WO2019076176A1 - 败血性巴氏杆菌毒素重组蛋白、其类病毒颗粒及其应用 - Google Patents

败血性巴氏杆菌毒素重组蛋白、其类病毒颗粒及其应用 Download PDF

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WO2019076176A1
WO2019076176A1 PCT/CN2018/106100 CN2018106100W WO2019076176A1 WO 2019076176 A1 WO2019076176 A1 WO 2019076176A1 CN 2018106100 W CN2018106100 W CN 2018106100W WO 2019076176 A1 WO2019076176 A1 WO 2019076176A1
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recombinant protein
pasteurella
toxin
protein
amino acids
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French (fr)
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杨滢臻
陈灿坚
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福又达生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/116—Polyvalent bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
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    • 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
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    • 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
    • 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
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/14—Disorders of ear, nose or throat

Definitions

  • the present invention relates to a recombinant protein of Pasteurella septicum toxin, in particular to a recombinant protein containing an epitope of a Pasteurella septicum toxin, and a viroid-like particle containing the recombinant protein.
  • Atrophic Rhinitis is one of the three major infectious diseases in the respiratory system of pigs.
  • Atrophic rhinitis in pigs can cause facial bone deformities and chronic suppurative rhinitis in infected pigs, causing atrophy of the nasal turbinates.
  • 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 will be infected.
  • Porcine atrophic rhinitis is caused by Bordetella bronchiseptica (Bb) and Pasteurella multocida type A (PmA) and D-type bacteria (PmD), especially Toxin produced by Pasteurella multocida type D (PmD) (Pasteurella multocida toxin, PMT).
  • Bb Bordetella bronchiseptica
  • PmA Pasteurella multocida type A
  • PmD D-type bacteria
  • PMT Pasteurella multocida type D
  • Inoculation of PMT toxins in the four-week-old piglets by intramuscular, intraperitoneal and nasal inoculation can cause pathological changes of the nasal axillary bone atrophy, and also affect the development of systemic bones and slow growth, even when inoculated 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 and increased production costs. 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 extremely urgent and extremely important to develop an effective porcine atrophic rhinitis vaccine to prevent pigs from suffering from atrophic rhinitis (AR).
  • the present invention provides, in the first part, a recombinant protein of Pasteurella multocida toxin comprising: an epitope (epitopes) of a Pasteurella multocida toxin protein having the amino acid sequence set forth in SEQ ID NO: 2.
  • An epitope of a S. septicum toxin protein having the amino acid sequence set forth in SEQ ID NO: 3 and an antigenic epitope of a S. septicum toxin protein having the amino acid sequence set forth in SEQ ID NO: Bit.
  • the invention provides a virus like particle (VLP) containing a recombinant protein of Pasteurella septicum toxin in the second part, comprising: a recombinant protein of Pasteurella septicum toxin and a type B as described above Hepatitis B virus core protein (HBc); wherein the recombinant protein of Pasteurella septicum toxin is inserted into a major immunodominant region (MIR) of the hepatitis B virus core protein.
  • VLP virus like particle
  • HBc Hepatitis B virus core protein
  • MIR major immunodominant region
  • the present invention provides, in a third part, a nucleic acid sequence encoding a recombinant protein of Pasteurella septicum toxin as described above.
  • the invention provides a nucleic acid sequence encoding a viroid-like particle comprising a recombinant protein of Pasteurella multocida toxin as described above.
  • the invention provides, in the fifth part, a porcine atrophic rhinitis immunological composition
  • a porcine atrophic rhinitis immunological composition comprising the recombinant protein of Pasteurella septicum toxin as described above and at least the viroid-like particle containing the recombinant protein of Pasteurella septicum toxin as described above.
  • a pharmaceutically acceptable carrier One of them, and a pharmaceutically acceptable carrier.
  • the invention provides the use of a porcine atrophic rhinitis immunological composition for the preparation of a medicament for combating atrophic rhinitis in pigs.
  • the present invention provides, in the seventh part, an antibody against the bacterium of the genus Pasteurella type D, which is a recombinant protein of Pasteurella septicum toxin as described above or a recombinant protein containing Pasteurella septicum as described above
  • the virus-like particles are prepared.
  • the invention provides a detection kit for porcine atrophic rhinitis in the eighth part, comprising a detecting unit, wherein the detecting unit is selected from the group consisting of at least one of the following groups: a septic cell as described above Recombinant protein of Bacillus toxin, a viroid-like particle containing a recombinant protein of Pasteurella septicum as described above, an antibody prepared by a recombinant protein of Pasteurella septicum as described above, and as before An antibody prepared by a viroid-like particle containing a recombinant protein of Pasteurella multocida toxin.
  • Figure 1 is an electron micrograph of a viroid-like particle (VLP) containing the recombinant protein of Pasteurella septicum toxin of the present invention in one embodiment. Arrows refer to a viroid-like particle of the invention. Scale bar: 50 ⁇ m.
  • Figure 2 shows the results of measuring the titer of anti-Septica toxin (PMT) antibody by enzyme-linked immunoassay (ELISA) in one example; the first group is the negative control group; the second group is the implementation The immunological composition of B. bronchiseptica (Bb), P. septicum type A (PmA) and P.
  • Bb B. bronchiseptica
  • PmA P. septicum type A
  • septicum D-type bacteria obtained in Example 2 (B.b+PmA+PmD) Group 3;
  • the third group is the viroid-like particle containing the recombinant protein of Pasteurella multocida toxin obtained in Example 1 (re-PmT VLP group);
  • the fourth group is the toxin-containing Pasteurella toxin obtained in the third embodiment
  • a porcine atrophic rhinitis immunocompetent composition of the virus-like particles of the recombinant protein (B.b+PmA+PmD+re-PmT VLP group);
  • Group 5 is a commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group).
  • the symbols * and ** represent significant differences from the first group (negative control group) (p ⁇ 0.05 and p ⁇ 0.01, respectively).
  • the symbols # and ## represent significant differences from the second group (B.b+PmA+PmD group) (p ⁇ 0.05 and p ⁇ 0.01, respectively).
  • the symbol ++ represents a significant difference (indicating p ⁇ 0.01) compared to the fifth group (commercially available vaccine group).
  • FIGS. 3A to 3C show the results of a neutralization antibody test analysis in one embodiment.
  • Figure 3A shows the cell morphology of the Vero cells cultured in DMEM containing fetal bovine serum (FBS) (negative control group);
  • Figure 3B shows the Pasteurella septicum containing 4 times the minimum toxic dose (MTD).
  • FBS fetal bovine serum
  • FIG. 3C shows the class with recombinant protein containing Pasteurella toxin Viral granules of porcine atrophic rhinitis immune composition (B.b+PmA+PmD+re-PmTVLP group) immunized mice were diluted 160-fold with 4-fold minimal toxic dose (MTD) of S. septicum toxin ( After neutralization of PMT), the cell morphology co-cultured with Vero cells was added; FIG.
