invention provides a method for combating atrophic rhinitis in pigs according to the fourth aspect, which comprises administering an effective amount of the above-mentioned immunological composition to the animal to enhance the immunity of the animal against atrophic rhinitis of the pig, thereby improving and improving Its clinical symptoms, survival rate, and weight gain trends.
  • the present invention provides an antibody against Pasteurella septicum D-type toxin (PMT) in the fifth part, which is prepared or derived by the recombinant protein of septicum Pasteurella toxin (rPMT) provided by the present invention.
  • PMT Pasteurella septicum D-type toxin
  • rPMT recombinant protein of septicum Pasteurella toxin
  • Such antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.
  • a test kit for porcine atrophic rhinitis which is used for detecting whether a sample contains a septicemia type D-toxin (PMT) or detecting whether a sample is in the test sample.
  • the porcine atrophic rhinitis immunological composition provided by the present invention has the following advantages when compared with other conventional techniques - the porcine atrophic rhinitis immunological composition provided by the present invention contains a recombinant protein of Pasteurella septicum toxin ( rPMT).
  • the S. septicum toxin recombinant protein (rPMT) comprises at least one antigenic epitope of the S. septicum toxin protein (PMT) and a partial amino acid sequence of the complement cleavage fragment C3d.
  • the S. septicum toxin recombinant protein (rPMT) is much shorter than the full-length amino acid sequence of the S. septicum toxin protein (PMT), is easier to express in the biological expression system, and has a higher yield of the recombinant protein. Reduce the cost of manufacturing vaccines.
  • the porcine atrophic rhinitis immunological composition provided by the invention comprises the recombinant protein of Pasteurella septicum toxin (rPMT) has a partial amino acid sequence of the complement cleavage fragment C3d, thereby increasing the specific immune response, and the test results show that the porcine atrophic rhinitis immunological composition provided by the invention can improve the immunity of the animal against porcine atrophic rhinitis. effectiveness.
  • rPMT Pasteurella septicum toxin
  • Figure 1 shows the results of measuring the valence of anti-Septic bacillus toxin (PMT) antibody by enzyme-linked immunoassay (ELISA);
  • Group 1 is a negative control group;
  • Group 2 is a bronchial septic group obtained in Example 2.
  • Immunocompetitive composition of Desmodium, S. septicum type A and S. septicum type D (Bb + PmA + PmD group);
  • Group 3 is the recombinant protein of Pasteurella septicum toxin obtained in Example 1.
  • rPMT group Group 4 is a porcine atrophic rhinitis immune composition containing B.
  • Figure 2 shows the results of the neutralization antibody test analysis;
  • Figure 2 shows the cell morphology of Vero cells cultured in DMEM containing fetal bovine serum (FBS) (negative control group);
  • Figure 2B shows the minimum toxic dose (MTD) of 4 times.
  • FBS fetal bovine serum
  • the septicemia pasteurin toxin (PMT) treated the cell morphology of Vero cells (positive control group), and the cells showed typical nodular morphology;
  • Figure 2C shows the atrophy of pigs immunized with recombinant protein containing Pasteurella septicum toxin
  • the mouse serum of the rhinitis immunological composition (Bb + PmA + PmD + rPMT group) was diluted 40-fold and neutralized with 4 times the minimum toxic dose (MTD) of S. septicum toxin (PMT), and then added to Vero cells. Cultured cell morphology.
  • FIG. 3 is a result of measuring the valence of anti-Septic bacillus toxin (PMT) antibody by enzyme-linked immunoassay (ELISA); the first group is a negative control group; the second group is a bronchial septic bolus obtained in the second embodiment.
  • Immune composition of Desmodium, S. septicum type A and S. septicum type D (Bb + PmA + PmD group);
  • Group 3 is porcine atrophy containing recombinant protein of S. septicum toxin Rhinitis immune composition (Bb + PmA + PmD + rPMT group).
  • the symbol ** represents a significant difference between the two groups (p ⁇ 0.01). detailed description
  • the present invention provides a S. septicum toxin recombinant protein (rPMT) comprising at least one epitope of a Pasteurella multocida toxin protein (PMT) for inducing an animal to produce a septic-resistant Pasteurella toxin protein (PMT) Antibodies.
  • rPMT S. septicum toxin recombinant protein
  • PMT Pasteurella multocida toxin protein
  • PMT septic-resistant Pasteurella toxin protein
  • the epitope of the S. septicum toxin protein has SEQ ID NOs: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, respectively.
  • the amino acid sequences shown in 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42, the epitope of the S. septicum toxin protein (PMT) may be from 1 to about
  • the order of the amino acid sequences of the epitopes from the N-terminus to the C-terminus of the protein is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • each of the three longer epitope amino acid sequences encompasses an epitope of a portion of the above-described Pasteurella septicum toxin protein (PMT), the three longer epitope-determining amino acid sequences.
  • PMT Pasteurella septicum toxin protein
  • SEQ ID NOs: 2, 3, 4 are shown.
  • the S. septicum toxin recombinant protein (rPMT) of the present invention comprises an epitope determined by the amino acid sequence set forth in SEQ ID NO: 2.
  • the S. septicum toxin protein (PMT) comprises an epitope determined by the amino acid sequence set forth in SEQ ID NO: 3.
  • the S. septicum toxin protein (PMT) comprises an epitope determined by the amino acid sequence set forth in SEQ ID NO: 4.
  • the S. septicum toxin protein (PMT) comprises an epitope determined by the amino acid sequence set forth in SEQ ID NOs: 2 and/or 3 and/or 4.
  • the S. septicum toxin recombinant protein (rPMT) of the present invention contains two or more of such longer epitope amino acid sequences.
  • the order of arrangement of the respective epitope amino acid sequences from the N-terminus to the C-terminus of the protein is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • each Pasteurella septicum when the epitope of the S. septicum toxin protein (PMT) and/or the longer epitope amino acid sequence are plural (2), each Pasteurella septicum
  • the epitope of the toxin protein (PMT) and/or the longer epitope amino acid sequence may be further linked by a linker comprising at least one glycine (Glycine, Gly), the linkage Subunits include but are not limited to: Gly-Gly, Gly-Ser, SEQ ID NOs: 5, 12, 13, 14, 15, 16, 17, 18, 19.
  • the linker has an amino acid sequence as set forth in SEQ ID NO: 5.
  • the antigenic epitope of the plurality of Pasteurella multocida toxin proteins (PMT) and/or the plurality of longer epitope-determining amino acid sequences are not necessarily linked by a linker.
  • the S. septicum toxin recombinant protein (rPMT) provided by the present invention further comprises a full-length or partial amino acid sequence of the complement cleavage fragment C3d to increase a specific immune response.
  • the full-length amino acid sequence of the complement cleavage fragment C3d is the full length sequence (mC3d) of the mouse complement cleavage fragment C3d, having the amino acid sequence set forth in SEQ ID NO: 8.
  • the partial amino acid sequence of the complement cleavage fragment C3d is the 211th to 238th amino acid fragment sequence (mC3d-p28) of the mouse complement cleavage fragment C3d, as shown in SEQ ID NO: 6.
  • the full length amino acid sequence of the complement cleavage fragment C3d is the full length sequence (pC3d) of the porcine complement cleavage fragment C3d, having the amino acid sequence set forth in SEQ ID NO: 9.
  • the partial amino acid sequence of the complement cleavage fragment C3d is the 201st amino acid to 231th amino acid fragment sequence (pC3d-p31) of the porcine complement cleavage fragment C3d, as shown in SEQ ID NO: 7. Amino acid sequence. The S.
  • septicum toxin recombinant protein provided by the present invention has a full-length or partial amino acid sequence of the above-described complement cleavage fragment C3d of 1 to 10 unit repeats, and the full-length or partial amino acid sequence of the complement cleavage fragment C3d is compared.
  • the number is from 1 to 10 units, and more preferably from 4 to 8 units.
  • the full-length or partial amino acid sequence of each complement cleavage fragment C3d may be linked by a linker containing at least one glycine (Gly).
  • Linkers include, but are not limited to, Gly-Gly, Gly-Ser, SEQ ID NOs: 5, 12, 13, 14, 15, 16, 17, 18, 19.
  • the amino acid sequence of the linker is Gly-Ser.
  • the full length or partial amino acid sequence of the plurality of complement cleavage fragments C3d is not necessarily linked by a linker.
  • the S. septicum toxin recombinant protein (rPMT) of the present invention can be represented by the following formula:
  • each of these A represents an epitope of an independent S. septicum toxin protein, and each A line is independently selected from SEQ ID NOs: 2, 3, 4, 20, 21, 22, 23, 24, Groups of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42;
  • Each of the C3d fragments represents an amino acid sequence of an independent complement cleavage fragment C3d, and each C3d fragment is independently selected from the group consisting of SEQ ID NOs: 6, 7, 8, and 9;
  • n is an integer representing from 1 to about 30;
  • n represents an integer from 0 to about 10.
  • each A is further joined by a linker, each of which is independently selected from Gly-Gly, Gly-Ser, SEQ ID NOs: 5, 12, 13, 14, 15 , 16, 17, 18, 19.
  • each C3d fragment is joined by a linker, each of which is independently selected from Gly-Gly, Gly-Ser, SEQ ID NOs: 5, 12, 13, 14, 15 , 16, 17, 18, 19.
  • the S. septicum toxin recombinant protein (rPMT) provided by the present invention has at least about 80% sequence homology with the amino acid sequence represented by the above formula (I), preferably, having about 85% sequence homology, more preferably, about 90% sequence homology, even about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, Approximately 98%, approximately 99% sequence homology.
  • the S. septicum toxin recombinant protein (rPMT) provided by the present invention has three epitopes covered by the amino acid sequences shown in SEQ ID NOs: 2, 3, and 4, respectively. And repeating the 6-unit mouse complement cleavage fragment C 3d from the 204th amino acid to the 231th amino acid fragment sequence (mC3d-p28), and the C-terminus of each epitope (epitopes) is a linker (sequence as SEQ ID NO : 5) The ligation of the S. septicum toxin recombinant protein (rPMT) has the amino acid sequence set forth in SEQ ID NO: 10.
  • the S. septicum toxin recombinant protein has three epitopes encompassed by the amino acid sequences set forth in SEQ ID NOs: 2, 3, 4, respectively, and repeats 6
  • the porcine complement cleavage fragment C3d from the 201st amino acid to the 231th amino acid fragment sequence (pC3d-p31), and the C-terminus of each epitope (epitopes) is ligated (the sequence is shown in SEQ ID NO: 5)
  • the S. septicum toxin recombinant protein has the amino acid sequence set forth in SEQ ID NO: 11.
  • the present invention also provides a nucleotide sequence encoding the recombinant S. septicum toxin (rPMT) of the present invention.
  • the S. septicum toxin recombinant protein (rPMT) comprises at least one epitope of the S. septicum toxin protein (PMT), and a partial amino acid sequence of the complement cleavage fragment C3d of 0 to 10 units.
  • the nucleotide sequence encoding the S. septicum toxin recombinant protein (rPMT) of the present invention is derived from the amino acid sequence of the S. septicum toxin recombinant protein (rPMT) of the present invention.
  • each amino acid of the amino acid sequence of the S. septicum toxin recombinant protein (rPMT) of the present invention into a nucleotide sequence encoding the amino acid listed in the genetic code table (including various degenerate codons) (degenerate codons, or synonymous codons), the nucleotide sequence provided by the present invention can be obtained.
  • serine on the amino acid sequence of the S. septicum toxin recombinant protein (rPMT) of the present invention can be encoded by nucleotide sequences such as TCT, TCC, TCA, TCG, AGT, AGC and the like.
  • Ben Each of the amino acids in the amino acid sequence of the S. septicum toxin recombinant protein (rPMT) of the invention may be encoded by the following nucleotide sequences:
  • the present invention also provides a porcine atrophic rhinitis immunological composition.
  • the porcine atrophic rhinitis immune composition contains a septicemia Pasteurella toxin recombinant protein (rPMT).
  • the S. septicum toxin recombinant protein (rPMT) comprises at least one epitope of the S. septicum toxin protein (PMT), and a partial amino acid sequence of the complement cleavage fragment C3d of 0 to 10 units.
  • the S. septicum toxin recombinant protein (rPMT) has the amino acid sequence set forth in SEQ ID NO: 10.
  • the S. septicum toxin recombinant protein has the amino acid sequence set forth in SEQ ID NO: 10.
  • the S. septicum toxin recombinant protein (rPMT) has the amino acid sequence set forth in SEQ ID NO:11.
  • the S. septicum toxin recombinant protein (rPMT) provided by the present invention includes, but is not limited to, a method of gene selection or synthesis by a peptide synthesizer; a method for obtaining the recombinant protein by gene selection This may be, but is not limited to, the selection of a DNA sequence encoding a recombinant protein of the septic Aspergillus toxin (rPMT) into a expression vector, each forming a nucleotide sequence containing a recombinant protein encoding the S. septicum toxin (rPMT) The plastid, which is then transferred to a biologically expressed host, and the antigenic protein obtained after protein expression.
  • the expression vector system includes, but is not limited to, a pET vector system and a pGEX vector system, etc.;
  • the biological expression system (host) includes but is not limited to: a prokaryotic expression system (eg, E. coli), a eukaryotic expression system (eg, : Animal cells (insect cells or mammalian cells), plant cells).
  • the porcine atrophic rhinitis immunological composition provided by the present invention further comprises B. bronchiseptica , ⁇ . bronchiseptica), Pasteurella septicum type A (PmA) and Pasteurella septicum D Type bacteria (PmD).
  • B. bronchiseptica ⁇ . bronchiseptica), Pasteurella multocida type A (PmA) and P. septicum D-type bacteria (PmD) are administered by the Executive Yuan Agricultural Committee livestock The Health Laboratory (Taiwan) gave way.
  • the source of B. bronchiseptica S.
  • Pasteurella multocida type A PmA
  • Pasteurella multocida type D PmD
  • Source strains from other sources may also be used in combination with the S. septicum toxin recombinant protein (rPMT) of the present invention.
  • rPMT S. septicum toxin recombinant protein
  • the B. bronchiseptica may also be a collection of standard biological products such as the United States ( American Type Culture Collection, ATCC) No.
  • the Pasteurella septicum type A may also be, for example, the National Collection of Type Cultures (NCTC) number NCTC 12177, and the septic The Pasteurella type D bacteria (PmD) may also be a strain such as the British National Standards Collection of Biological Products (NCTC) number NCTC 12178, or a strain derived from field isolation.