  • 3D shows the dilution of the serum of the mouse immunized with the commercially available porcine atrophic rhinitis vaccine (commercial vaccine group) by 160-fold with a 4-fold minimum toxic dose ( After neutralizing the septic bacillary toxin (PMT) of MTD), the morphology of the cells co-cultured with Vero cells can still be seen in the cells showing typical nodule (as indicated by the arrow).
  • PMT septic bacillary toxin
  • the present invention provides a recombinant protein of Resin septicum toxin (re-PmT) comprising three epitopes of P. septicum toxin protein (PmT) to induce an anti-Septic bacillus An antibody to the toxin protein (PmT).
  • re-PmT Resin septicum toxin
  • the three epitopes are:
  • Epitope A SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO: 2);
  • Epitope B SISPDDPPREITD (SEQ ID NO: 3);
  • Epitope C LNSTPGTGRPMP (SEQ ID NO: 4).
  • the amino acid sequences of each of said epitopes may be further linked by a linker comprising at least one glycine (Glycine, Gly), said linkage Subunits include, but are not limited to, Gly-Gly, Gly-Ser, or sequences as set forth in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21.
  • the linker has the amino acid sequence set forth in SEQ ID NO:11. However, between the individual epitopes, the amino acid sequences are not necessarily linked by a linker.
  • recombinant S. septicum toxin (re-PmT) provided by the present invention can be represented by the following formula:
  • One of the epitopes 1, epitope 2, and epitope 3 has the amino acid sequence of SEQ ID NO: 2, and the other two epitopes have SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • Amino acid sequence; the linker 1 and the linker 2 are each independently selected from Gly-Gly, Gly-Ser, SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19;
  • n is an integer representing from 0 to about 10;
  • n represents an integer from 0 to about 10.
  • the S. septicum toxin recombinant protein (re-PmT) provided by the present invention has at least one of the amino acid sequences set forth in SEQ ID NOs: 5, 22, 23, 24, 25, and 26.
  • the recombinant S. septicum toxin recombinant protein (re-PmT) provided by the present invention has at least about 80% sequence homology with the amino acid sequence represented by the above formula (I), preferably, 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% , about 98%, about 99% sequence homology.
  • the present invention also provides a viroid-like particle (re-PmT VLP) containing a recombinant protein of Pasteurella septicum toxin, which is an insertion or substitution of an epitope (epitopes) of three P. septicum toxin proteins (PmT) Go to a major immunodominant region (MIR) of Hepatitis B virus core protein (HBc) to form a viroid-like particle containing recombinant protein of Pasteurella septicum (re-PmT VLP) ).
  • MIR major immunodominant region
  • HBc Hepatitis B virus core protein
  • Epitope A SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO: 2);
  • Epitope B SISPDDPPREITD (SEQ ID NO: 3);
  • Epitope C LNSTPGTGRPMP (SEQ ID NO: 4).
  • the hepatitis B virus core protein (HBc) has the amino acid sequence set forth in SEQ ID NO: 6.
  • the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 73 to 94 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 73 to 82 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at the 75th to 81th amino acid positions of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 78 to 79 of the protein.
  • the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 78 to 81 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 78 to 82 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 78 to 86 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 78 to 89 of the protein.
  • the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 78 to 94 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 81 to 82 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is at positions 82 to 83 of the protein.
  • the amino acid sequences of each of the epitopes and between the amino acid sequence of the epitope and the hepatitis B virus core protein (HBc) sequence may be further linked by a linker
  • the linker contains at least one glycine (Glycine, Gly) including but not limited to: Gly-Gly, Gly-Ser, SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21.
  • the linker has an amino acid sequence as set forth in SEQ ID NOs: 11 and/or 12.
  • each of said antigens is positioned between amino acid sequences and is not necessarily linked by a linker.
  • the virus-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin provided by the present invention can be represented by the following formula:
  • epitopes 1, epitope 2, and epitope 3 has the amino acid sequence of SEQ ID NO: 2, and the other two epitopes have SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Amino acid sequence;
  • the linker 1, the linker 2, the linker 3, and the linker 4 are each independently selected from Gly-Gly, Gly-Ser, SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21;
  • n is an integer representing from 0 to about 10;
  • n is an integer representing from 0 to about 10;
  • p is an integer representing from 0 to about 10;
  • q is an integer representing from 0 to about 10.
  • the N-terminal portion of the hepatitis B virus core protein (HBc-N-terminal segment) has the sequence of amino acid 1 to 73 amino acids
  • the C-terminal portion of hepatitis B virus core protein (HBc- The C-terminal segment has a sequence of 83 to 144 amino acids of the HBc protein.
  • the HBc-N terminus has a sequence of from 1 to 75 amino acids of the HBc protein
  • the HBc-C terminus has a sequence of from 82 to 144 amino acids of the HBc protein.
  • the HBc-N terminus has a sequence of from 1 to 78 amino acids of the HBc protein, and the HBc-C terminus has a sequence of from 79 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has a sequence of from 1 to 78 amino acids of the HBc protein, and the HBc-C terminus has a sequence of from 82 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has a sequence of 1 to 78 amino acids of the HBc protein, and the HBc-C terminus has a sequence of 83 to 144 amino acids of the HBc protein.
  • the HBc-N terminus has a sequence of 1 to 78 amino acids of the HBc protein, and the HBc-C terminus has a sequence of 87 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has a sequence of from 1 to 78 amino acids of the HBc protein, and the HBc-C terminus has a sequence of from 90 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has a sequence of from 1 to 78 amino acids of the HBc protein, and the HBc-C terminus has a sequence of from 95 to 144 amino acids of the HBc protein.
  • the HBc-N terminus has a sequence of from 1 to 81 amino acids of the HBc protein, and the HBc-C terminus has a sequence of from 82 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has a sequence of 1 to 82 amino acids of the HBc protein, and the HBc-C terminus has a sequence of 83 to 144 amino acids of the HBc protein.
  • a viroid-like particle comprising a recombinant protein of Pasteurella multocida toxin provided by the present invention has SEQ ID NOs: 9, 10, 27, 28, 29, 30, 31 At least one of the amino acid sequences shown in 32, 33, 34, 35, and 36.
  • the viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin provided by the present invention has at least about 80% sequence homology with the amino acid sequence represented by the above formula (II). , preferably, has about 85% sequence homology, more preferably, has about 90% sequence homology, even about 91%, about 92%, about 93%, about 94%, about 95%, Approximately 96%, approximately 97%, approximately 98%, approximately 99% sequence homology.
  • the present invention also provides a nucleic acid sequence encoding the recombinant protein of Resin septicum toxin (re-PmT) of the present invention and a viroid-like particle (re-PmT) encoding the recombinant protein of Pasteurella multocida toxin of the present invention.
  • Nucleic acid sequence of VLP Nucleic acid sequence of VLP.