  • NCTC National Collection of Type Cultures
  • PMD The Pasteurella type D bacteria
  • the porcine atrophic rhinitis immunological composition provided by the present invention may further comprise other pathogenic antigens selected from the group consisting of porcine circovirus type 2 (PCV2) antigen, swine influenza virus (SIV). Antigen, Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) antigen, Mycoplasma, Parvovirus (PPV), Erysipelas, Aujeszky's disease, and Actinobacillus pleuropneumoniae Actinobacillus pleuropneumonia , ⁇ ).
  • PCV2 porcine circovirus type 2
  • SIV swine influenza virus
  • PRRSV Porcine Reproductive and Respiratory Syndrome Virus
  • PSV Parvovirus
  • Erysipelas Erysipelas
  • Aujeszky's disease and Actinobacillus pleuropneumoniae Actinobacillus pleuropneumonia , ⁇ ).
  • porcine atrophic rhinitis immunological composition may further comprise one or more selected from the following pharmaceutically acceptable carriers, including: a solvent, an emulsifier, a suspending agent, a decomposing agent, a binder, and an agent.
  • a solvent including: a solvent, an emulsifier, a suspending agent, a decomposing agent, a binder, and an agent.
  • the pharmaceutically acceptable carrier comprises one or more reagents selected from the group consisting of: a solvent ( so ven t), an emulsifier, a suspension ([suspending agent] a decomposer) , (binding agent) [I (excipient), stabilizing agent, chelating agent, diluent, gelling agent, preservative Preservative, lubricant, surfactant, adjuvant, and other carriers similar or suitable for use in the present invention.
  • the pharmaceutically acceptable excipient can be a pharmaceutically acceptable organic or inorganic carrier material suitable for parenteral, enteral or intranasal administration, and the excipient does not deleteriously react with the active composition.
  • Suitable excipients include, but are not limited to, water, Salt solution, vegetable oil, polyethylene glycol, gelatin, amylose, lactose, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglyceride and glycerin, fatty acid ester, hydroxymethyl cellulose , polyvinylpyrrolidone and the like.
  • the pharmaceutically acceptable adjuvants include, but are not limited to, aqueous aluminum hydroxide gel, alum, Freimd's incomplete adjuvant, oily adjuvant, water soluble adjuvant, or water-in-oil-in-water dual phase adjuvant (water- In-oil-in-water, W/0/W);
  • the adjuvant is an aqueous aluminum hydroxide gel.
  • the present invention provides a method for combating atrophic rhinitis in pigs, comprising administering an effective amount of the above-mentioned immunological composition to an animal to enhance the immunity of the animal against atrophic rhinitis of the pig, thereby improving and improving the clinical condition thereof. Symptoms, survival rates, and trends in weight gain.
  • the present invention also provides an anti-Septica bacillus type D toxin (PMT) antibody, which is prepared or derived by the recombinant S. septicum toxin recombinant protein (rPMT) provided by the present invention; However, it is not limited to: monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.
  • the anti-system is a multi-strain antibody obtained by administering the S. septicum toxin recombinant protein (rPMT) provided by the present invention to an animal.
  • the invention also provides a test kit for porcine atrophic rhinitis, which is used for detecting whether a test sample contains Pasteurella multocida D-toxin (PMT) or detecting whether the test sample contains anti-septic An antibody to Pasteurella D-type toxin (PMT).
  • the test kit includes, but is not limited to: (1) an antigen, which is a recombinant protein of septicum bacillus toxin (rPMT) provided by the present invention. In one embodiment, the antigen is placed in an antigenic disk. And / or (2) - an antibody which is a monoclonal antibody or a plurality of antibodies which are produced by the recombinant S. septicum toxin recombinant protein (rPMT) provided by the present invention.
  • test kit includes, but is not limited to, an enzyme-linked immu sorbent assay (ELISA) kit, a microchip test kit (Microchip kit), and an immunofluorescence assay (IMA) assay. Kits, or other test kits made by the S. septicum toxin recombinant protein (rPMT).
  • the test kit comprises at least one antigenic disk containing the recombinant protein of septicum bacillus toxin (rPMT) provided by the present invention, which can be used to test whether the sample contains anti-Septic bacillus toxin (PMT). ) antibodies.
  • the full-length amino acid sequence of P. septicum toxin protein (as shown in SEQ ID NO: 1) is used as a template.
  • the epitope is predicted using an epitope (eg, but not limited to BCPred (http:/'/ailab.cs.iastate.edu/bcpredsA) to predict the epitope of the S. septicum toxin protein (PMT).
  • Epi-predicted epitopes are shown in Table 1 (SEQ ID NOs: 20-42), and these epitopes are divided into three segments, numbered Epi 1, Epi 2 and Epi 3, respectively. As shown in SEQ ID NOs: 2, 3, and 4; Epi 1, Epi 2, and Epi 3 respectively include the respective epitopes shown in Table 1.
  • the recombinant protein of Pasteurella septicum toxin was designed with the amino acid sequence of different combinations of Epi 1, Epi 2 and Epi 3 (rPMT in Epi 1 (SEQ ID NO: 2), Epi 2 (SEQ ID NO: 3), Epi 3
  • the amino acid sequence of the amino acid sequence of (SEQ ID NO: 4) is each added with a linker sequence having the amino acid sequence shown in SEQ ID NO: 5, and the N-terminal to the C-terminal are sequentially linked to Epi 1 and Epi. 2.
  • S. septicum toxin recombinant protein (rPMT) amino acid sequence is shown in SEQ ID NO: 10, and the amino acid sequence is synthesized by a synthesizer or by gene colonization. 2. Septic Design of amino acid sequence of recombinant protein 2 (rPMT2)
  • the prediction of epitopes (epitopes) of S. septicum toxin protein (PMT) is as described above.
  • the S. septicum toxin recombinant protein (rPMT) was designed based on the amino acid sequences of Epi 1, Epi 2 and Epi 3 . Adding a linker sequence to the C-terminus of the amino acid sequences of Epi 1 (SEQ ID NO: 2), Epi 2 (SEQ ID NO: 3), Epi 3 (SEQ ID NO: 4), respectively, the linker having SEQ.
  • B. bronchiseptica (S. 6ra «c/ ⁇ £ ⁇ rica) is administered by the Executive Yuan Agricultural Committee The Health Laboratory (Taiwan) gave way.
  • the B. bronchiseptica wwc/ ⁇ e ⁇ 'ca) was first inoculated on TSB solid medium [containing 5% (v/v) yeast extract, 10% (v/v) serum Soybean protein kainate medium (tryptic soy broth, TSB, BD, USA)], after overnight incubation at 37 ° C, select a single colony inoculated into brain heart extract (BHI) liquid medium (BD, USA), shake culture overnight at 37 ° C; then the inoculum was inoculated into BHI liquid medium, shake culture overnight at 37 ° C, and calculate the colony forming unit (CFU) Value; Finally add formaldehyde (formaldehyde), shake at room temperature for 24 to 36 hours, the bacteria liquid is not activated.
  • TSB solid medium containing 5% (v/v) yeast extract, 10% (v/v) serum Soybean protein kainate medium (tryptic soy broth, TSB, BD, USA)
  • BHI brain
  • the Pasteurella multocida type A bacteria (PmA) and the Pasteurella multocida type D bacteria (PmD) are distributed from the Animal Health Laboratory of the Executive Yuan Agricultural Committee (Taiwan), and the septic Pasteurella type D bacteria (PmD) have the ability to produce toxins.
  • Pasteurella multocida type A bacteria (PmA) and the Pasteurella multocida type D bacteria (PmD) were inoculated on the TSB solid medium [containing 5% (v/v) yeast extract, 10% (v /v) Serum, soy protein-degraded protein casein medium (TSB, BD, USA), after overnight incubation at 37 ° C, a single colony was selected and inoculated into brain heart extract (BHI) liquid medium (BD company, US), shake culture overnight at 37 ° C; then take 0.1% (v / v) bacterial solution and then inoculated in BHI liquid medium, shake culture overnight at 37 ° C, and calculate colony forming unit (CFU) value Finally, formaldehyde is added to inactivate the bacterial solution.
  • BHI brain heart extract
  • CFU colony forming unit
  • Example 3 Preparation of porcine atrophic rhinitis immunocompetent composition containing recombinant S. septicum toxin recombinant protein (rPMT)
  • S. septicum toxin recombinant protein (rPMT) obtained in Example 1 (SEQ ID NO: 10 or 11) (The final concentration is 65 g/ml) and the inactivated B.
  • bronchiseptica obtained in Example 2 (A bronchiseptica final concentration is lxlO 9 CFU/ml), inactivated Pasteurella septicum Type A bacteria (PmA) (; final concentration of lxlO 9 CFU / ml) and non-activated Pasteurella septicum type D bacteria (PmD) (; final concentration of lxlO 9 CFU / ml) with phosphate buffer solution (phosphate The buffered solution (PBS) was uniformly mixed, and an aluminum gel [final concentration of 30% (v/v)] was added as an adjuvant to prepare an immunological composition for porcine atrophic rhinitis.
  • PBS buffered solution
  • an aluminum gel [final concentration of 30% (v/v)] was added as an adjuvant to prepare an immunological composition for porcine atrophic rhinitis.
  • mice Twenty-six healthy 4-week-old BALB/c male mice (National Experimental Animal Center, Taiwan) were used as experimental animals, and all mice were treated with Pasteurella multocida toxin (PMT) and B. bronchiseptica ⁇ . Bronchiseptica), the enzyme-binding immunosorbent assay (ELISA) antibody of S. septicum type A (PmA) and S. septicum type D (PmD) were negative.
  • PMT Pasteurella multocida toxin
  • ELISA enzyme-binding immunosorbent assay
  • mice were randomly divided into 5 groups, the first group (negative control group) had 6 mice, and the second to fifth groups had 5 mice per group. Each mouse was injected with 0.2 ml of the following substances by intraperitoneal injection (ip.):
  • Group 1 PBS buffer solution containing 30% ( ⁇ / ⁇ ) aluminum gel (negative control group);
  • Group 2 S. bronchiseptica ( ⁇ x ⁇ 0 9 CFU/ml) obtained in Example 2 ⁇ P. septicum type A (PmA) (lxl0 9 CFU/ml) And an immunological composition of P. septicum type D bacteria (PmD) (lxlO 9 CFU/ml) (containing 30% (v/v) aluminum gel adjuvant) (Bb + PmA + PmD group);
  • Group 3 The recombinant protein of Pasteurella septicum toxin obtained in Example 1 (sequence as shown in SEQ ID NO: 10, concentration 65 g/ml) (rPMT group);
  • Group 4 the porcine atrophic rhinitis immunocomb composition (Bb + PmA + PmD + rPMT group) containing the recombinant protein of septicum Pasteurella toxin (rPMT, sequence as shown in SEQ ID NO: 10) obtained in Example 3; as well as
  • Group 5 Commercially available atrophic rhinitis vaccine for pigs (Bayovac®, Bayer, Taiwan, Taiwan) (Bayovac group).
  • Each mouse was collected for blood collection 24 hours before the first immunization, and then collected on the 13th day after the first immunization.
  • the second immunization was performed at the same immunization dose on the 14th day after the first immunization, and the blood was collected on the 10th day after the second immunization. And separating serum from the blood sample for Enzyme-linked immunosorbent assay (ELISA).
  • ELISA Enzyme-linked immunosorbent assay
  • Pasteurella multocida toxin was used as an antigen, and the antigen was coated on a 96-well plate (Thermo Corporation, USA) for ELISA, and allowed to stand at 4 ° C for 16 hours. . After removing excess antigen, add washing buffer (wash buffer; 0.9% NaCl; 0.1% Tween20), wash 3 times, and then dry. Then, blocking buffer (wash buffer containing 1% BS A) was added, and after standing at room temperature for 1 hour, it was washed with a washing buffer, and then serum samples collected from the above groups of mice were used as PBS buffer solution.
  • washing buffer 0.9% NaCl; 0.1% Tween20
  • blocking buffer was added, and after standing at room temperature for 1 hour, it was washed with a washing buffer, and then serum samples collected from the above groups of mice were used as PBS buffer solution.
  • mice serum After dilution, diluted mouse serum was added to each well, and after standing at room temperature for 1 hour, serum samples were removed, washed with washing buffer, and then goat anti-small by horseradish peroxidase (HRP) was added.
  • HRP horseradish peroxidase
  • Rat anti-mouse conjugated HRP (Gene Tex, USA)
  • this secondary antibody was diluted 5000 times with blocking buffer and then added to a 96-well plate (100 ⁇ /well) and allowed to stand at room temperature. After 1 hour, the secondary antibody was removed and washed with washing buffer, and 100 ⁇ M of 3, 3 ', 5, 5 '-tetramethylbenzidine dihydrochloride (3, 3 ', 5, 5 '-) was added to each well.
  • the tetramethylbenzidine, TMB, KPL, USA was protected from light for 10 minutes and the absorbance at 650 nm was read with an enzyme-linked immunoassay reader (SpectraMax® M2/M2 ELISA Reader, Molecular Devices, USA).
  • the results of enzyme-linked immunoassay are shown in Figure 1.
  • the mice immunized with the porcine atrophic rhinitis immunological composition containing the recombinant protein of Pasteurella septicum toxin contained serum.
  • the anti-Septic Aspergillus toxin (PMT) antibody has the highest titer, followed by the mouse serum of the immunized commercial atrophic rhinitis vaccine (Group 5, Bayovac group); and the septicemia obtained only by immunization Example 1.
  • the mouse serum of the Pasteurella toxin recombinant protein (Group 3, ie, the rPMT group) also contains an anti-Septic Pasteurin toxin (PMT) antibody, and the antibody price thereof is higher than that obtained by the immunization Example 2 B. bronchiseptica, Pasteurella multocida type A and S. septicum D-type bacteria (p. Mouse sera of group 2, ie, Bb + PmA + PmD group. From this, it can be seen that the recombinant S. septicum toxin (rPMT) provided by the present invention can efficiently induce anti-Septic bacillus toxin (PMT) antibodies in an animal, and is immunogenic.
  • PMT anti-Septic Pasteurin toxin
  • Vero cells were used as test materials to test the antiserum contained in the serum of mice immunized with porcine atrophic rhinitis immunosuppressant containing recombinant protein of S. septicum toxin (Group 4, ie Bb + PmA + PmD + rPMT group)
  • a septic bacillus toxin (PMT) antibody is a neutralizing antibody that neutralizes PMT toxicity.
  • the minimum toxin dose (MTD) of Vera cells was measured.