  • the S. septicum toxin recombinant protein (re-PmT) comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4.
  • viroid-like particle comprising the recombinant protein of Pasteurella septicum toxin comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4 and a SEQ ID NO: 6 Hepatitis B virus core protein (HBc), wherein the antigen is positioned to insert or replace the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc).
  • MIR major immunodominant region
  • the nucleic acid sequence is the amino acid sequence of the recombinant protein of Resin septicum toxin (re-PmT) of the present invention, and the amino acid of the viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella multocida toxin of the present invention, respectively.
  • the sequence is derived.
  • the present invention can be obtained by a nucleotide sequence encoding the amino acid listed in the genetic code table (including various degenerate codons, or synonymous codons).
  • septicum toxin recombinant protein (re-PmT) of the present invention and the serine of the amino acid sequence of the viroid-like particle (re-PmT VLP) containing the recombinant protein of the septicum Pasteurella toxin of the present invention are serine. It can be encoded by nucleotide sequences such as TCT, TCC, TCA, TCG, AGT, AGC. Each of the amino acids in the amino acid sequence of the S.
  • septicum toxin recombinant protein (rPMT) of the present invention and the viroid-like particle (re-PmT VLP) of the recombinant protein of the septicum Pasteurella toxin of the present invention may be each of the following nucleosides
  • the acid sequence is encoded:
  • the present invention also provides a porcine atrophic rhinitis immunological composition.
  • the porcine atrophic rhinitis immunocomplex comprises a S. septicum toxin recombinant protein (re-PmT) and/or a viroid-like particle (re-PmT VLP) containing a recombinant protein of S. septicum toxin.
  • the S. septicum toxin recombinant protein (re-PmT) comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4.
  • viroid-like particle comprising the recombinant protein of Pasteurella septicum toxin comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4 and a SEQ ID NO: 6 Hepatitis B virus core protein (HBc), wherein the antigen is positioned to insert or replace the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc).
  • MIR major immunodominant region
  • the S. septicum toxin recombinant protein has the amino acid sequence set forth in SEQ ID NOs: 5, 22, 23, 24, 25 or 26.
  • the viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella septicum has the amino acid sequence set forth in SEQ ID NO: 9.
  • the viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella multocida toxin has the amino acid sequence set forth in SEQ ID NO: 10.
  • the viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella multocida toxin has SEQ ID NOs: 27, 28, 29, 30, 31, 32, 33, 34, One of the amino acid sequences shown in 35 and 36.
  • the recombinant septicum toxin recombinant protein (re-PmT) provided by the present invention and the viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin include, but are not limited to, genetically selected or succeeded Synthesized by a peptide synthesizer; the manner of obtaining the recombinant protein by gene selection may be, but not limited to, a nucleic acid sequence or encoding encoding a recombinant protein of Resin septicum toxin (re-PmT)
  • the nucleic acid sequence of the viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella multocida toxin is selected into a expression vector, each of which forms a nucleic acid sequence containing a recombinant protein (re-PmT) encoding a Pasteurella septicum toxin a
  • 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), true Nuclear expression systems (eg animal cells (insect cells or mammalian cells), plant cells).
  • the porcine atrophic rhinitis immune composition provided by the present invention further comprises B. bronchiseptica, P. septicum type A (PmA), and Pasteurella septicum Type D bacteria (PmD).
  • the source of the B. bronchiseptica may be, for example, but not limited to, the American Type Culture Collection (ATCC) number ATCC 31437, the Pasteurella septicum type A
  • the source of the bacterium (PmA) may be, for example, but not limited to, the National Collection of Type Cultures (NCTC) number NCTC 12177, and the source of the S. septicum D-type bacterium (PmD). It may be, for example, but not limited to, a strain such as the British National Standards Collection of Biological Products (NCTC) number NCTC 12178, or a strain derived from field isolation.
  • the porcine atrophic rhinitis immune composition provided by the present invention may further comprise other pathogenic antigens, including but not limited to: porcine circovirus type 2 (PCV2) antigen, swine influenza virus (SIV) antigen, pig Reproductive and Respiratory Syndrome Virus (PRRSV) antigen, Mycoplasma, Parvovirus (PPV), Erysipelas, Aujeszky's disease, and/or Actinobacillus pneumoniae (actinobacillus) Pleuropneumonia, APP).
  • PCV2 porcine circovirus type 2
  • SIV swine influenza virus
  • PRRSV pig Reproductive and Respiratory Syndrome Virus
  • PSV Parvovirus
  • Erysipelas Erysipelas
  • Aujeszky's disease and/or Actinobacillus pneumoniae (actinobacillus) Pleuropneumonia, APP).
  • 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 a futon.
  • pharmaceutically acceptable carriers including: a solvent, an emulsifier, a suspending agent, a decomposing agent, a binder, and a futon.
  • the pharmaceutically acceptable carrier comprises one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, binding agents. , excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, interfacial activity 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 produce harmful effects with the active composition. reaction.
  • Suitable excipients include, but are not limited to, water, salt solutions, vegetable oils, polyethylene glycol, gelatin, amylose, lactose, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycan Esters and glycerol, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.
  • the pharmaceutically acceptable adjuvants include, but are not limited to, aqueous aluminum hydroxide gum, alum, Freund's incomplete adjuvant, oil adjuvant, water soluble adjuvant, or water-in-water-in-water two-phase adjuvant (water-in-oil-in-water, W/O/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 antibody against Pasteurella septicum D-type toxin (PmT) which is a recombinant protein of septice septicum toxin (re-PmT) and/or contains The virus-like particles (re-PmT VLP) of the recombinant protein of the Pasteurella toxin are prepared or derived; and the antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.
  • the antibody is a polyclonal 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, wherein the test kit is used for detecting whether a test sample contains a septicemia type D-toxin (PmT) or detecting whether a test sample contains a resistance An antibody to Pasteurella multocida D-type toxin (PmT).
  • PmT septicemia type D-toxin
  • the detection kit comprises, but is not limited to: (1) an antigen which is a recombinant protein of septice septicum toxin (re-PmT) provided by the present invention and/or recombinant with septicemia a virus-like particle of a protein (re-PmT VLP), in one embodiment, the antigen is placed on an antigenic disk; and/or (2) an antibody that is septically provided by the present invention A monoclonal antibody or a polyclonal antibody derived from a recombinant protein of the Pasteurella toxin (re-PmT) and/or a viroid-like particle (re-PmT VLP) containing the recombinant protein of the Pasteurella septicum toxin.
  • an antigen which is a recombinant protein of septice septicum toxin (re-PmT) provided by the present invention and/or recombinant with septicemia a virus-like particle of a protein (re-P
  • the form of the detection kit includes, but is not limited to, an enzyme-linked immu sorbent assay (ELISA) kit, a microchip assay kit (Microchip kit), and an immunofluorescence assay (IFA) assay.