  • the Vero cells were seeded in a 96-well culture dish, and after the cells were grown into a single layer, the culture solution was removed and added to a PMT containing 2 times dilution of DMEM medium (GIBCO, USA) containing no serum. DMEM medium containing fetal bovine serum (FBS) was used as a negative control group. After culture, the type change of the cells was observed to induce the lowest PMT concentration of cytopathic effect (CPE) in Vero cells to be the minimum toxic dose ( MTD). The test results showed that the minimum toxic dose of Septicemia toxin (PMT) in Vera cells was 60 ng.
  • DMEM medium containing fetal bovine serum
  • the method for neutralizing antibody titer is based on the method proposed by Liao et al. (2006) and Lee et al. (2012) and appropriately modified (Liao, CM, Huang, C, Hsuan, S. L" Chen, ZW, Lee , W. C, Liu, CI, Winton, JR, and Chien, MS (2006). Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs. Vaccine. 24, 27-35; Lee, J Kang, HE, and Woo, HJ (2012). Protective immunity conferred by the C-terminal fragment of recombinant Pasteurella multocida toxin.
  • Vero cells were treated with S. septicum toxin (PMT) containing 4 times the minimum toxic dose (MTD) as a positive control group, and the cell morphology was as shown in Fig. 2B, and the cells showed typical nodule-like appearance.
  • PMT S. septicum toxin
  • MTD minimum toxic dose
  • Vero cells were cultured in DMEM containing fetal bovine serum (FBS) as a negative control group, and the cell morphology was as shown in Fig. 2A; and the atrophy of pigs containing the recombinant protein of Pasteurella septicum toxin was immunized.
  • FBS fetal bovine serum
  • mice of the rhinitis immunocompetitor composition (group 4, ie, Bb + PmA + PmD + rPMT group) were diluted 40-fold in serum and neutralized with 4 times the minimum toxic dose (MTD) of S. septicum toxin (PMT).
  • MTD minimum toxic dose
  • PMT S. septicum toxin
  • porcine atrophic rhinitis immunological composition containing the recombinant protein of Pasteurella septicum toxin (Group 4) , ie Bb + PmA + PmD + rPMT group) serum contains anti-septic Neutralizing antibodies to Pasteurella toxin (PMT).
  • porcine atrophic rhinitis immunocompetent composition containing recombinant S. septicum toxin recombinant protein (rPMT) 1- Taiwan pig atrophic rhinitis bacterin test standard (Pap Pasteur efficacy test)
  • Group 1 PBS buffer solution containing 30% ( ⁇ / ⁇ ) aluminum gel (control group);
  • Group 2 the porcine atrophic rhinitis immunocomb composition (Bb + PmA + PmD + rPMT group) containing the recombinant protein of septicum Pasteurella toxin (rPMT, sequence as shown in SEQ ID NO: 10) obtained in Example 3; as well as
  • Group 3 Commercially available atrophic rhinitis vaccine for pigs (Bayovac®, Bayer, Taiwan, Taiwan) (Bayovac group).
  • the immunoassay group (Group 2 and Group 3) was divided into 3 groups on the 14th day after immunization, and the virulent strains of Pasteurella multocida D (PmD) with the ability to produce toxin were respectively Example 2, from the Animal Health Laboratory of the Executive Yuan Agricultural Committee (Taiwan), lxlO 3 CFU/ml, lxlO 4 CFU/ml, lxlO 5 CFU/ml, three concentrations of live bacteria, 0.1 ml, were injected intraperitoneally.
  • mice 12 mice were also divided into three groups, according to the order of the three strains of the Pasteurella virulent strain lxlO 2 CFU / ml, lxlO 3 CFU / ml, lxlO 4 CFU / ml Intravenous injection of 0.1 ml of bacterial solution.
  • Observed 10 days after each immunization test group and control group respectively Bekaert two's method (Beherens-Karber) calculated LD 5Q, and the immune defense indices in each test group to be higher than 1 ⁇ 10 ⁇ ⁇ 5.
  • Beka II method of defense index calculation is as follows:
  • LD 50 minimum dilution factor for the challenge dose - [(sum of mortality for each group / 100) - 0.5] X 1
  • the results are shown in Table 2.
  • the porcine atrophic rhinitis immunological composition containing the recombinant protein of Pasteurella multocida toxin did induce protective efficacy in mice and was resistant.
  • the attack of the virulent strain of Pasteurella multocida type D (PmD), and its defense index is as high as 10 2 ⁇ ⁇ 6 , far higher than the Taiwan test standard (10 ⁇ ⁇ 5 ) and the commercial atrophic rhinitis vaccine ( Bayovac®, Taiwan Bayer, Taiwan) (Bayovac group) defense index (1 ( ⁇ 74 ).
  • mice National Experimental Animal Center, Taiwan
  • mice with negative antibody to Pasteurella septicum and weighing 15-20 g
  • mice there were 5 mice.
  • Groups 2 and 3 were immunoassay groups with 30 mice per group.
  • Each immunoassay was subdivided into 3 groups of 10 mice per group; each group was injected intraperitoneally with the following substances:
  • Group 2-1 Each mouse was injected with 0.2 ml of the porcine atrophic rhinitis immunological composition stock solution containing the recombinant protein of septicum Pasteurella toxin (rPMT, sequence as shown in SEQ ID NO: 10) obtained in Example 3 ( Bb + PmA + PmD + rPMT group);
  • Group 2-2 Each mouse was injected with 0.2 ml of 5-fold diluted recombinant protein containing septicemia (rPMT) obtained in Example 3, and the sequence is SEQ ID NO: 10 shows the porcine atrophic rhinitis immune composition (1/5 Bb + PmA + PmD + rPMT group);
  • Groups 2-3 Each mouse was injected with 0.2 ml of a 25-fold diluted 25-fold porcine atrophic rhinitis immunization with a recombinant protein containing septicemia (initial SEQ ID NO: 10).
  • Composition 1/25 Bb + PmA + PmD + rPMT group);
  • Group 3-1 0.2 ml of commercially available porcine atrophic rhinitis vaccine stock (Bayovac®, Taiwan Bayer, Taiwan) (Bayovac group);
  • Group 3-2 Each mouse was injected with 0.2 ml of a 5-fold diluted commercial atrophic rhinitis vaccine (Bayovac®, Taiwan Bayer, Taiwan) (1/5 Bayovac group);
  • Groups 3-3 Each mouse was injected with 0.2 ml of a 25-fold diluted commercial atrophic rhinitis vaccine (Bayovac®, Taiwan Bayer, Taiwan) (1/25 Bayovac group).
  • Boyovac® commercial atrophic rhinitis vaccine
  • the immunization test group (groups 2 and 3) was immunized twice on the 14th day after the first immunization, and the dose was the same as the first immunization; the control group (group 1) was injected with 0.2 ml of PBS buffer solution again; after the second immunization 10 days of challenge test, each mouse was injected intraperitoneally with 0.2 ml of a virulent strain of Pasteurella pneumoniae type D (PmD) with the ability to produce toxins [same example 2, by the Executive Yuan Agricultural Committee Animal Health The laboratory (Taiwan) was given a] 100 LD 5Q live bacterial solution, and the survival rate was observed for 10 days.
  • PmD Pasteurella pneumoniae type D
  • the survival rate of the stock immunization group (Group 2-1, Section 3-1) must be higher than 80%, and the survival rate of the 5-fold diluted immunization group (Group 2-2, Group 3-2) must be higher than 50%.
  • the survival rate of the 25-fold diluted immunized group (Groups 2-3, 3-3) must be higher than 20%, and the control group must all die.
  • the porcine atrophic rhinitis immunological composition (Group 2-1, Group 2-2, Group 2-3) containing the recombinant protein of Pasteurella septicum toxin was able to induce sufficient production of the mouse.
  • the protective efficacy, the survival rate of the mouse with the immunogenic vaccine is 90%, which is in line with the survival rate of 80% or more.
  • the survival rate of the mouse diluted with the 5-fold vaccine is 50%, which is in line with the survival rate of 50% or more.
  • the survival rate of mice with a 25-fold vaccine was 30%, which was consistent with a survival rate of 20% or more.
  • Table 3 Immunogenicity and protective efficacy analysis of porcine atrophic rhinitis immunocomplex containing S. septicum toxin recombinant protein (rPMT) 2 Results
  • Group 3-1 1 0 10 0 8 80% ⁇ 80% (Bayovac group) 2 2 8 2 (Qualified) Group 3-2 1 2 8 2 6 60% ⁇ 50%
  • the virulent strain of Pasteurella multocida D (PmD) with the ability to produce toxins is inoculated into the brain heart extract (the same as in the second embodiment, assigned by the Animal Health Laboratory of the Executive Yuan Agricultural Committee (Taiwan)].
  • BHI in liquid medium (BD, USA), after overnight incubation at 37 ° C, 100 ⁇ M of bacterial solution was applied to 7% blood medium on average, cultured overnight at 37 ° C, and bacteria were scraped with PBS.
  • mice The median lethal dose (LD 5Q ) test of crude extract of Pasteurella septicum in mice
  • mice National Experimental Animal Center, Taiwan
  • the crude extract toxins of Pasteurella septicum prepared above was thawed. After, according to the following toxin dose for each group Only 0.5 ml of crude toxin was injected into mice:
  • mice were observed for 10 days and the number of deaths was recorded to calculate the median lethal dose (LD 5Q ) of the crude extract of Pasteurella septicum to mice.
  • the LD 5Q was based on Calcu-Syn software (Biosoft, British) calculated.
  • mice challenged with the above-mentioned crude extract of Pasteurella septicum was 2 ⁇ 10 8 CFU/ml.
  • mice National Experimental Animal Center, Taiwan
  • Group 1 was the control group
  • Groups 2 to 5 were the immune group.
  • Test group each group of each group was injected intraperitoneally (ip.) with 0.2 ml of the following substances:
  • Group 1 PBS buffer solution containing 30% ( ⁇ / ⁇ ) aluminum gel (negative control group);
  • Group 2 S. bronchiseptica ( ⁇ x ⁇ 0 9 CFU/ml) obtained in Example 2 ⁇ P. septicum type A (PmA) (lxl0 9 CFU/ml) And an immunological composition of P. septicum type D bacteria (PmD) (lxlO 9 CFU/ml) (containing 30% (v/v) aluminum gel adjuvant) (Bb + PmA + PmD group);
  • Group 3 the porcine atrophic rhinitis immunocomb composition (Bb + PmA + PmD + rPMTl group) containing the recombinant protein of septicum Pasteurella toxin (rPMT1, sequence as shown in SEQ ID NO: 10) obtained in Example 3;
  • Group 4 the porcine atrophic rhinitis immunocomb composition (Bb + PmA + PmD + rPMT2 group) containing the recombinant protein of septicum Pasteurella toxin (rPMT2, sequence as shown in SEQ ID NO: 11) obtained in Example 3; as well as Group 5: Commercially available porcine atrophic rhinitis vaccine (Bayovac®, Taiwan Bayer, Taiwan) (Bayovac group).
  • Each mouse was further immunized with the same immunization dose on the 14th day after the first immunization, and the crude extract toxin prepared by the above-mentioned S. septicum was taken for the challenge test on the 10th day after the second immunization.
  • Each mouse was injected with 0.5 ml of LD 85 dose (ie 6 ⁇ 10 8 CFU/ml) of the crude extract of Pasteurella septicum. After the crude toxin was injected, the mice were observed for 10 days and the number of deaths was recorded to calculate the survival of each group. rate.
  • the porcine atrophic rhinitis immunological composition i.e., Groups 3 and 4 containing the recombinant protein of septic Pasteurella toxin (rPMTl or rPMT2) did induce the protective effect of the mouse and was resistant.
  • the challenge of the crude extract of Pasteurella multocida, and its survival rate is greater than the negative control group and only B. septicum (S. /wwc/ ⁇ eprica), Pasteurella septicum type A (PmA) and immunocompetent composition of Pasteurella multocida type D bacteria (PmD).
  • Table 5 Results of mouse immunization and challenge test of Pasteurella septicum crude extract toxin
  • the above pigs were randomly divided into 3 groups, 10 pigs in each group, and each pig was intramuscularly injected (2 ml/mouse) at the beginning of the test (first immunization on day 0) and on day 21 (secondary immunization).
  • Group 1 PBS buffer solution containing 30% ( ⁇ / ⁇ ) aluminum gel (negative control group);
  • Group 2 S. bronchiseptica ( ⁇ x ⁇ 0 9 CFU/ml) obtained in Example 2 ⁇ P. septicum type A (PmA) (lxl0 9 CFU/ml) And an immunological composition of P. septicum type D bacteria (PmD) (lxlO 9 CFU/ml) (containing 30% (v/v) aluminum gel adjuvant) (Bb + PmA + PmD group);
  • Group 3 The porcine atrophic rhinitis immunocomb composition (B.b + PmA + PmD + rPMT group) containing the recombinant protein of Pasteurella multocida toxin (rPMT, sequence as shown in SEQ ID NO: 11) obtained in Example 3.
  • Each pig was collected before the first immunization, before the second immunization, and on the 14th day after the second immunization, and the body weight was recorded, and the serum in the blood sample was separated for enzyme-linked immunoassay (ELISA) of the toxin antibody. .
  • ELISA enzyme-linked immunoassay
  • Septicemia Pasteurin toxin (PMT) (Merck, USA) was used as an antigen, and the antigen was coated on a 96-well plate (Thermo, USA) for ELISA, and allowed to stand at 4 ° C for 16 hours. After removing excess antigen, add a cleaning buffer (0.9% NaCl; 0.1% Tween20), rinse and dry. Then, a blocking buffer (washing buffer containing 1% BSA) was added, and after standing at room temperature for 1 hour, it was washed with a washing buffer, and then the serum samples collected from the respective groups of pigs were diluted with a PBS buffer solution, and each was diluted.
  • PMT Septicemia Pasteurin toxin
  • HRP horseradish peroxidase
  • Gene Tex horseradish peroxidase
  • the secondary antibody is diluted 1000 times in blocking buffer and then added to a 96-well plate (100 ⁇ /well). After standing at room temperature for 1 hour, the secondary antibody is removed and washed with a washing buffer. After 6 times, 100 ⁇ 3, 3 ', 5, 5 '-tetramethylbenzidine dihydrochloride (KPL, USA) solution was added to each well for 10 minutes in the dark, and the enzyme-linked immunoassay instrument was used. Labsystem multiskan MCC/340 Microplate Reader, Labsystem, USA) Read absorbance at 650 nm.
  • the result of the enzyme-linked immunoassay is shown in Figure 3.
  • the pigs of the porcine atrophic rhinitis immunological composition containing the recombinant protein of Pasteurella septicum toxin were immunized (Group 3, ie Bb + PmA) + PmD + rPMT group)
  • the serum-resistant Pasteurella toxin (PMT) antibody contained in the serum was the most expensive, and compared with the negative control group pigs and the B.