  • ELISA enzyme-linked immu sorbent assay
  • Microchip kit microchip assay kit
  • IFA immunofluorescence assay
  • a kit, or other test kit prepared by the recombinant S. septicum toxin recombinant protein (re-PmT) and/or a viroid-like particle (re-PmTVLP) containing a recombinant protein of S. septicum toxin.
  • the detection kit comprises at least one virus-like particle comprising a recombinant protein of septice septicum toxin (re-PmT) provided by the present invention and/or a recombinant protein containing Pasteurella septicum toxin ( The antigenic disk of re-PmT VLP) can be used to test whether the sample contains antibodies against P. septicum toxin (PmT).
  • re-PmT septice septicum toxin
  • PmT VLP recombinant protein containing Pasteurella septicum toxin
  • PmT P. septicum toxin protein
  • Epitope A SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO: 2);
  • Epitope B SISPDDPPREITD (SEQ ID NO: 3);
  • Epitope C LNSTPGTGRPMP (SEQ ID NO: 4).
  • each of the epitopes A (SEQ ID NO: 2), epitope B (SEQ ID NO: 3), and epitope C (SEQ ID NO: 4) are each joined by a linker, the linkage
  • the subunit has the amino acid sequence shown as SEQ ID NO:11.
  • the amino acid sequence of the recombinant septicum toxin recombinant protein (re-PmT) is SEQ ID NOs: 5, 22, respectively.
  • the amino acid sequence is synthesized by a synthesizer or obtained by gene expression.
  • S. septicum toxin recombinant protein (SEQ ID NO: 5) was inserted into the 78th and 79th amino acids of hepatitis B virus core protein (HBc) (SEQ ID NO: 6). Between the N-terminal segment of hepatitis B virus core protein (HBc) (1st to 78th amino acids; SEQ ID NO: 7) and the recombinant protein of septicemia toxin (re-PmT) (SEQ ID NO) :5), and the S.
  • septicum toxin recombinant protein (SEQ ID NO: 5) and the hepatitis B virus core protein (HBc) C-terminal segment (79-144 amino acids; SEQ ID Between NO: 8), each is ligated with a linker having the amino acid sequence set forth in SEQ ID NO: 12.
  • the amino acid sequence of the viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella multocida toxin is shown in SEQ ID NO: 9.
  • the amino acid sequence can be synthesized by a synthesizer, or a nucleic acid sequence encoding the amino acid sequence can be synthesized first, and the nucleic acid sequence can be selected into a expression vector, which is expressed and purified in a biological expression host.
  • the above-mentioned viroid-like particles (re-PmT VLP) containing the recombinant protein of Pasteurella multocida toxin can also be obtained by gene colonization expression.
  • a nucleic acid sequence encoding a viroid-like particle (re-PmT VLP) containing a recombinant protein of Pasteurella septicum toxin is constructed by gene selection, each fragment can be ligated using a restriction enzyme cleavage site.
  • MCS multiple restriction sites
  • a restriction enzyme cleavage site such as HindIII and KpnI is ligated to each of the above nucleic acid sequences to construct a nucleic acid sequence encoding a viroid-like particle (re-PmT VLP) encoding a recombinant protein containing Pasteurella septicum toxin, and then containing the nucleic acid sequence.
  • the vector is transfected into a biological expression host, and the protein-like expression results in a viroid-like particle (re-PmT VLP) containing a recombinant protein of Pasteurella septicum toxin, the amino acid sequence of which is shown in SEQ ID NO: 10.
  • virus-like particles containing the recombinant protein of Pasteurella septicum toxin were purified, and the protein concentration was adjusted to 1 mg/ml, and then filtered through a 0.22 mm filter to obtain a JEM-1400 electron microscope (JEOL Co., Ltd.). , Japan) for negative staining observation.
  • a carbon-coated copper grid of 300 mesh was prepared, and then 8 ml of the sample was dropped on each copper wire, and after standing for 3-5 minutes, the excess liquid was sucked off with a filter paper, followed by a 1% uranium acetate solution.
  • the viroid-like particles (re-PmT VLP) containing the recombinant protein of Pasteurella septicum in the present invention exhibit a virus-like particle shape (as indicated by an arrow in Fig. 1).
  • B. bronchiseptica was inoculated on TSB solid medium [containing 5% (v/v) yeast extract, 10% (v/v) serum, soy protein) Casein medium (tryptic soy broth, TSB, BD, USA)], after overnight incubation at 37 ° C, select a single colony inoculated in brain heart infusion (BHI) liquid medium (BD company, USA) ), incubated at 37 ° C overnight; then the inoculum was inoculated in BHI liquid medium, shake culture overnight at 37 ° C, and calculate the colony forming unit (CFU) value; finally added formaldehyde (formaldehyde ), the bacteria liquid is inactivated by shaking at room temperature for 24 to 36 hours.
  • BHI brain heart infusion
  • CFU colony forming unit
  • Pasteurella multocida type A bacteria (PmA) and the septic-producing Pasteurella type D-type bacteria (PmD) were inoculated on 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, select a single colony inoculated in brain heart extract (BHI) liquid medium (BD, USA), 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 units (CFU Value; finally added formaldehyde to inactivate the bacterial liquid.
  • TSB solid medium containing 5% (v/v) yeast extract) , 10% (v/v) serum, soy protein degraded protein casein medium (TSB, BD, USA)
  • BHI brain heart extract
  • the recombinant S. septicum toxin recombinant protein (re-PmT) (SEQ ID NO: 5) obtained in Example 1 (final concentration is 250 ⁇ g/ml) or viroid-like particles containing the recombinant protein of S. septicum toxin ( re-PmT VLP) (SEQ ID NO: 10) (final concentration 250 ⁇ g/ml) and inactivated B.
  • bronchiseptica obtained in Example 2 (final concentration 1 ⁇ 10 9 CFU/ M), inactivated Pasteurella multocida type A (PmA) (final concentration 1x10 9 CFU/ml) and inactivated Pasteurella septicum type D (PmD) (final concentration 1x10 9 CFU/ Ml) was uniformly mixed with a phosphate buffered solution (PBS), and an aluminum gel [final concentration of 30% (v/v)] was added as an adjuvant to prepare a porcine atrophic rhinitis immunological composition.