  • the anti-Septica toxin (PMT) antibody titer contained in the serum of the septicum type A bacillus and the septicum type B bacterium (group 2, ie, Bb + PmA + PmD group) , with significant differences (**, 0.01). In addition, there was no significant difference in body weight change between the three groups of pigs (results not shown).
  • the recombinant S. septicum toxin recombinant protein (rPMT) provided by the present invention can effectively induce anti-Septic bacillus toxin (PMT) antibody in an animal, particularly a target animal pig, and It has no adverse effects on the growth of pigs and is safe.
  • Example 9 Preparation of anti-Septica Pasteurella toxin recombinant protein (rPMT) antibody
  • the recombinant S. septicum toxin recombinant protein (rPMT) obtained in Example 1 is mixed with a suitable adjuvant (eg, aluminum gel) and then administered to an animal (eg, mouse, rat, pig, goat, rabbit)
  • a suitable adjuvant eg, aluminum gel
  • an animal eg, mouse, rat, pig, goat, rabbit
  • a second immunization may be administered as needed.
  • the serum of immunized animals such as: mice, rats, pigs, goats, rabbits
  • rPMT recombinant protein of anti-Septica toxin
  • the multi-strain antibody of the anti-Septic bacillus toxin recombinant protein may be combined with a developer or a fluorescent dye as needed.
  • the animal may increase the number of immunizations as needed to increase the antibody titer.
  • the animals to be administered include, but are not limited to, mice, rats, rabbits, poultry (eg eggs), pigs, goats, cattle, aquatic animals.
  • the recombinant S. septicum toxin recombinant protein (rPMT) obtained in Example 1 is mixed with a suitable adjuvant (eg, aluminum gel) and then administered to an animal (eg, mouse, rat, pig, goat, rabbit)
  • a suitable adjuvant eg, aluminum gel
  • an animal eg, mouse, rat, pig, goat, rabbit
  • a second immunization may be administered as needed.
  • the serum of the immunized animal eg, mouse
  • the serum of the immunized animal is collected to assess mice suitable for collecting spleen cells.
  • Spleen cells and myeloma cells are collected from the suitable mouse for cell fusion with PEG (Polyethylene Glycol, e.g., PEG 1500).
  • PEG Polyethylene Glycol, e.g., PEG 1500.
  • rPMT S. septicum toxin recombinant protein
  • the antibody obtained by the above preparation can be used for an immunoassay reagent, a therapeutic agent, or added to a food or a feed to make the consumer immune.

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Abstract

本发明提供了一种败血性巴氏杆菌毒素重组蛋白，包含至少一个败血性巴氏杆菌毒素蛋白的抗原决定簇；当该抗原决定簇为复数个时，各抗原决定簇之间可由连接子连接。该重组蛋白进一步可包含至少一个单元的补体裂解片段C3d的氨基酸序列，且该抗原决定簇与该补体裂解片段C3d的氨基酸序列可以连接子连接。本发明还提供了一种编码上述败血性巴氏杆菌毒素重组蛋白的核苷酸序列，以及一种含有上述败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物。

Description

败血性巴氏杆菌毒素重组蛋白及其应用 技术领域
本发明关于败血性巴氏杆菌毒素重组蛋白在动物保健领域上的应用， 特别是关于含有败 血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物在动物保健领域上的应用。
说
背景技术
猪萎缩性鼻炎 (Atrophic Rhinitis, AR)为猪只呼吸系统的三大主要传染病之一。猪萎缩性鼻 炎会造成受感染的猪只的颜面骨畸形及慢性化脓性鼻炎， 而引起鼻甲介骨的萎缩。 在严重感 染时， 鼻腔、 颔骨、 上颔骨亦会发生病变。 下鼻甲骨的下游窝状部位最常受感染。 上鼻甲、 下鼻甲骨、 鼻中膈或筛骨等部位均会被感染。 书
猪萎缩性鼻炎 (AR)是由支气管败血性博德氏杆菌 (Sortfete//a bronchiseptica)以及败血性巴 氏杆菌 CPaWeMre//a multocida) 型菌 (PmA)及 D型菌 (PmD)所引起， 尤其是败血性巴氏杆菌 D 型菌 (PmD)所产生的毒素 0PaW£¾/re//a multocida toxin, PMT)。取 PMT毒素分别以肌肉、腹腔及 鼻腔接种等方式接种于四周龄小猪， 均可以引致鼻甲介骨萎缩的病变产生， 此外也会波及全 身性骨骼发育而使生长迟缓， 甚至高剂量接种时， 会导致肝脏受损而造成猪只黄疸及死亡。
猪萎缩性鼻炎 (AR)遍及世界各养猪地区， 患猪生长缓慢， 饲料利用效率降低。 单独感染 时虽然死亡率不高， 但污染率却很大， 而且容易诱发其他合并症或病原的感染， 造成高死亡 率， 增加生产成本。 在猪萎缩性鼻炎 (AR)严重感染的养猪场， 其经济损失约为 15-38%， 且在 重度感染的养猪场有较明显的生长障碍情形， 平均日增重约较正常猪只少 5-8%。 因此开发有 效的猪萎缩性鼻炎疫苗， 以预防猪只罹患猪萎缩性鼻炎 (AR)可以说是刻不容缓且极为重要的 事。 发明内容
本发明于第一部分提供一种败血性巴氏杆菌毒素重组蛋白 (recombinant PMT, rPMT), 该 重组蛋白包含至少一个败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位 (epitopes) , 以诱导动物 产生抗败血性巴氏杆菌毒素蛋白 (PMT)的抗体。 该败血性巴氏杆菌毒素重组蛋白 (rPMT)可进 一步含有补体裂解片段 C3d的全长或部分氨基酸序列， 以增加特异性免疫反应。 且各个该败 血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位之间、 各个该补体裂解片段 C3d的全长或部分氨 基酸序列之间、 以及败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位与补体裂解片段 C3d的全 长或部分氨基酸序列之间可进一步由连接子连接。
本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)， 可以由下式所表示： (A)m-(C3d片段) n;
其中每一个 A代表一个独立的败血性巴氏杆菌毒素蛋白的抗原决定位，每一个 A系各自 独立选自于由 SEQ ID N0s: 2、 3、 4、 20、 21、 22、 23、 24、 25、 26、 27、 28、 29、 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41及 42所组成的群组；
其中每一个 C3d片段代表一个独立的补体裂解片段 C3d的氨基酸序列，每一个 C3d片段 系各自独立选自于 SEQ ID N0s: 6、 7、 8及 9所组成的群组；
其中 m是代表从 1至约 30的整数；
其中 n是代表从 0至约 10的整数。
本发明于第二部分提供一种编码一败血性巴氏杆菌毒素重组蛋白 (rPMT)的核苷酸序列。 该败血性巴氏杆菌毒素重组蛋白 (rPMT)包含至少一个败血性巴氏杆菌毒素蛋白 (PMT)的抗原 决定位， 以及重复 0至 10个单元的补体裂解片段 C3d的部分氨基酸序列。
本发明于第三部分提供一种猪萎缩性鼻炎免疫组合物。 该猪萎缩性鼻炎免疫组合物含有 一败血性巴氏杆菌毒素重组蛋白 (rPMT)以及一药学上可接受的载剂。 该败血性巴氏杆菌毒素 重组蛋白 (rPMT)包含至少一个败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位 (epitopes), 以及 重复 0至 10个单元的补体裂解片段 C3d的部分氨基酸序列。 该猪萎缩性鼻炎免疫组合物可 进一步含有支气管败血性博德氏杆菌 Οδ. wwc/^e ^'ca)、败血性巴氏杆菌 A型菌 (PmA)以及败 血性巴氏杆菌 D型菌 (PmD)。
本发明于第四部分中提供一种动物对抗猪萎缩性鼻炎的方法， 包含使用有效量的上述免 疫组合物以施予动物， 以增强该动物对抗猪萎缩性鼻炎的免疫力， 进而提升、 改善其临床症 状、 存活率、 及增重趋势。
本发明于第五部分中提供一种抗败血性巴氏杆菌 D型菌毒素 (PMT)的抗体， 系藉由本发 明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)所制备或衍生而得； 该抗体包括但不限于： 单株抗体、 多株抗体， 以及经基因重组的抗体。
本发明于第六部分中提供一种猪萎缩性鼻炎的检测套组， 该检测套组系用以侦测检验样 本是否含有败血性巴氏杆菌 D型菌毒素 (PMT)或侦测检验样本内是否含有抗败血性巴氏杆菌 D型菌毒素 (PMT)的抗体。
本发明所提供的猪萎缩性鼻炎免疫组合物， 与其他习用技术相互比较时， 更具有下列的 优点- 本发明所提供的猪萎缩性鼻炎免疫组合物含有一败血性巴氏杆菌毒素重组蛋白 (rPMT)。 该败血性巴氏杆菌毒素重组蛋白 (rPMT)包含至少一个败血性巴氏杆菌毒素蛋白 (PMT)的抗原 决定位 (epitopes)以及补体裂解片段 C3d的部分氨基酸序列。 该败血性巴氏杆菌毒素重组蛋白 (rPMT)远比败血性巴氏杆菌毒素蛋白 (PMT)的全长氨基酸序列短， 在生物表现系统中较容易 表达， 且重组蛋白的产率较高， 可降低制造疫苗的成本。
此外， 本发明所提供的猪萎缩性鼻炎免疫组合物所含的败血性巴氏杆菌毒素重组蛋白 (rPMT)具有补体裂解片段 C3d的部分氨基酸序列， 因此可以增加特异性免疫反应， 经试验结 果显示， 本发明所提供的猪萎缩性鼻炎免疫组合物可提高动物对抗猪萎缩性鼻炎的免疫性及 效率。
本发明系以下面的实施例予以示范阐明， 但本发明不受下述实施例所限制。 附图说明
图 1为以酵素连结免疫分析 (ELISA)测定抗败血性巴氏杆菌毒素 (PMT)抗体力价的结果； 第 1组为负对照组； 第 2组为含有实施例二所得的支气管败血性博德氏杆菌、 败血性巴氏杆 菌 A型菌及败血性巴氏杆菌 D型的免疫组合物 (B.b + PmA + PmD组)； 第 3组为实施例一所 得的败血性巴氏杆菌毒素重组蛋白 (rPMT组)； 第 4组为含有败血性巴氏杆菌毒素重组蛋白的 猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT组)； 第 5组为市售猪萎缩性鼻炎疫苗 (Bayovac组)。 符号 **代表两组之间具有显著差异 (/ 0.01)
图 2为中和抗体试验分析结果； 图 2Α为以含胎牛血清 (FBS)的 DMEM培养液培养 Vero 细胞的细胞形态 (负对照组)； 图 2B 为以含 4倍最小毒性剂量 (MTD)的败血性巴氏杆菌毒素 (PMT)处理 Vero细胞的细胞形态 (；正对照组)， 可见到细胞呈现典型的结节样； 图 2C为以免疫 含有败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT组) 的小鼠血清稀释 40倍后与 4倍最小毒性剂量 (MTD)的败血性巴氏杆菌毒素 (PMT)中和后， 加 入 Vero细胞共培养的细胞形态。
图 3为以酵素连结免疫分析 (ELISA)测定抗败血性巴氏杆菌毒素 (PMT)抗体力价的结果； 第 1组为负对照组； 第 2组为含有实施例二所得的支气管败血性博德氏杆菌、 败血性巴氏杆 菌 A型菌及败血性巴氏杆菌 D型的免疫组合物 (B.b + PmA + PmD组)； 第 3组为含有败血性 巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT组)。 符号 **代 表两组之间具有显著差异 (p<0.01)。 具体实施方式
本发明提供一种败血性巴氏杆菌毒素重组蛋白 (rPMT)， 包含至少一个败血性巴氏杆菌毒 素蛋白 (PMT)的抗原决定位， 以诱导动物产生抗败血性巴氏杆菌毒素蛋白 (PMT)的抗体。
于一实施例中， 该败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位系分别具有如 SEQ ID NOs: 20、 21、 22、 23、 24、 25、 26、 27、 28、 29、 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41及 42所示的氨基酸序列， 该败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位可为 1 至约 30个， 且各抗原决定位氨基酸序列自蛋白质 N端到 C端的排列顺序并不限于依照序列 辨识号 (SEQ ID NO)的顺序排列。
于一较佳实施例中， 分别以三段较长的抗原决定位氨基酸序列涵盖部分上述败血性巴氏 杆菌毒素蛋白 (PMT)的抗原决定位， 该三段较长的抗原决定位氨基酸序列系分别如 SEQ ID NOs: 2、 3、 4所示。于一实施例中，本发明的败血性巴氏杆菌毒素重组蛋白 (rPMT)含有如 SEQ ID NO: 2所示的氨基酸序列所涵盖的抗原决定位。 于另一实施例中， 该败血性巴氏杆菌毒素 蛋白 (PMT)含有如 SEQ ID NO: 3所示的氨基酸序列所涵盖的抗原决定位。 于另一实施例中， 该败血性巴氏杆菌毒素蛋白 (PMT)含有如 SEQ ID NO: 4所示的氨基酸序列所涵盖的抗原决定 位。 于再一实施例中， 该败血性巴氏杆菌毒素蛋白 (PMT)含有如 SEQ ID NOs: 2及 /或 3及 /或 4 所示的氨基酸序列所涵盖的抗原决定位。 于一较佳实施例中， 本发明的败血性巴氏杆菌毒 素重组蛋白 (rPMT)含有二个以上该较长的抗原决定位氨基酸序列。 此外， 各抗原决定位氨基 酸序列自蛋白质 N端到 C端的排列顺序并不限于依照序列辨识号 (SEQ ID NO)的顺序排列。