  • PBS phosphate buffered solution
  • mice (Laboratory Animal Center, Taiwan), which were negative for 3 weeks old with Pasteurella septicum antibody, were randomly divided into 5 groups; Group 1 was the control group, and Groups 2-5 were the immunoassay group; Each mouse was injected with 0.2 ml of the following substances by intraperitoneal injection (ip.):
  • Group 1 PBS buffer solution containing 30% (v/v) aluminum gel (negative control group);
  • Group 2 B.bronchiseptica (1x10 9 CFU/ml), Pasteurella multocida type A (PmA) (1x10 9 CFU/ml) and defeated in Example 2 Immunological composition of Pasteurella pneumoniae type D (PmD) (1x10 9 CFU/ml) (containing 30% (v/v) aluminum gel adjuvant) (B.b+PmA+PmD group);
  • Group 3 viroid-like particles (SEQ ID NO: 10) of the obtained recombinant protein of Pasteurella septicum toxin obtained in Example 1 (concentration: 250 ⁇ g/ml) (re-PmT VLP group);
  • Group 4 Porcine atrophic rhinitis immunological composition (B.b+PmA+PmD+re-PmT) of the viroid-like particle (SEQ ID NO: 10) containing the recombinant protein of Pasteurella septicum toxin obtained in Example 3. VLP group); and
  • Group 5 Commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group); each dose (2 ml) contains: B. burgdorferi Bb-1 type NO. 12-1 strain inactivated bacteria (before inactivation) ⁇ 1x109 CFU , Inactivated bacteria of Pasteurella multocida type A Pm-A-8 strain (before inactivation) ⁇ 1x10 9 CFU, Pasteurella septicum D-type strain Pm-D-8 strain inactivated bacteria (inactivated Pre)) ⁇ 1x10 9 CFU, porcine septicum toxin gene Tox1 transformed E. coli BL21/rsPMT/Tox1 strain recombinant toxin (P.
  • Each mouse was collected for blood collection 24 hours before the first immunization (Day 0). After the initial immunization (Day 1), blood was collected again on the 13th day, and the second immunization was performed at the same immunization dose on the 14th day. Blood was collected again on the 24th day. The serum in the blood sample is separated for Enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody assay.
  • ELISA Enzyme-linked immunosorbent assay
  • S. septicum toxin (PMT) (Abcam, USA) 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. hour. After removing excess antigen, add washing buffer (wash buffer; 0.9% NaCl; 0.1% Tween 20), wash 3 times, and then dry. Then, blocking buffer (wash buffer containing 1% BSA) was added, and after standing at room temperature for 1 hour, it was washed with washing buffer, and then the serum samples collected from the above groups of mice were diluted with PBS buffer solution.
  • washing buffer 0.9% NaCl; 0.1% Tween 20
  • blocking buffer (wash buffer containing 1% BSA) was added, and after standing at room temperature for 1 hour, it was washed with washing buffer, and then the serum samples collected from the above groups of mice were diluted with PBS buffer solution.
  • mice 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-mouse calibrated with horseradish peroxidase (HRP) was added.
  • Secondary antibody goat anti-mouse conjugated HRP, Gene Tex, USA
  • HRP horseradish peroxidase
  • the secondary antibody was diluted 5,000 times in blocking buffer and then added to a 96-well plate (100 ⁇ l/well) and allowed to stand at room temperature. After the hour, the secondary antibody was removed and washed with washing buffer, and 100 ⁇ l of 3,3',5,5'-tetramethylbenzidine dihydrochloride (3,3',5,5'-tetramethylbenzidine was added to each well.
  • TMB TMB, KPL, USA
  • enzyme-linked immunoassay tester M2/M2 ELISA Reader, Molecular Devices, Inc., USA
  • mice immunized with a porcine atrophic rhinitis immunocompetent composition (group 4, ie, B.b+PmA+PmD+re-PmT VLP group) containing viroid-like particles of the recombinant protein of Pasteurella toxin
  • the serum containing the anti-Septic Pasteurin toxin (PMT) antibody has the highest titer, followed by the viroid-like particles of the recombinant protein of the Pasteurella septicum toxin obtained in the first embodiment (Group 3, ie, the re-PmT VLP group).
  • mice serum; and immunosuppressed porcine atrophic rhinitis vaccine (Group 5, commercially available vaccine group) containing anti-Septic Pasteurin toxin (PMT) antibody titer, and immunization
  • the mouse serum of B. bronchiseptica, B. septicum type A and S. septicum D type bacteria (group 2, namely B.b+PmA+PmD group) obtained in Example 2
  • the titer of anti-Septic Pasteurin toxin (PMT) antibody contained in the serum of each immunoassay group (Groups 2-5) was higher than that of the mouse serum of Group 1 (negative control group).
  • virus-like particle (re-PmT VLP) of the recombinant protein of Pasteurella septicum toxin provided by the present invention can effectively induce anti-Septic Pasteurella toxin (PMT) antibody in an animal, and has an immunogen Sex, and the immune effect is better than the commercial atrophic rhinitis vaccine.
  • a green aphid kidney cell (Vero cell) was used as a test material to test a porcine atrophic rhinitis immune composition that immunizes a viroid-like particle containing a recombinant protein of Pasteurella multocida toxin (Group 4, ie B.b+PmA+PmD+ Whether the anti-Septic Pasteurin toxin (PMT) antibody contained in the mouse serum of the re-PmT VLP group is a neutralizing antibody capable of neutralizing PMT toxicity.
  • the minimum toxin dose (MTD) of Vero cells was determined prior to the neutralization antibody titer test.
  • the Vero cells were seeded in a 96-well culture dish. After the cells were grown into a single layer, the culture solution was removed, and the septicemia pasteurin toxin (PMT, diluted with serum-free DMEM medium (GIBCO, USA) was added. Abcam) (0, 20, 50, 100 ng) for processing.
  • the DMEM medium containing fetal bovine serum (FBS) was used as a negative control group, and the type change of the cells was observed after culture to induce the lowest septic Pasteurella toxin of Vero cells producing cytopathic effect (CPE).
  • the PMT) concentration is the minimum toxic dose (MTD). The results of the test showed that the minimal toxic dose of S. septicum toxin (PMT) of Vero cells was 50 ng.
  • mice sera of a porcine atrophic rhinitis immunological composition (Group 4, B.b+PmA+PmD+re-PmT VLP group) immunized with viroid-like particles containing a recombinant protein of Pasteurella septicum toxin, respectively
  • the mouse serum of the immunized commercial atrophic rhinitis vaccine (Group 5, commercially available vaccine group) was diluted 10-fold, and then serially diluted (40, 80, 120, 160, 200, 240 times).
  • the above diluted mouse serum was separately added to a 96-well culture dish, and a septicemia pasteurin toxin (PMT) containing 4 times the minimum toxic dose (MTD) was added to each well, and then placed at 37 ° C for reaction 1 hour.
  • PMT septicemia pasteurin toxin
  • MTD minimum toxic dose
  • the above reaction solution was added to Vero cells cultured in a 96-well plate, and after culturing at 37 ° C in a 5% CO 2 incubator, it was observed whether the serum could inhibit Vero cell-producing cytopathic effect (CPE).
  • CPE Vero cell-producing cytopathic effect
  • Vero cells were treated with S. septicum toxin (PMT) containing 4 times the minimum toxic dose (MTD) as a positive control group.
  • the cell morphology was as shown in Fig. 3B. It can be seen that the cells showed typical nodule (Fig. 3B arrow)).