于一较佳实施例中， 当该败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位及 /或该较长的抗 原决定位氨基酸序列为复数个（ 2)时， 各败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位及 / 或该较长的抗原决定位氨基酸序列之间可进一步由连接子 (linker)连接， 该连接子至少含有一 个以上的甘氨酸 (Glycine, Gly)， 该连接子包含但不限于： Gly-Gly、 Gly-Ser、 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、 19。 于一实施例中， 该连接子具有如 SEQ ID NO: 5所示的氨 基酸序列。 然而， 该复数个败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位及 /或该复数个较长 的抗原决定位氨基酸序列之间， 不必然要由连接子所连接。
于一实施例中， 本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)进一步含有补体裂 解片段 C3d的全长或部分氨基酸序列， 以增加特异性免疫反应。 于一实施例中， 该补体裂解 片段 C3d的全长氨基酸序列为小鼠补体裂解片段 C3d的全长序列 (mC3d)，具有如 SEQ ID NO: 8所示的氨基酸序列。 于一较佳实施例中， 该补体裂解片段 C3d的部分氨基酸序列为小鼠补 体裂解片段 C3d第 211个氨基酸至第 238个氨基酸片段序列 (mC3d-p28)，具有如 SEQ ID NO: 6所示的氨基酸序列。 于另一实施例中， 该补体裂解片段 C3d的全长氨基酸序列为猪补体裂 解片段 C3d的全长序列 (pC3d)， 具有如 SEQ ID NO: 9所示的氨基酸序列。于另一较佳实施例 中， 该补体裂解片段 C3d的部分氨基酸序列为猪补体裂解片段 C3d第 201个氨基酸至第 231 个氨基酸片段序列 (pC3d-p31)， 具有如 SEQ ID NO: 7所示的氨基酸序列。 本发明所提供的败 血性巴氏杆菌毒素重组蛋白 (rPMT)具有 1至 10个单元重复的上述补体裂解片段 C3d的全长 或部分氨基酸序列， 该补体裂解片段 C3d的全长或部分氨基酸序列较佳为 1至重复 10个单 元， 更佳为重复 4至 8个单元。
当该补体裂解片段 C3d的全长或部分氨基酸序列为数个时， 各补体裂解片段 C3d的全长 或部分氨基酸序列之间可由连接子连接， 该连接子至少含有一个以上的甘氨酸 (Gly)， 该连接 子包含但不限于： Gly-Gly、 Gly-Ser、 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、 19。 于一实施例中， 该连接子的氨基酸序列为 Gly-Ser。然而， 该复数个补体裂解片段 C3d的全长 或部分氨基酸序列之间， 不必然要由连接子所连接。
本发明的败血性巴氏杆菌毒素重组蛋白 (rPMT)， 可以由下式所表示：
(A)m-(C3d片段) n; 式 (I) 其中每一个 A代表一个独立的败血性巴氏杆菌毒素蛋白的抗原决定位，每一个 A系各自 独立选自于由 SEQ ID N0s: 2、 3、 4、 20、 21、 22、 23、 24、 25、 26、 27、 28、 29、 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41及 42所组成的群组；
其中每一个 C3d片段代表一个独立的补体裂解片段 C3d的氨基酸序列，每一个 C3d片段 系各自独立选自于 SEQ ID N0s: 6、 7、 8及 9所组成的群组；
其中 m是代表从 1至约 30的整数；
其中 n是代表从 0至约 10的整数。
于一实施例中， 每一个 A之间进一步以一个连接子连接， 该每一个连接子系各自独立选 自于 Gly-Gly、 Gly-Ser、 SEQ ID N0s: 5、 12、 13、 14、 15、 16、 17、 18、 19。
于一实施例中， 每一个 C3d片段之间以一个连接子连接， 该每一个连接子系各自独立选 自于 Gly-Gly、 Gly-Ser、 SEQ ID N0s: 5、 12、 13、 14、 15、 16、 17、 18、 19。
于部分实施态样中， 本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)与上述式 (I)所 表示的氨基酸序列具有至少大约 80%序列同源性， 较佳者， 具有大约 85%序列同源性， 更佳 者， 具有大约 90%序列同源性， 甚至是大约 91%、 大约 92%、 大约 93%、 大约 94%、 大约 95%、 大约 96%、 大约 97%、 大约 98%、 大约 99%序列同源性。
于一较佳实施例中， 本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)具有三个分别 如 SEQ ID NO: 2、 3、 4所示的氨基酸序列所涵盖的抗原决定位， 以及重复 6个单元的小鼠补 体裂解片段 C3d第 204个氨基酸至第 231 个氨基酸片段序列 (mC3d-p28)， 且各抗原决定位 (epitopes)的 C端以连接子 (序列如 SEQ ID NO: 5所示)连接， 该败血性巴氏杆菌毒素重组蛋白 (rPMT)具有如 SEQ ID NO: 10所示的氨基酸序列。
于另一较佳实施例中， 该败血性巴氏杆菌毒素重组蛋白 (rPMT)具有三个分别如 SEQ ID NO: 2、 3、 4所示的氨基酸序列所涵盖的抗原决定位， 以及重复 6个单元的猪补体裂解片段 C3d第 201个氨基酸至第 231个氨基酸片段序列 (pC3d-p31)， 且各抗原决定位 (epitopes)的 C 端以连接子 (序列如 SEQ ID NO: 5所示)连接， 该败血性巴氏杆菌毒素重组蛋白 (rPMT)具有如 SEQ ID NO: 11所示的氨基酸序列。
本发明并提供一种编码本发明的败血性巴氏杆菌毒素重组蛋白 (rPMT)的核苷酸序列。 该 败血性巴氏杆菌毒素重组蛋白 (rPMT)包含至少一个败血性巴氏杆菌毒素蛋白 (PMT)的抗原决 定位， 以及重复 0至 10个单元的补体裂解片段 C3d的部分氨基酸序列。 该编码本发明的败 血性巴氏杆菌毒素重组蛋白 (rPMT)的核苷酸序列， 系由本发明的败血性巴氏杆菌毒素重组蛋 白 (rPMT)的氨基酸序列衍生而来。 将本发明的败血性巴氏杆菌毒素重组蛋白 (rPMT)氨基酸序 列上的各个氨基酸置换为遗传密码表 (genetic code table)所列的编码该氨基酸的核苷酸序列 (包含各种简并密码子 (degenerate codons, 或称同义密码子， synonymous codons))， 即可得到 本发明所提供的该核苷酸序列。 例如， 本发明的败血性巴氏杆菌毒素重组蛋白 (rPMT)氨基酸 序列上的丝胺酸 (serine)可由 TCT、 TCC、 TCA、 TCG、 AGT、 AGC等核苷酸序列所编码。 本 发明的败血性巴氏杆菌毒素重组蛋白 (rPMT)氨基酸序列上的各个氨基酸， 可由以下各核苷酸 序列所编码：
本发明的败血性巴氏杆菌毒素重组蛋白
可编码该氨基酸的核苷酸序列
(rPMT)氨基酸序列上的氨基酸
丙氨酸 (Alanine) GCA、 GCC、 GCG、 GCT
胱胺酸 (Cysteine) TGC、 TGT
天门冬胺酸 (Aspartic acid) GAC、 GAT
麸胺酸 (Glutamic acid) GAA、 GAG
苯丙胺酸 (Phenylalanine) TTC、 TTT
甘胺酸 (Glycine) GGA、 GGC、 GGG、 GGT
组胺酸 (Histidine) CAC、 CAT
异白胺酸 (Isoleucine) ATA、 ATC、 ATT
离胺酸 (Lysine) AAA、 AAG
白胺酸 (Leucine) CTA、 CTC、 CTG、 CTT、 TTA、 TTG 甲硫胺酸 (Methionine) ATG
天冬酰胺 (Asparagine AAC、 AAT
脯胺酸 (Praline) CCA、 CCC、 CCG、 CCT
谷胺酰胺 (Glutamine) CAA、 CAG
精胺酸 (Arginine) AGA、 AGG、 CGA、 CGC、 CGG、 CGT 丝胺酸 (Serine) AGC、 AGT、 TCA、 TCC、 TCG、 TCT 羟丁胺酸 (Threonine) ACA、 ACC、 ACG、 ACT
缬草胺酸 （Valine) GTA、 GTC、 GTG、 GTT
色胺酸 (Tryptophan) TGG
酪胺酸 (Tyrosine) TAC、 TAT
此外， 本发明亦提供一种猪萎缩性鼻炎免疫组合物。 该猪萎缩性鼻炎免疫组合物含有一 败血性巴氏杆菌毒素重组蛋白 (rPMT)。 该败血性巴氏杆菌毒素重组蛋白 (rPMT)包含至少一个 败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位 (epitopes), 以及重复 0至 10个单元的补体裂解 片段 C3d的部分氨基酸序列。 于一实施例中， 该败血性巴氏杆菌毒素重组蛋白 (rPMT)具有如 SEQ ID NO: 10所示的氨基酸序列。于另一实施例中，该败血性巴氏杆菌毒素重组蛋白 (rPMT) 具有如 SEQ ID NO: 11所示的氨基酸序列。 本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)包含但不限于由基因选殖方式或以 胜肽合成仪 (peptide synthesizer)合成而得； 以基因选殖方式获得该重组蛋白的方式可为， 但不 限于： 将编码败血性巴氏杆菌毒素重组蛋白 (rPMT)的 DNA序列选殖到表现载体中， 各形成 含有编码败血性巴氏杆菌毒素重组蛋白 (rPMT)的核苷酸序列的质体， 再将该质体转殖到生物 表现宿主中， 经蛋白质表现后而得到的抗原蛋白。
该表现载体系统包含但不限于 pET载体系统以及 pGEX载体系统等；该生物表现系统 (宿 主)包含但不限于： 原核表现系统 (如： 大肠杆菌 (E. coli)), 真核表现系统 (如： 动物细胞 (昆虫 细胞或哺乳类动物细胞)、 植物细胞)。
于一实施例中， 本发明所提供的猪萎缩性鼻炎免疫组合物进一步含有支气管败血性博德 氏杆菌 Οδ. bronchiseptica), 败血性巴氏杆菌 A型菌 (PmA)以及败血性巴氏杆菌 D型菌 (PmD)。 于一较佳实施例中， 该支气管败血性博德氏杆菌 Οδ. bronchiseptica), 败血性巴氏杆菌 A型菌 (PmA)以及败血性巴氏杆菌 D 型菌 (PmD)由行政院农业委员会家畜卫生试验所 (台湾)分让而 来。然而， 该支气管败血性博德氏杆菌 (S. 6rw7c/^e ^'ca)、败血性巴氏杆菌 A型菌 (PmA)以及 败血性巴氏杆菌 D型菌 (PmD)的来源不限于上述来源， 其他来源的菌株亦可搭配本发明的败 血性巴氏杆菌毒素重组蛋白 (rPMT)， 例如， 该支气管败血性博德氏杆菌 (B. bronchiseptica)亦 可为如美国标准生物品收藏中心 (American Type Culture Collection, ATCC)编号 ATCC31437 , 该败血性巴氏杆菌 A型菌 (PmA)亦可为如英国国家标准生物品收藏中心 (National Collection of Type Cultures, NCTC)编号 NCTC 12177， 以及该败血性巴氏杆菌 D型菌 (PmD)亦可为如英国 国家标准生物品收藏中心 (NCTC)编号 NCTC 12178等菌株， 或源自野外分离所得的菌株。
本发明所提供的猪萎缩性鼻炎免疫组合物可进一步包含其他病原抗原， 该病原抗原系选 自由下列群组所组成者： 猪环状病毒第二型 (PCV2)抗原、 猪流感病毒 (SIV)抗原、 猪繁殖与呼 吸症候群病毒PRRSV)抗原、 猪霉桨菌 (Mycoplasma)、 猪小病毒 （Parvovirus, PPV)、 猪丹毒 ( Erysipelas )、 伪狂犬病（Aujeszky's disease) , 以及猪胸膜肺炎放线杆菌（actinobacillus pleuropneumonia , ΑΡΡ)。
另， 本发明所提供的猪萎缩性鼻炎免疫组合物可进一步包含一或多种选自于下列药学上 可接受的载剂， 包括： 溶剂、 乳化剂、 悬浮剂、 分解剂、 黏结剂、 赋形剂、 安定剂、 螯合剂、 稀释剂、 胶凝剂、 防腐剂、 润滑剂、 界面活性剂、 佐剂、 生物型载体等。
该药学上可接受的载剂包含一或多种选自于下列的试剂： 溶剂 (soivent)、 乳化剂 (emulsifier)、 悬浮齐 [((suspending agent) 分角军齐 lJ(decomposer)、 黍占结齐 [((binding agent) 赋形齐 [I (excipient)、安定剂 (stabilizing agent)、螯合剂 (chelating agent)、稀释剂 (diluent)、胶凝剂 (gelling agent)、 防腐剂 (preservative)、 润滑齐 lj (lubricant)、 界面活性齐 lj(surfactant)、 佐齐 Ij(adjuvant), 及 其他类似或适用本发明的载剂。
该药学上可接受的赋形剂可为适合于肠外、 肠内或滴鼻施用的药学上可接受的有机或无 机载体物质， 且该赋形剂不会与活性组合物产生有害的反应。适合的赋形剂包含但不限于水、 盐类溶液、 蔬菜油、 聚乙二醇、 明胶、 直链淀粉、 乳糖、 硬脂酸镁、 滑石、 硅酸、 黏性石蜡、 脂肪酸单甘酯和甘油、 脂肪酸酯、 羟甲基纤维素、 聚乙烯吡咯垸酮等。
该药学上可接受的佐剂包含但不限于水性氢氧化铝胶、 明矾、 Freimd氏不完全佐剂、 油 质佐剂、 水溶性佐剂、 或水包油包水双相佐剂 (water-in-oil-in-water, W/0/W); 于一实施例中， 该佐剂为水性氢氧化铝胶。
进一步地， 本发明提供一种动物对抗猪萎缩性鼻炎的方法， 包含使用有效量的上述免疫 组合物以施予动物， 以增强该动物对抗猪萎缩性鼻炎的免疫力， 进而提升、 改善其临床症状、 存活率、 及增重趋势。
本发明并提供一种抗败血性巴氏杆菌 D型菌毒素 (PMT)的抗体， 系藉由本发明所提供的 败血性巴氏杆菌毒素重组蛋白 (rPMT)所制备或衍生而得； 该抗体包括但不限于： 单株抗体、 多株抗体， 以及经基因重组的抗体。 于一实施例中， 该抗体系为经由将本发明所提供的败血 性巴氏杆菌毒素重组蛋白 (rPMT)施打于一动物体内而得到的多株抗体。 本发明并提供一种猪 萎缩性鼻炎的检测套组， 该检测套组系用以侦测检验样本是否含有败血性巴氏杆菌 D型菌毒 素 (PMT)或侦测检验样本内是否含有抗败血性巴氏杆菌 D 型菌毒素 (PMT)的抗体。 该检测套 组包含但不限于： （1)一抗原， 该抗原系为本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT), 于一实施例中， 该抗原系置于一抗原盘上； 及 /或 (2)—抗体， 该抗体系由该本发明所 提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)所衍生、 制备而得的单株抗体或多株抗体。
该检测套组的形式包含但不限于：酵素连结免疫分析 (enzyme-linked immuNOsorbent assay, ELISA)套组、微晶片检验套组 (Microchip kit)、免疫萤光分析法 (immuNO fluorescent assay, IFA) 检测套组、 或其他藉由该败血性巴氏杆菌毒素重组蛋白 (rPMT)所制得的检测套组。 于一实施 例中， 该检测套组至少包含一含有本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)的抗 原盘， 可用以检验样本中是否含有抗败血性巴氏杆菌毒素 (PMT)的抗体。
于本发明中所使用的单数形式「一」、及「该」包含复数形式， 除非文中另有清楚指明者。 因此， 例如， 当提及 「一样本」 时， 包含复数个该等样本及对该领域具有通常技艺者所知的 同等物。
本文所使用的 「约」、 「大约」 或 「近乎」 一词实质上代表所述的数值或范围位于 20%以 内， 较佳为于 10%以内， 以及更佳者为于 5%以内。 于本文所提供的数字化的量为近似值， 意旨若术语 「约」、 「大约」 或 「近乎」 没有被使用时亦可被推得。
本说明书中所述的所有技术性及科学术语， 除非另外有所定义， 皆为该所属领域具有通 常技艺者可共同了解的意义。
本发明系以下面的实施例予以示范阐明， 但本发明不受下述实施例所限制。
实施例一败血性巴氏杆菌毒素重组蛋白 (rPMT)的构筑
1. 败血性巴氏杆菌毒素重组蛋白 l (rPMTl)氨基酸序列的设计
首先以败血性巴氏杆菌毒素蛋白 (PMT)全长氨基酸序列 (如 SEQ ID NO: 1所示)为模板， 利用抗原决定位 (epitopes)预测网站 (；例如，但不限于 BCPred (http:/'/ailab.cs.iastate.edu/bcpredsA 预测败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位。
Epi预测出的抗原决定位如表 1所示 (SEQ ID NOs: 20 - 42)， 将这些抗原决定位划分为三 个区段， 分别编号为 Epi 1、 Epi 2及 Epi 3， 其氨基酸序列分别如 SEQ ID NO: 2、 3、 4所示； Epi 1、 Epi 2及 Epi 3分别含括了表 1所示的各个抗原决定位。
表 1 败血性巴氏杆菌毒素蛋白 (PMT)的抗原决定位预测结果
划分区段 序列 SEQ ID NO.