  • Vero cells were cultured in DMEM medium containing fetal bovine serum (FBS) as a negative control group, and the cell morphology was as shown in Fig. 3A; and the virus-like particles containing the recombinant protein of Pasteurella septicum toxin were immunized.
  • FBS fetal bovine serum
  • the porcine atrophic rhinitis immune composition (Group 4, ie B.b+PmA+PmD+re-PmT VLP group) was diluted 160-fold in serum and 4 times the minimum toxic dose (MTD) of septic Pap. After neutralization of the bacillus toxin (PMT), the morphology of the cells co-cultured with Vero cells is shown in Figure 3C. The mice were incubated with the sera of the porcine atrophic rhinitis vaccine (Group 5, the commercially available vaccine group) diluted 160-fold and neutralized with 4 times the minimum toxic dose (MTD) of S. septicum toxin (PMT).
  • MTD minimum toxic dose
  • Fig. 3D the morphology of the cells co-cultured with Vero cells was as shown in Fig. 3D.
  • the cells shown in Fig. 3A and Fig. 3C did not show a typical nodule as shown in Fig. 3B, and Fig. 3D still had a few nodular cell morphology, showing that the virus containing the recombinant protein of Pasteurella septicum was immunized.
  • porcine atrophic rhinitis immunological composition Group 4, ie B.b+PmA+PmD+re-PmT VLP group
  • porcine atrophic rhinitis vaccine Group 5, ie market
  • the mouse serum of the vaccine group contains neutralizing antibodies against Pasteurella multocida (PMT), and the former has a higher neutralizing antibody content than the latter.
  • PMT Pasteurella multocida
  • Example 5 Immunogenicity and Protective Efficacy of Porcine Atrophic Rhinitis Immunological Composition Containing Reticulum-like Particles of Recombinant Protein of Pasteurella toxin (re-PmT VLP) 1 - Taiwan Pig Atrophic Rhinitis Vaccine Test Standard ( Pasteurella efficacy test)
  • the BALB/c mice (Laboratory Animal Center, Taiwan) with negative antibody to Pasteurella septicum were randomly divided into 3 groups, the first group was the control group, and the second group was the second group.
  • the third group was the commercially available vaccine immunoassay group; each mouse was injected intraperitoneally (ip.) with 0.5 ml of 10-fold diluted test substance.
  • Each group is:
  • Group 1 PBS buffer solution containing 30% (v/v) aluminum gel (control group);
  • Group 2 Porcine atrophic rhinitis immunocomb composition (B.b+PmA+PmD+re-PmT VLP) of the viroid-like particle (SEQ ID NO: 10) containing the recombinant protein of Pasteurella septicum toxin obtained in Example 3. Group); and
  • Group 3 Commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group), and the composition is as described in Example 4.
  • 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 pneumoniae type D (PmD) with the ability to produce toxin were respectively
  • PmD Pasteurella pneumoniae type D
  • Example 2 0.1 ml of a live bacterial solution of 1 ⁇ 10 6 CFU/ml, 1 ⁇ 10 7 CFU/ml, and 1 ⁇ 10 8 CFU/ml was intraperitoneally injected.
  • mice were also divided into three groups, according to the order of the Pasteurella virulent strains 1x10 5 CFU / ml, 1x10 6 CFU / ml, 1x10 7 CFU / ml three concentrations of live bacteria 0.1 ml intraperitoneal injection.
  • the LD 50 of each immunoassay group and the control group were calculated by Bekas's method (Beherens-Karber), and the defense index of each immunoassay group was higher than that of the control group by 1 ⁇ 10 0.5 or more.
  • Beka II method of defense index calculation is as follows:
  • LD 50 minimum dilution factor for the challenge dose - [(sum of deaths per group / 100) - 0.5] x 1
  • the porcine atrophic rhinitis immunological composition containing the viroid-like particles of the recombinant protein of Pasteurella multocida (group 2, namely, B.b+PmA+PmD+re-PmT VLP group) was able to Induces the protective effect of mice, and is resistant to the virulent strain of Pasteurella multocida type D (PmD), and its defense index is greater than 1x10 2.8 , higher than the Taiwan test standard (1x10 0.5 ) and the shrinking of commercial pigs.
  • the defensive index of the rhinitis vaccine (commercial vaccine group) (1x10 2.5 ).
  • mice (Laboratory Animal Center, Taiwan) with negative antibody to Pasteurella septicum and weighing 15-20 g were randomly divided into 3 groups, the first group was the control group, and the second and third groups were the immunoassay group.
  • Each immunoassay was subdivided into 3 groups; 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 viroid-like particles of the recombinant protein of Pasteurella septicum (SEQ ID NO: 10) obtained in Example 3 (B. b+PmA+PmD+re-PmT VLP group);
  • Group 2-2 Each mouse was injected with 0.2 ml of the porcine atrophic rhinitis immunological composition containing the viroid-like particle (SEQ ID NO: 10) containing the recombinant protein of Pasteurella septicum toxin obtained in Example 3, which was diluted 5 times. (1/5B.b+PmA+PmD+re-PmT VLP group);
  • Group 2-3 Each mouse was injected with 0.2 ml of a 25-fold diluted porcine atrophic rhinitis immunological composition containing the viroid-like particle of the recombinant protein of Pasteurella septicum (SEQ ID NO: 10) obtained in Example 3. (1/25B.b+PmA+PmD+re-PmT VLP group);
  • Group 3-1 Each mouse was injected with 0.2 ml of a commercially available porcine atrophic rhinitis vaccine stock solution as described in Example 4 (commercially available vaccine group);
  • Group 3-2 Each mouse was injected with 0.2 ml of a 5-fold diluted commercial atrophic rhinitis vaccine, and the composition was as described in Example 4 (1/5 commercial vaccine group);
  • Groups 3-3 Each mouse was injected with 0.2 ml of a 25-fold diluted commercial atrophic rhinitis vaccine, and the composition was as described in Example 4 (1/25 commercially available vaccine group).
  • the immunization test group (groups 2 and 3) was immunized twice on the 14th day after the initial 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
  • the challenge test was carried out for 10 days.
  • Each mouse was injected intraperitoneally with 0.2 ml of a virulent strain of Pasteurella multocida strain D (PmD) with the ability to produce toxin [Same Example 2] 100LD 50 live bacteria. The survival rate was recorded for 10 days.
  • PmD Pasteurella multocida strain D
  • the survival rate of the stock immunization group (group 2-1, group 3-1) must be higher than 80%, and the survival rate of the 5-fold diluted immunization group (groups 2-2, 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.
  • porcine atrophic rhinitis immunocomplexes containing viroid-like particles of the recombinant protein of Pasteurella toxin (Group 2-1, Groups 2-2, 2 Group 3) did induce sufficient protective efficacy in mice, and the survival rate of mice immunized with vaccines, mice immunized with 5-fold vaccine, and mice immunized with 25-fold vaccine were all 100%. The standard of survival.
  • the virus-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin obtained in Example 1 is mixed with a suitable adjuvant (for example, aluminum glue) and then administered to an animal (eg, mouse, rat) , pigs, goats, rabbits) for primary immunization, after appropriate time interval (eg: 2 to 3 weeks), secondary immunization may be required as needed. After appropriate time interval (eg, 2 to 3 weeks), the serum of immunized animals (eg, mice, rats, pigs, goats, rabbits) is collected to obtain more anti-Septica toxin (PmT) Cloning antibodies.
  • a suitable adjuvant for example, aluminum glue
  • the polyclonal antibody of the anti-Septic Pasteurella toxin (PmT) may be combined with a developer or fluorescence as needed.
  • the number of immunizations may be increased as needed to increase the antibody titer.
  • mice mice, rats, rabbits, poultry (eggs), pigs, goats, cattle, and aquatic animals.
  • the virus-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin obtained in Example 1 is mixed with a suitable adjuvant (for example, aluminum glue) and then administered to an animal (eg, mouse, rat) , pigs, goats, rabbits) for primary immunization, after appropriate time interval (eg: 2 to 3 weeks), secondary immunization may be required as needed. After appropriate time intervals (eg, 2 to 3 weeks), serum from immunized animals (eg, mice) is collected for evaluation of mice suitable for collecting spleen cells.
  • a suitable adjuvant for example, aluminum glue
  • the spleen cells and the myeloma cells are collected from the suitable mouse for cell fusion with PEG (Polyethylene Glycol, such as PEG 1500). After screening for a secreted fusion tumor from a fused cell and monoculture, a conjugated cell strain suitable for producing a monoclonal antibody against P. septicum toxin (PmT) can be obtained.
  • PEG Polyethylene Glycol, such as PEG 1500
  • the antibody obtained by the above preparation can be used in an immunoassay reagent, a therapeutic agent, or added to foods and feeds to make the consumer immune.

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Abstract

提供一种败血性巴氏杆菌毒素重组蛋白，其具有三个败血性巴氏杆菌毒素蛋白的抗原决定位。所述三个抗原决定位分别具有如SEQ ID NOs:2、3以及4所示的序列。还提供一种含有所述败血性巴氏杆菌毒素重组蛋白的类病毒颗粒、编码所述败血性巴氏杆菌毒素重组蛋白或编码所述含有所述败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的核酸序列，以及含有所述败血性巴氏杆菌毒素重组蛋白及/或所述含有所述败血性巴氏杆菌毒素重组蛋白的类病毒颗粒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、其类病毒颗粒及其应用 技术领域
本发明关于败血性巴氏杆菌毒素重组蛋白，特别是关于含有败血性巴氏杆菌毒素抗原决定位的重组蛋白，以及含有所述重组蛋白的类病毒颗粒。
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。
猪萎缩性鼻炎(AR)是由支气管败血性博德氏杆菌(Bordetella bronchiseptica,B.b)以及败血性巴氏杆菌(Pasteurella multocida)A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(Pasteurella 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
本发明在第一部分提供一种败血性巴氏杆菌(Pasteurella multocida)毒素重组蛋白，包含：一具有如SEQ ID NO:2所示氨基酸序列的败血性巴氏杆菌毒素蛋白的抗原决定位(epitopes)、一具有如SEQ ID NO:3所示氨基酸序列的败血性巴氏杆菌毒素蛋白的抗原决定位，以及一具有如SEQ ID NO:4所示氨基酸序列的败血性巴氏杆菌毒素蛋白的抗原决定位。
本发明在第二部分提供一种含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(virus like particle,VLP)，包含：一如前所述的败血性巴氏杆菌毒素重组蛋白以及一乙型肝炎病毒核心蛋白(Hepatitis B virus core protein,HBc)；其中所述的败血性巴氏杆菌 毒素重组蛋白插入所述乙型肝炎病毒核心蛋白的主要免疫显性区域(Major immunodominant region,MIR)。
本发明在第三部分提供一种编码如前所述的败血性巴氏杆菌毒素重组蛋白的核酸序列。
本发明在第四部分提供一种编码如前所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的核酸序列。
本发明在第五部分提供一种猪萎缩性鼻炎免疫组合物，包含如前所述的败血性巴氏杆菌毒素重组蛋白以及如前所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒至少其中一种，以及一药学上可接受的载体。
本发明在第六部分提供一种猪萎缩性鼻炎免疫组合物用于制备动物对抗猪萎缩性鼻炎的药物的应用。
本发明在第七部分提供一种抗败血性巴氏杆菌D型菌毒素的抗体，是通过如前所述的败血性巴氏杆菌毒素重组蛋白或如前述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒所制备而得。
本发明在第八部分提供一种猪萎缩性鼻炎的检测试剂盒，包含一侦测单元，所述侦测单元选自于下列群组所组成中至少一者：一如前所述的败血性巴氏杆菌毒素重组蛋白、一如前所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒、一如前所述的败血性巴氏杆菌毒素重组蛋白所制备的抗体，以及一如前所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒所制备的抗体。
本发明以下面的实施例及所附图式予以示范阐明，但本发明不受下述实施例所限制。
附图说明
图1所示为在一实施例中，含有本发明的败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(VLP)的电子显微镜照片。箭头所指为本发明的一个类病毒颗粒。比例尺：50μm。
图2所示为在一实施例中，以酵素连结免疫分析(ELISA)测定抗败血性巴氏杆菌毒素(PMT)抗体效价的结果；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(B.b)、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(B.b+PmA+PmD组)；第3组为实施例一所得的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP组)；第4组为实施例三所得的具有含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(B.b+PmA+PmD+re-PmT VLP组)；第5组为市售猪萎缩性鼻炎疫苗(市售疫苗组)。符号*与**代表与第1组(负对照组)相比具有显著差异(分别表示p<0.05与p<0.01)。符号#与##代表与第2组(B.b+PmA+PmD组)相比具有显著差异(分别表示p<0.05与p<0.01)。符号++代表与第5组(市售疫苗组)相比具有显著差异(表示p<0.01)。
图3A至图3C所示为在一实施例中，中和抗体试验分析结果。图3A所示为以含有胎牛血清(FBS)的DMEM培养液培养Vero细胞的细胞形态(负对照组)；图3B所示为以含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(如箭头所示)；图3C所示为以具有含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(B.b+PmA+PmD+re-PmTVLP组)免疫的小鼠血清稀释160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3D所示为以市售猪萎缩性鼻炎疫苗(市售疫苗组)免疫的小鼠血清稀释160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，依然可以见到细胞呈现典型的结节样(如箭头所示)。