Epi 1 SSDSSDGN 20
Epi 1 YSVGKEGAYYPDHDYGPEYNPVWGPNEQI 21
Epi 1 FSSSDSSDGNVFHYNSLSES 22
Epi 1 GKEGAYYPDHDYGPEYNPVW 23
Epi 1 LMNIFVERYFDDWDLLNSLA 24
Epi 1 SSSDSSDGNVFHYN 25
Epi 1 GKEGAYYPDHDYGP 26
Epi 1 VERYFDDWDLLNSL 27
Epi 1 YNPVWGPNEQIYHS 28
Epi 1 VGKEGAYYPDHDYGPEYNPV 29
Epi 2 HSDNSVPPFNNPYK 30
Epi 2 LLNSTPGTGRPMP 31
Epi 2 TVVDSDGA 32
Epi 2 TPGTGRPMPGLVQYLKIPAT 33
Epi 2 H SDNS VPPFNNP YKSLYYKG 34
Epi 2 LNSTPGTGRPMPGL 35
Epi 2 STPGTGRPMPGLVQYL IPA 36
Epi 3 LLNSTPGTGRPMP 31
Epi 3 TVVDSDGA 32
Epi 3 VASSR 37
Epi 3 VLTPPQG 38 划分区段 序列 SEQ ID NO.
Epi 3 TPGTGRPMPGLVQYLK PAT 33
Epi 3 TELENWQVLTPPQG ILGLK 39
Epi 3 PDVASS VPIEVTE 40
Epi 3 LNSTPGTGR PMPGL 35
Epi 3 NWQVLTPPQGKILG 41
Epi 3 STPGTGRPMPGLVQYLKIPA 36
Epi 3 RVPI EVTELENWQVLTPPQG 42
接着以 Epi 1、 Epi 2、 Epi 3 不同组合的氨基酸序列设计败血性巴氏杆菌毒素重组蛋白 (rPMT 分别在 Epi 1 (SEQ ID NO: 2)、 Epi 2 (SEQ ID NO: 3)、 Epi 3 (SEQ ID NO: 4)的氨基酸 序列 C端各加上一段连接子序列， 该连接子具有如 SEQ ID NO: 5所示的氨基酸序列，再由 N 端至 C端依序连接 Epi 1、 Epi 2、 Epi 3； 并且在 Epi 3的连接子序列 C端加上六个 mC3d-p28 分子佐剂 (bioadjuvant)序列 (SEQ ID NO: 6)，每个 mC3d-p28分子佐剂序列皆以 GS连接子连接； 得到的败血性巴氏杆菌毒素重组蛋白 (rPMT)氨基酸序列如 SEQ ID NO: 10所示， 并以合成仪 合成该氨基酸序列或以基因选殖表现方式进行之。 2. 败血性巴氏杆菌毒素重组蛋白 2 (rPMT2)氨基酸序列的设计
败血性巴氏杆菌毒素蛋白 (PMT)的的抗原决定位 (epitopes)的预测如上述。以 Epi 1、 Epi 2、 Epi 3的氨基酸序列设计败血性巴氏杆菌毒素重组蛋白 (rPMT)。 分别在 Epi 1 (SEQ ID NO: 2)、 Epi 2 (SEQ ID NO: 3)、 Epi 3 (SEQ ID NO: 4)的氨基酸序列 C端各加上一段连接子序列， 该连 接子具有如 SEQ ID NO: 5所示的氨基酸序列，再由 N端至 C端依序连接 Epi 1、 Epi 2、 Epi 3 ; 并且在 Epi 3的连接子序列 C端加上六个 pC3d-p31分子佐剂 (bioadjuvant)序列 (如 SEQ ID NO: 7所示)， 每个 pC3d-p31分子佐剂序列皆以 GS连接子连接； 得到的败血性巴氏杆菌毒素重组 蛋白 2 (rPMT2)氨基酸序列如 SEQ ID NO: 11所示，并以合成仪合成该氨基酸序列或以基因选 殖表现方式进行之。 实施例二 支气管败血性博德氏杆菌 及败血性巴氏杆菌Pasteurella m ocida)的培养
1. 支气管败血性博德氏杆菌 Οδ. /wwc/^e ^'ca)的培养
于本实施例中， 支气管败血性博德氏杆菌 (S. 6ra«c/^£^rica)由行政院农业委员会家畜卫 生试验所 (台湾)分让而来。
先将该支气管败血性博德氏杆菌 wwc/^e ^'ca)接种于 TSB固体培养基上 [含 5% (v/v) 酵母抽出液 (yeast extract)、 10% (v/v)血清、 大豆分解蛋白质干酪素培养基 (tryptic soy broth, TSB, BD公司， 美国)]， 于 37°C下培养隔夜后， 挑选单一菌落接种于脑心浸出液 (brain heart infosion, BHI)液体培养基内 (BD公司， 美国)， 于 37°C下震荡培养隔夜； 接着取菌液再接种 于 BHI液体培养基内，于 37°C下震荡培养隔夜，并计算菌落形成单位 (colony forming unit, CFU) 值； 最后加入甲醛 (formaldehyde), 于室温下震荡 24至 36小时， 对菌液进行不活化作用。
2. 败血性巴氏杆菌 OP ww/tocWa)的培养
于本实施例中， 败血性巴氏杆菌 A型菌 (PmA)及败血性巴氏杆菌 D型菌 (PmD)由行政院 农业委员会家畜卫生试验所 (台湾)分让而来，且该败血性巴氏杆菌 D型菌 (PmD)具有产生毒素 能力。
先将败血性巴氏杆菌 A型菌 (PmA)及败血性巴氏杆菌 D型菌 (PmD)分别接种于 TSB固体 培养基上 [含 5% (v/v)酵母抽出液、 10% (v/v)血清、 大豆分解蛋白质干酪素培养基 (TSB， BD 公司，美国)]，于 37°C下培养隔夜后，挑选单一菌落接种于脑心浸出液 (BHI)液体培养基内 (BD 公司， 美国)， 于 37°C下震荡培养隔夜； 接着取 0.1% (v/v)菌液再接种于 BHI液体培养基内， 于 37°C下震荡培养隔夜， 并计算菌落形成单位 (CFU)值； 最后加入甲醛对菌液进行不活化作 用。 实施例三含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的配制 将实施例一所得到的败血性巴氏杆菌毒素重组蛋白 (rPMT)(SEQ ID NO: 10或 11) (；最终浓 度为 65 g/ml)与实施例二所得到的不活化的支气管败血性博德氏杆菌 (A bronchiseptica 最终 浓度为 lxlO9 CFU/ml)、 不活化的败血性巴氏杆菌 A型菌 (PmA) (；最终浓度为 lxlO9 CFU/ml)及 不活化的败血性巴氏杆菌 D 型菌 (PmD) (；最终浓度为 lxlO9 CFU/ml)以磷酸盐缓冲溶液 (phosphate buffered solution, PBS)混和均匀， 并加入铝胶 [最终浓度为 30% (v/v)]作为佐剂， 以 配制成为猪萎缩性鼻炎免疫组合物。 实施例四 含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫 原性 (immimogenicity)分析 -小鼠免疫试验
1. 试验小鼠
取 26只 4周龄健康的 BALB/c雄性小鼠 (国家实验动物中心， 台湾)作为试验动物， 所有 小鼠的败血性巴氏杆菌毒素蛋白 (PMT)、 支气管败血性博德氏杆菌 Οδ. bronchiseptica), 败血性 巴氏杆菌 A型菌 (PmA)及败血性巴氏杆菌 D型菌 (PmD)的酵素结合免疫吸附分析 (ELISA)抗体 皆呈阴性。
2. 免疫计画 将这些小鼠随机分为 5组， 第 1组 (负对照组)有 6只小鼠， 而第 2至 5组则每组有 5只 小鼠。 每只小鼠分别以腹腔注射 (intraperitoneal injection, ip.)注射 0.2 ml以下物质：
第 1组： 含 30% (ν/ν)铝胶的 PBS缓冲溶液 (负对照组)；
第 2组： 实施例二所得的支气管败血性博德氏杆菌 (S. bronchiseptica)(\x\09 CFU/ml)^ 败 血性巴氏杆菌 A型菌 (PmA)(lxl09 CFU/ml)及败血性巴氏杆菌 D型菌 (PmD)(lxl09 CFU/ml)的 免疫组合物 (含 30% (v/v)铝胶佐剂) (B.b + PmA + PmD组)；
第 3组： 实施例一所得的败血性巴氏杆菌毒素重组蛋白 (序列如 SEQ ID NO: 10所示， 浓 度为 65 g/ml)(rPMT组)；
第 4组：实施例三所得的含有败血性巴氏杆菌毒素重组蛋白 (rPMT，序列如 SEQ ID NO: 10 所示)的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT组)； 以及
第 5组： 市售猪萎缩性鼻炎疫苗 (Bayovac®, 台湾拜耳公司， 台湾) (Bayovac组)。
每只小鼠分别于首次免疫前 24小时采血保存， 首次免疫后第 13天再进行采血， 首次免 疫后第 14天以相同免疫剂量再进行第二次免疫， 第二次免疫后第 10天采血， 并分离血液样 本中的血清， 以进行毒素抗体的酵素连结免疫分析 (Enzyme-linked immunosorbent assay, ELISA)。
3. 毒素抗体的酵素连结免疫分析 (ELISA)
以商品化的败血性巴氏杆菌毒素 (PMT Merck 公司， 美国)作为抗原， 并将抗原涂布 (coating)于 ELISA用 96孔盘 (Thermo公司， 美国)， 于 4°C下静置 16小时。 去除多余抗原后 加入清洗缓冲液 (wash buffer; 0.9% NaCl; 0.1 % Tween20), 清洗 3次后倒干。 接着加入阻隔 缓冲液 (blocking buffer; 含有 1% BS A的 wash buffer), 于室温下静置 1小时后， 以清洗缓冲 液清洗， 接着将上述各组小鼠采集到的血清样品以 PBS缓冲溶液稀释后， 每孔加入稀释的小 鼠血清， 于室温下静置 1小时后， 去除血清样品， 并以清洗缓冲液清洗， 然后加入辣根过氧 化酵素 (Horseradish peroxidase, HRP)标定的山羊抗小鼠的二级抗体 (goat anti-mouse conjugated HRP， Gene Tex公司， 美国)， 该二级抗体先以阻隔缓冲液稀释 5000倍后再加入 96孔盘 (100 μΐ/孔)， 于室温下静置 1小时后， 去除二级抗体， 并以清洗缓冲液清洗后， 每孔加入 100 μΐ 3, 3 ', 5, 5 '-四甲基联苯胺二盐酸 (3, 3 ', 5, 5 '-tetramethylbenzidine, TMB， KPL公司， 美国)溶液避 光呈色 10分钟，并以酵素连结免疫分析测读仪 (SpectraMax® M2/M2 ELISA Reader, Molecular Devices公司， 美国)读取波长 650 nm的吸光值。
酵素连结免疫分析结果如图 1所示， 免疫含有败血性巴氏杆菌毒素重组蛋白的猪萎缩性 鼻炎免疫组合物的小鼠 (第 4组， 即 B.b + PmA + PmD + rPMT组)血清中含有的抗败血性巴氏 杆菌毒素 (PMT)抗体力价最高， 其次为免疫市售猪萎缩性鼻炎疫苗 (第 5 组， 即 Bayovac组) 的小鼠血清； 而只免疫实施例一所得的败血性巴氏杆菌毒素重组蛋白 (第 3组， 即 rPMT组) 的小鼠血清中也含有的抗败血性巴氏杆菌毒素 (PMT)抗体， 且其所含的抗体力价高于免疫实 施例二所得的支气管败血性博德氏杆菌、败血性巴氏杆菌 A型菌及败血性巴氏杆菌 D型菌 (第 2组， 即 B.b + PmA + PmD组)的小鼠血清。 由此可知， 本发明所提供的败血性巴氏杆菌毒素 重组蛋白 (rPMT)可以在动物体内有效地诱导出抗败血性巴氏杆菌毒素 (PMT)抗体， 具免疫原 性。
4. 中和抗体力价试验
以 Vero细胞作为试验材料，测试免疫含有败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎 免疫组合物的小鼠 (；第 4组， 即 B.b + PmA+ PmD + rPMT组)血清中所含的抗败血性巴氏杆菌 毒素 (PMT)抗体， 是否为可中和 PMT毒性的中和抗体。 进行中和抗体力价测试的前， 先进行 Vera细胞的最小毒性剂量 (minimum toxin dose, MTD)测定。
(a) Vera细胞的最小毒性剂量 (MTD)测定
将 Vero细胞接种于 96孔培养盘中， 待细胞长成单层后， 去除培养液， 并加入以不含血 清的 DMEM培养液 (GIBCO公司， 美国)连续 2倍稀释的 PMT处理。 以含胎牛血清 (FBS)的 DMEM培养液作为负对照组， 培养后并观察细胞的型态变化， 以能诱导 Vero细胞产生细胞 病变 (cytopathic effect, CPE)的最低 PMT浓度为最小毒性剂量 (MTD)。 试验结果显示， Vera 细胞的败血性巴氏杆菌毒素 (PMT)的最小毒性剂量为 60 ng。
(b) 中和抗体力价试验
中和抗体力价试验方法系参考 Liao等人 (2006)与 Lee等人 (2012)所提出的方法并进行适 当修饰 (Liao, C. M.， Huang, C, Hsuan, S. L" Chen, Z. W.， Lee, W. C, Liu, C. I., Winton, J. R.， and Chien, M. S. (2006). Immunogenicity and efficacy of three recombinant subunit Pasteurella multocida toxin vaccines against progressive atrophic rhinitis in pigs. Vaccine. 24, 27-35； Lee, J., Kang, H. E.， and Woo, H. J. (2012). Protective immunity conferred by the C -terminal fragment of recombinant Pasteurella multocida toxin. Clin Vaccine Immunol. 19, 1526- 1531.)。首先将免疫含有 败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物的小鼠 (；第 4组， 即 B.b + PmA + PmD + rPMT组)血清稀释 10倍后， 再做连续两倍的稀释 (^1〜—6)。 于 96孔培养盘中， 分别加 入上述稀释的小鼠血清， 并于各孔中加入含 4倍最小毒性剂量 (MTD)的败血性巴氏杆菌毒素 (PMT),然后置于 37°C中反应 1小时。接着将上述反应液加入培养于 96孔盘的 Vero细胞中， 于 37°C、 5% C02培养箱中培后， 并观察该血清是否能够抑制 Vero细胞产生细胞病变 (CPE)。
结果如图 2所示。 以含 4倍最小毒性剂量 (MTD)的败血性巴氏杆菌毒素 (PMT)处理 Vero 细胞作为正对照组， 其细胞形态如图 2B 所示， 可见到细胞呈现典型的结节样。 相较之下， 以含胎牛血清 (FBS)的 DMEM培养液培养 Vero细胞作为负对照组，其细胞形态如图 2A所示； 而以免疫含有败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物的小鼠 (；第 4 组， 即 B.b + PmA + PmD + rPMT组)血清稀释 40倍后与 4倍最小毒性剂量 (MTD)的败血性巴氏杆菌 毒素 (PMT)中和后， 加入 Vero细胞共培养的细胞形态如图 2C所示。 图 2A与图 2C所示的细 胞皆未出现如图 2B 所示的典型的结节样， 显示免疫含有败血性巴氏杆菌毒素重组蛋白的猪 萎缩性鼻炎免疫组合物的小鼠 (第 4组， 即 B.b + PmA+ PmD + rPMT组)血清中含有抗败血性 巴氏杆菌毒素 (PMT)的中和抗体。 实施例五含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫 原性及保护效力分析 1-台湾猪萎缩性鼻炎菌苗检验标准 (巴氏杆菌效力试验）
根据台湾猪萎缩性鼻炎菌苗检验标准， 选取败血性巴氏杆菌抗体阴性 3周龄 BALB/c小 鼠 (国家实验动物中心， 台湾） 32只， 随机分为 3组， 第 1组为对照组 (n = 9)， 第 2组为 rPMT 免疫试验组 (n = 12)， 第 3组为 Bayovac®免疫试验组 (n = 11)； 每只小鼠分别以腹腔注射 (ip.) 注射 0.5 ml的 10倍稀释待测物， 各组分别为：
第 1组： 含 30% (ν/ν)铝胶的 PBS缓冲溶液 (对照组)；
第 2组：实施例三所得的含有败血性巴氏杆菌毒素重组蛋白 (rPMT，序列如 SEQ ID NO: 10 所示)的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT组)； 以及
第 3组： 市售猪萎缩性鼻炎疫苗 (Bayovac®, 台湾拜耳公司， 台湾) (Bayovac组)。
免疫试验组 (第 2组及第 3组)于免疫后第 14日各分为 3小组， 依组序分别以具有生产毒 素能力的败血性巴氏杆菌 D型菌 (PmD)强毒菌株 [同实施例二， 由行政院农业委员会家畜卫生 试验所 (台湾)分让而来] lxlO3 CFU/ml、 lxlO4 CFU/ml、 lxlO5 CFU/ml三种浓度活菌液 0.1 ml 腹腔注射。 同时对照组 (第 1 组） 12 只小鼠亦分为三小组， 依组序分别以巴氏杆菌强毒菌株 lxlO2 CFU/ml、 lxlO3 CFU/ml、 lxlO4 CFU/ml三种浓度活菌液 0.1 ml腹腔注射。观察 10天后， 各免疫试验组与对照组分别以贝卡二氏法 (Beherens-Karber)计算其 LD5Q， 且各免疫试验组的 防御指数须高于对照组 1χ10ϋ·5以上。 贝卡二氏法防御指数计算法如下：
LD50 = 攻毒剂量的最低稀释倍数 - [(各组死亡率的总和 /100) - 0.5] X 1
防御指数=【对照组攻毒剂量的最低稀释倍数 - [(；各组死亡率的总和 /100) - 0.5] X 1】 -【免 疫组攻毒剂量的最低稀释倍数 - [(；各组死亡率的总和 /100) - 0.5] X 1】。
结果如表 2所示，含有败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物 (第 2组， 即 B.b + PmA + PmD + rPMT组)确实能诱发小鼠产生保护效力， 并耐过败血性巴氏杆菌 D型 菌 (PmD)强毒菌株的攻毒，且其防御指数高达 102·ϋ6，远高于台湾检验标准 (10ϋ·5)以及市售猪萎 缩性鼻炎疫苗 (Bayovac®, 台湾拜耳公司， 台湾) (Bayovac组)的防御指数 (1(^74)。 表 2含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫原性及 保护效力分析 1结果
Figure imgf000017_0001
实施例六含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫 原性及保护效力分析 2
选取败血性巴氏杆菌抗体阴性、 体重 15~20 g的 BALB/c小鼠 (；国家实验动物中心， 台湾) 65只， 随机分为 3组， 第 1组为对照组， 共有 5只小鼠， 第 2、 3组为免疫试验组， 每组 30 只小鼠。 分别将各免疫试验再细分为 3小组， 每小组 10只小鼠； 各组小鼠分别以腹腔注射方 式注射以下物质：
第 1组： 每只小鼠注射 0.2 ml PBS缓冲溶液 (对照组)；
第 2-1组:每只小鼠注射 0.2 ml实施例三所得的含有败血性巴氏杆菌毒素重组蛋白 (rPMT， 序列如 SEQ ID NO: 10所示)的猪萎缩性鼻炎免疫组合物原液 (B.b + PmA + PmD + rPMT组)； 第 2-2组： 每只小鼠注射 0.2 ml稀释 5倍的实施例三所得的含有败血性巴氏杆菌毒素重 组蛋白 (rPMT,序列如 SEQ ID NO: 10所示)的猪萎缩性鼻炎免疫组合物 (1/5 B.b + PmA + PmD + rPMT组）；
第 2-3组：每只小鼠注射 0.2 ml稀释 25倍的实施例三所得的含有败血性巴氏杆菌毒素重 组蛋白 (rPMT, 序列如 SEQ ID NO: 10所示)的猪萎缩性鼻炎免疫组合物 (1/25 B.b + PmA + PmD + rPMT组)；
第 3-1组： 每只小鼠注射 0.2 ml市售猪萎缩性鼻炎疫苗原液 (Bayovac®, 台湾拜耳公司， 台湾) (Bayovac组)；
第 3-2组： 每只小鼠注射 0.2 ml稀释 5倍的市售猪萎缩性鼻炎疫苗 (Bayovac®, 台湾拜耳 公司， 台湾 )(1/5 Bayovac组)；
第 3-3组： 每只小鼠注射 0.2 ml稀释 25倍的市售猪萎缩性鼻炎疫苗 (Bayovac®, 台湾拜 耳公司， 台湾) (1/25 Bayovac组)。
免疫试验组 (第 2、 3组)于首次免疫后第 14日进行二次免疫，剂量同首次免疫；对照组 (第 1组)小鼠则再次注射 0.2 ml PBS缓冲溶液； 二次免疫后第 10天进行攻毒试验， 每只小鼠以 腹腔注射方式注射 0.2 ml具有生产毒素能力的败血性巴氏杆菌 D型菌 (PmD)强毒菌株 [同实施 例二， 由行政院农业委员会家畜卫生试验所 (台湾)分让而来] 100 LD5Q活菌液， 观察 10天记 录存活率。 原液免疫组 (第 2-1组、 第 3-1组)存活率须高于 80%、 5倍稀释免疫组 (第 2-2组、 第 3-2组)存活率须高于 50%、 25倍稀释免疫组 (第 2-3组、 第 3-3组)存活率须高于 20%， 对 照组则须全部死亡。
结果如表 3所示， 含有败血性巴氏杆菌毒素重组蛋白的猪萎缩性鼻炎免疫组合物 (第 2-1 组、 第 2-2组、 第 2-3组)确实能诱发小鼠产生足够的保护效力， 免疫原液疫苗的小鼠存活率 为 90%， 符合存活率 80%以上的标准， 免疫稀释 5倍疫苗的小鼠存活率为 50%， 符合存活率 50%以上的标准， 免疫稀释 25倍疫苗的小鼠存活率为 30%， 符合存活率 20%以上的标准。 表 3含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫原性及 保护效力分析 2结果
组别 攻 死 存 累 最 存 效力
毒 亡 活 计 后 活 判定
后 数 数 死 存 率
天 亡 活
数 数 数
第 1组 1 2 3 2 0 0% 0%
(对照组） 2 3 0 5 (合格） 第 2-1组 （B.b+PmA+ 1 0 10 0 9 90% ^ 80%
PmD+rPMT组） 2 1 9 1 (合格） 第 2-2组 （1/5 1 2 8 2 5 50% ^ 50%
B.b+PmA+ 2 3 5 5 (合格） 组别 攻 死 存 累 最 存 效力
毒 亡 活 计 后 活 判定
后 数 数 死 存 率
天 亡 活
数 数 数
PmD+rPMT组）
第 2-3组 （1/25 1 3 7 3 3 30%
B.b+PmA+ 2 3 4 6
PmD+rPMT组） 3 1 3 7
第 3-1组 1 0 10 0 8 80% ^ 80% (Bayovac组) 2 2 8 2 (合格） 第 3-2组 1 2 8 2 6 60% ^ 50%
(1/5 Bayovac组） 2 2 6 4 (合格） 第 3-3组 1 2 8 2 2 20%
(1/25 Bayovac组） 2 4 4 6
3 2 2 8 w 实施例七含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫 原性及保护效力分析 3 - 以败血性巴氏杆菌毒素进行攻毒的小鼠试验
1. 败血性巴氏杆菌粗萃毒素的制备
先将具有生产毒素能力的败血性巴氏杆菌 D型菌 (PmD)强毒菌株 [同实施例二， 由行政院 农业委员会家畜卫生试验所 (台湾)分让而来]接种于脑心浸出液 (BHI)液体培养基内 (BD公司， 美国)， 于 37°C下培养隔夜后， 取 100 μΐ菌液平均涂布于 7%血液培养基， 于 37°C下培养隔 夜， 以 PBS刮洗细菌并置于离心管中， 以超音波振荡器 (SONOPULS, Bandelin公司， 德国) 处理以将细菌击碎， 再以高速离心机 (KUBOTA公司， 日本)离心后， 取上清液以 0.45 μηι孔 径过滤膜 (Millipore公司，美国)过滤后，此过滤液即为败血性巴氏杆菌粗萃毒素 (crude extracted PMT)， 分装后保存于 -20°C备用。
2. 败血性巴氏杆菌粗萃毒素对小鼠的半数致死剂量 (LD5Q)试验
取败血性巴氏杆菌抗体阴性 3周龄健康的 BALB/c小鼠 (；国家实验动物中心，台湾） 76只， 随机分为 5组； 将上述制得的败血性巴氏杆菌粗萃毒素解冻后， 依照下列毒素剂量对每组每 只小鼠注射 0.5 ml粗萃毒素：
第 1组： lxl08 CFU/ml萃取的毒素蛋白 (n = 14);
第 2组： 2xl08 CFU/ml萃取的毒素蛋白 (n = 8);
第 3组： 4xl08 CFU/ml萃取的毒素蛋白 (n = 12);
第 4组： 6xl08 CFU/ml萃取的毒素蛋白 (n = 20);
第 5组： 8xl08 CFU/ml萃取的毒素蛋白 (n = 22);
注射粗萃毒素后， 观察小鼠 10天并纪录死亡数， 以计算败血性巴氏杆菌粗萃毒素对小鼠 的半数致死剂量 (LD5Q)， LD5Q是以 Calcu-Syn软体 (Biosoft公司， 英国)计算得出。
结果如表 4所示，以上述败血性巴氏杆菌粗萃毒素对小鼠进行攻毒的半数致死剂量 (LD5()) 为 2xl08 CFU/ml。
表 4 败血性巴氏杆菌粗萃毒素对小鼠的半数致死剂量 (LD5Q)试验结果
Figure imgf000020_0001
3. 小鼠免疫及以败血性巴氏杆菌粗萃毒素攻毒试验
取败血性巴氏杆菌抗体阴性 3周龄健康的 BALB/c小鼠 (；国家实验动物中心，台湾） 24只， 随机分为 5组； 第 1组为对照组， 第 2至 5组为免疫试验组； 各组每只小鼠分别以腹腔注射 (ip.)注射 0.2 ml的以下物质：
第 1组： 含 30% (ν/ν)铝胶的 PBS缓冲溶液 (负对照组)；
第 2组： 实施例二所得的支气管败血性博德氏杆菌 (S. bronchiseptica)(\x\09 CFU/ml)^ 败 血性巴氏杆菌 A型菌 (PmA)(lxl09 CFU/ml)及败血性巴氏杆菌 D型菌 (PmD)(lxl09 CFU/ml)的 免疫组合物 (含 30% (v/v)铝胶佐剂) (B.b + PmA + PmD组)；
第 3组：实施例三所得的含有败血性巴氏杆菌毒素重组蛋白 (rPMTl ,序列如 SEQ ID NO: 10所示)的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMTl组)；
第 4组：实施例三所得的含有败血性巴氏杆菌毒素重组蛋白 (rPMT2，序列如 SEQ ID NO: 11所示)的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT2组)； 以及 第 5组： 市售猪萎缩性鼻炎疫苗 (Bayovac®, 台湾拜耳公司， 台湾) (Bayovac组)。
每只小鼠分别于首次免疫后第 14天以相同免疫剂量再进行第二次免疫，第二次免疫后第 10天取上述制得的败血性巴氏杆菌粗萃毒素进行攻毒试验， 对每只小鼠注射 0.5 ml的 LD85 剂量 (即 6xl08 CFU/ml)的败血性巴氏杆菌粗萃毒素， 注射粗萃毒素后， 观察小鼠 10天并纪录 死亡数， 以计算各组存活率。
结果如表 5所示， 含有败血性巴氏杆菌毒素重组蛋白 (rPMTl或 rPMT2)的猪萎缩性鼻炎 免疫组合物 (即第 3组及第 4组)确实能诱发小鼠产生保护效力， 并耐过败血性巴氏杆菌粗萃 毒素的攻毒， 且其存活率皆大于负对照组及仅免疫支气管败血性博德氏杆菌 (S. /wwc/^eprica)、败血性巴氏杆菌 A型菌 (PmA)及败血性巴氏杆菌 D型菌 (PmD)的免疫组合物。 表 5 小鼠免疫及以败血性巴氏杆菌粗萃毒素攻毒试验的结果
Figure imgf000021_0001
实施例八含有败血性巴氏杆菌毒素重组蛋白 (rPMT)的猪萎缩性鼻炎免疫组合物的免疫 原性分析 -猪只免疫试验
1. 试验猪只 自一猪场 (宜兰， 台湾)随机选 3~4周龄健康的哺乳猪 30只作为试验猪只。
2. 免疫计画
将上述猪只随机分为 3组， 每组 10只猪， 每只猪分别于试验开始 (第 0天首次免疫)与第 21天 (二次免疫)各进行一次肌肉注射 (2 ml/只)下列物质：
第 1组： 含 30% (ν/ν)铝胶的 PBS缓冲溶液 (负对照组)；
第 2组： 实施例二所得的支气管败血性博德氏杆菌 (S. bronchiseptica)(\x\09 CFU/ml)^ 败 血性巴氏杆菌 A型菌 (PmA)(lxl09 CFU/ml)及败血性巴氏杆菌 D型菌 (PmD)(lxl09 CFU/ml)的 免 疫组合物 (含 30% (v/v)铝胶佐剂) (B.b + PmA + PmD组)； 以及
第 3组：实施例三所得的含有败血性巴氏杆菌毒素重组蛋白 (rPMT，序列如 SEQ ID NO: 11 所示)的猪萎缩性鼻炎免疫组合物 (B.b + PmA + PmD + rPMT组)。
每只猪分别于首次免疫前、 二次免疫前， 以及在二次免疫后第 14天采血保存， 并且记录 体重， 并分离血液样本中的血清， 以进行毒素抗体的酵素连结免疫分析 (ELISA)。
3. 酵素连结免疫分析 (ELISA)
以败血性巴氏杆菌毒素 (PMT)(Merck 公司， 美国)作为抗原， 并将抗原涂布 (coating)于 ELISA用 96孔盘 (Thermo公司， 美国)， 于 4°C下静置 16小时。 去除多余抗原后加入清洗缓 冲液 (0.9% NaCl; 0.1 % Tween20), 清洗后倒干。 接着加入阻隔缓冲液 (含有 1% BSA的清洗 缓冲液)， 于室温下静置 1小时后， 以清洗缓冲液清洗， 接着将上述各组猪采集到的血清样品 以 PBS缓冲溶液稀释后， 每孔加入稀释的猪血清， 于室温下静置 1小时后， 去除血清样品， 并以清洗缓冲液清洗， 然后加入辣根过氧化酵素 (HRP)标定的山羊抗小鼠的二级抗体 (Gene Tex公司， 美国)， 该二级抗体先以阻隔缓冲液稀释 1000倍后再加入 96孔盘 (100 μΐ/孔)， 于室 温下静置 1小时后， 去除二级抗体， 并以清洗缓冲液清洗 6次后， 每孔加入 100 μΐ 3, 3 ', 5, 5 '- 四甲基联苯胺二盐酸 (KPL公司， 美国)溶液避光呈色 10分钟， 并以酵素连结免疫分析测读仪 (Labsystem multiskan MCC/340 Microplate Reader, Labsystem公司， 美国）读取波长 650 nm的 吸光值。
酵素连结免疫分析结果如图 3所示， 在二次免疫后第 14天时， 免疫含有败血性巴氏杆菌 毒素重组蛋白的猪萎缩性鼻炎免疫组合物的猪只 (第 3组， 即 B.b + PmA + PmD + rPMT组)血 清中含有的抗败血性巴氏杆菌毒素 (PMT)抗体力价最高， 且其相较于负对照组猪只与免疫实 施例二所得的支气管败血性博德氏杆菌、败血性巴氏杆菌 A型菌及败血性巴氏杆菌 D型菌的 猪只 (第 2组， 即 B.b + PmA + PmD组)血清中含有的抗败血性巴氏杆菌毒素 (PMT)抗体力价， 分别具有显著差异 (**, 0.01)。此外，这三组猪只之间的体重变化并无显著差异 (结果未显示)。
由此可知， 本发明所提供的败血性巴氏杆菌毒素重组蛋白 (rPMT)在动物体内， 特别是目 标动物猪只体内， 可有效地诱导出抗败血性巴氏杆菌毒素 (PMT)抗体， 而且对猪只的生长无 不良影响， 具有安全性。 实施例九抗败血性巴氏杆菌毒素重组蛋白 (rPMT)抗体的制备
1.抗败血性巴氏杆菌毒素重组蛋白 (rPMT)的多株抗体
将实施例一所得到的败血性巴氏杆菌毒素重组蛋白 (rPMT)与适用的佐剂 (如： 铝胶)混合 后施与动物 (；如： 小鼠、 大鼠、 猪、 山羊、 兔)以进行初级免疫， 经适当时间间隔后 (如： 2~3 周)， 视需要可进第二次免疫。 经适当时间间隔后 (如： 2~3周)， 采集免疫动物 (；如： 小鼠、 大 鼠、 猪、 山羊、 兔)的血清， 即制得抗败血性巴氏杆菌毒素重组蛋白 (rPMT)的多株抗体。
其中该抗败血性巴氏杆菌毒素重组蛋白 (rPMT)的多株抗体， 可视需要与显色剂或萤光结 合。
其中该动物经施予初级免疫及第二次免疫后， 可视需要增加免疫次数， 以提高抗体力价。 其中施与的动物包含， 但不限于： 小鼠、 大鼠、 兔、 禽 (；蛋)、 猪、 山羊、 牛、 水产动物。
2.抗败血性巴氏杆菌毒素重组蛋白 (rPMT)的单株抗体
将实施例一所得到的败血性巴氏杆菌毒素重组蛋白 (rPMT)与适用的佐剂 (如： 铝胶)混合 后施与动物 (；如： 小鼠、 大鼠、 猪、 山羊、 兔)以进行初级免疫， 经适当时间间隔后 (如： 2~3 周)， 视需要可进第二次免疫。 经适当时间间隔后 (如： 2~3周)， 采集免疫动物 (；如： 小鼠)的血 清，用以评估适合用以采集脾脏细胞的小鼠。从该适用的小鼠采集脾脏细胞与骨髓瘤细胞 (如： FO细胞株、 NS细胞株)以 PEG( Polyethylene Glycol, 如 PEG1500)进行细胞融合。 从融合细 胞中筛选出具分泌能力的融合瘤并单株化后， 可得一适合用以产制抗败血性巴氏杆菌毒素重 组蛋白 (rPMT)的单株抗体的融合细胞系。
经上述制备所得的抗体， 可用于免疫检测试剂、 治疗剂、 或加入食品、 饲料中使食用者 具有免疫力等。
上列详细说明系针对本发明的一可行实施例的具体说明， 惟该实施例并非用以限制本发 明的专利范围， 凡未脱离本发明技艺精神所为的等效实施或变更， 均应包含于本案的专利范 围中。

Claims

权 利 要 求 书
Figure imgf000024_0001
ww/toc t a)毒素重组蛋白， 包含至少一个败血性 巴氏杆菌毒素蛋白的抗原决定位 (epitopes)。
2.如权利要求 1所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该至少一个败 血性巴氏杆菌毒素蛋白的抗原决定位系选自于由 SEQ ID NOs: 2、 3、 4、 20、 21、 22、
23、 24、 25、 26、 27、 28、 29、 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41及 42所组成的群组中至少一个。
3.如权利要求 1所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 当该至少一个 败血性巴氏杆菌毒素蛋白的抗原决定位为复数个时， 各该败血性巴氏杆菌毒素蛋白的抗 原决定位之间由连接子连接。
4.如权利要求 3所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该连接子系各 自独立选自于由 Gly-Gly、 Gly-Ser及 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、 19所组成的群组。
5.如权利要求 1所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该败血性巴氏 杆菌毒素重组蛋白进一步包含至少一个单元的补体裂解片段 C3d 的部分氨基酸序列或 全长氨基酸序列。
6.如权利要求 5所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该至少一个败 血性巴氏杆菌毒素蛋白的抗原决定位与该补体裂解片段 C3d 的部分氨基酸序列或全长 氨基酸序列系以连接子连接。
7.如权利要求 6所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该连接子系各 自独立选自于由 Gly-Gly、 Gly-Ser及 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、 19所组成的群组。
8.如权利要求 5所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 当该至少一个 单元的补体裂解片段 C3d的氨基酸序列为复数个时，各该补体裂解片段 C3d的氨基酸序 列之间由连接子连接。
9.如权利要求 8所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该连接子系各 自独立选自于由 Gly-Gly、 Gly-Ser及 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、
19所组成的群组。
10.如权利要求 5所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该补体裂解片 段 C3d系选自于由 SEQ ID NOs: 6、 7、 8及 9所组成的群组。
11.如权利要求 5所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该败血性巴氏 杆菌毒素重组蛋白包含如 SEQ ID NO: 10或 11所示的氨基酸序列。
12.—种败血性巴氏杆菌 OPaWeMre//a ww/toc t a)毒素重组蛋白， 其系以下式表示： (A)m-(C3d片段) n; 式 (I)
其中每一个 A代表一个独立的败血性巴氏杆菌毒素蛋白的抗原决定位；
其中每一个 C3d片段代表一个独立的补体裂解片段 C3d的氨基酸序列；
其中 m是代表从 1至约 30的整数；
其中 n是代表从 0至约 10的整数。
13.如权利要求 12所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 每一个 A系 各自独立选自于由 SEQ ID NOs: 2、 3、 4、 20、 21、 22、 23、 24、 25、 26、 27、 28、 29、 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41及 42所组成的群组。
14.如权利要求 12所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 每一个 C3d 片段系各自独立选自于由 SEQ ID NOs: 6、 7、 8及 9所组成的群组。
15.如权利要求 12所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 每一个 A之 间以一个连接子连接， 每一个连接子系各自独立选自于由 Gly-Gly、 Gly-Ser及 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、 19所组成的群组。
16.如权利要求 12所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 每一个 C3d 片段之间以一个连接子连接，每一个连接子系各自独立选自于由 Gly-Gly、 Gly-Ser及 SEQ ID NOs: 5、 12、 13、 14、 15、 16、 17、 18、 19所组成的群组。
17.如权利要求 12所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， A与 C3d片 段的间以一连接子连接， 该连接子系选自于由 Gly-Gly、 Gly-Ser及 SEQ ID NOs: 5、 12、
13、 14、 15、 16、 17、 18、 19所组成的群组。
18.如权利要求 12所述的败血性巴氏杆菌毒素重组蛋白， 其特征在于， 该败血性巴 氏杆菌毒素重组蛋白包含与式 (I)具有至少 80%序列同源性的氨基酸序列。
19.一种编码如权利要求 1或 12所述的败血性巴氏杆菌毒素重组蛋白的核苷酸序列。
20.—种猪萎缩性鼻炎免疫组合物， 包含如权利要求 1或 12所述的败血性巴氏杆菌 毒素重组蛋白以及一药学上可接受的载剂。
21.如权利要求 20所述的猪萎缩性鼻炎免疫组合物， 其特征在于， 该猪萎缩性鼻炎 免疫组合物进一步包含一支气管败血性博德
Figure imgf000026_0001
bronchiseptica), 一败血性 巴氏杆菌 A型菌 (Pastet/fv a？ ?w/toc t a Type A)以及一败血性巴氏杆菌 D型菌 (Paste multocida Type D)。
22.如权利要求 20所述的猪萎缩性鼻炎免疫组合物， 其特征在于， 该猪萎缩性鼻炎 免疫组合物进一步包含其他病原抗原， 该病原抗原系选自由下列群组所组成者： 猪环状 病毒第二型 (PCV2)抗原、猪流感病毒 (SIV)抗原、猪繁殖与呼吸症候群病毒 (PRRSV)抗原、 猪霉桨菌 (Mycoplasma)、 猪小病毒 （Parvovirus, PPV)、 猪丹毒 (Erysipelas)、 猪胸膜肺炎 方文线杆菌 (actinobacillus pleuropneumonia, APP), 以及伪狂犬病 (Aujeszky's disease)。
23.—种动物对抗猪萎缩性鼻炎的方法， 包含使用如权利要求 20所述的免疫组合物 以施予一动物， 以增强该动物对抗猪萎缩性鼻炎的免疫力。
24.—种抗败血性巴氏杆菌 D型菌毒素的抗体， 系藉由如权利要求 1或 12所述的败 血性巴氏杆菌毒素重组蛋白所制备而得。
25.如权利要求 24所述的抗体， 该抗体包含至少下列其中一种： 一单株抗体、 一多 株抗体， 以及一经基因重组的抗体。
26.—种猪萎缩性鼻炎的检测试剂盒， 包含一侦测单元， 该侦测单元系选自于下列群 组所组成中至少一者： 一如权利要求 1或 12所述的败血性巴氏杆菌毒素重组蛋白以及 一藉由如权利要求 1或 12所述的败血性巴氏杆菌毒素重组蛋白所制备的抗体。
27.如权利要求 26所述的检测套组， 其中该败血性巴氏杆菌毒素重组蛋白系置于一 盘上。
28.如权利要求 26所述的检测套组， 其中该抗体包含至少下列其中一种： 一单株抗 体、 一多株抗体， 以及一经基因重组的抗体。
PCT/CN2014/073851 2013-03-22 2014-03-21 败血性巴氏杆菌毒素重组蛋白及其应用 WO2014146603A1 (zh)

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