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(re-PmT)，包含三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。所述三个抗原决定位分别是：
抗原决定位A:SVGKEGAYYPDHDYGPEYNPVWGPNEQI(SEQ ID NO:2)；
抗原决定位B:SISPDDPPREITD(SEQ ID NO:3)；以及
抗原决定位C:LNSTPGTGRPMP(SEQ ID NO:4)。
在某些优选的实施例中，各个所述的抗原决定位的氨基酸序列之间可以进一步由连接子(linker)连接，所述连接子至少含有一个以上的甘氨酸(Glycine,Gly)，所述连接子包含但不限于：Gly-Gly、Gly-Ser，或如SEQ ID NOs:11、12、13、14、15、16、17、18、19、20、21所示的序列。在一实施例中，所述连接子具有如SEQ ID NO:11所示的氨基酸序列。然而，各个所述抗原决定位氨基酸序列之间，不必然由连接子所连接。
本发明所提供的败血性巴氏杆菌毒素重组蛋白(re-PmT)，可以由下式表示：
(抗原决定位1)-(连接子1) m-(抗原决定位2)-(连接子2) n-(抗原决定位3)  式(I)
所述抗原决定位1、抗原决定位2、抗原决定位3其中一个具有如SEQ ID NO:2的氨基酸序列，其他二个抗原决定位分别具有如SEQ ID NO:3及SEQ ID NO:4的氨基酸序列；所述连接子1以及连接子2各自独立选自于Gly-Gly、Gly-Ser、SEQ ID NOs:11、12、13、14、15、16、17、18、19；
其中m是代表从0至约10的整数；
其中n是代表从0至约10的整数。
在某些实施例中，本发明提供的败血性巴氏杆菌毒素重组蛋白(re-PmT)具有如SEQ ID NOs:5、22、23、24、25以及26所示的氨基酸序列至少一种。
在部分实施态样中，本发明所提供的败血性巴氏杆菌毒素重组蛋白(re-PmT)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，优选的，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。
本发明并且提供一种含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)，是将三个败血性巴氏杆菌毒素蛋白(PmT)的抗原决定位(epitopes)插入或取代到一个乙型肝炎病毒核心蛋白(Hepatitis B virus core protein,HBc)的主要免疫显性区域(Majorimmunodominant region,MIR)，以形成含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)。所述三个败血性巴氏杆菌毒素蛋白(PmT)的抗原决定位分别为：
抗原决定位A:SVGKEGAYYPDHDYGPEYNPVWGPNEQI(SEQ ID NO:2)；
抗原决定位B:SISPDDPPREITD(SEQ ID NO:3)；以及
抗原决定位C:LNSTPGTGRPMP(SEQ ID NO:4)。
在某些优选的实施例中，所述乙型肝炎病毒核心蛋白(HBc)具有如SEQ ID NO:6所示的氨基酸序列。
在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第73～94个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第73～82个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第75～81个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第78～79个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第78～81个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第78～82个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第78～86个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第78～89个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第78～94个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第81～82个氨基酸位置上。在某些实施例中，所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)位在所述蛋白的第82～83个氨基酸位置上。
在某些优选的实施例中，各所述抗原决定位的氨基酸序列之间以及抗原决定位的氨基酸序列与乙型肝炎病毒核心蛋白(HBc)序列之间可进一步由连接子(linker)连接，所述连接子至少含有一个以上的甘氨酸(Glycine,Gly)，所述连接子包含但不限于：Gly-Gly、Gly-Ser、SEQ ID NOs:11、12、13、14、15、16、17、18、19、20、21。在一实施例中，所述连接子具有如SEQ ID NOs:11及/或12所示的氨基酸序列。然而，各个所述抗原决 定位氨基酸序列之间，不必然由连接子所连接。
本发明所提供的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)，可以由下式所表示：
(HBc-N端段)-(连接子3) p-(抗原决定位1)-(连接子1) m-(抗原决定位2)-(连接子2) n-(抗原决定位3)-(连接子4) q-(HBc-C端段)式(II)
所述抗原决定位1、抗原决定位2、抗原决定位3其中一个具有如SEQ ID NO:2的氨基酸序列，其他二个抗原决定位分别具有如SEQ ID NO:3及SEQ ID NO:4的氨基酸序列；
所述连接子1、连接子2、连接子3以及连接子4为各自独立选自于Gly-Gly、Gly-Ser、SEQ ID NOs:11、12、13、14、15、16、17、18、19、20、21；
其中m是代表从0至约10的整数；
其中n是代表从0至约10的整数；
其中p是代表从0至约10的整数；
其中q是代表从0至约10的整数。
在某些实施例中，所述乙型肝炎病毒核心蛋白N端段(HBc-N端段)具有HBc蛋白第1～73个氨基酸的序列，而乙型肝炎病毒核心蛋白C端段(HBc-C端段)具有HBc蛋白第83～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～75个氨基酸的序列，而HBc-C端段具有HBc蛋白第82～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～78个氨基酸的序列，而HBc-C端段具有HBc蛋白第79～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～78个氨基酸的序列，而HBc-C端段具有HBc蛋白第82～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～78个氨基酸的序列，而HBc-C端段具有HBc蛋白第83～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～78个氨基酸的序列，而HBc-C端段具有HBc蛋白第87～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～78个氨基酸的序列，而HBc-C端段具有HBc蛋白第90～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～78个氨基酸的序列，而HBc-C端段具有HBc蛋白第95～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～81个氨基酸的序列，而HBc-C端段具有HBc蛋白第82～144个氨基酸的序列。在某些实施例中，所述HBc-N端段具有HBc蛋白第1～82个氨基酸的序列，而HBc-C端段具有HBc蛋白第83～144个氨基酸的序列。
在某些实施例中，本发明所提供的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)具有如SEQ ID NOs:9、10、27、28、29、30、31、32、33、34、35、36所示的氨基酸序列的至少一种。
在部分实施态样中，本发明所提供的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)与上述式(II)所表示的氨基酸序列具有至少大约80％序列同源性，优选的，具有大约85％序列同源性，更为优选的，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。
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(re-PmT)的核酸序列以及一种编码本发明的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的核酸序列。所述败血性巴氏杆菌毒素重组蛋白(re-PmT)包含如SEQ ID NOs:2、3以及4所示的抗原决定位。而所述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)包含如SEQ ID NOs:2、3以及4所示的抗原决定位以及一如SEQ ID NO:6所示的乙型肝炎病毒核心蛋白(HBc)，其中所述抗原定位插入或取代所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)。
所述编码本发明的败血性巴氏杆菌毒素重组蛋白(re-PmT)的核苷酸序列以及所述编码本发明的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的核酸序列，是分别由本发明的败血性巴氏杆菌毒素重组蛋白(re-PmT)的氨基酸序列以及本发明的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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(re-PmT)氨基酸序列以及本发明的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的氨基酸序列上的各个氨基酸置换为遗传密码表(genetic code table)所列的编码所述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所述核苷酸序列。例如，本发明的败血性巴氏杆菌毒素重组蛋白(re-PmT)以及本发明的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的氨基酸序列上的丝氨酸(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)以及本发明的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的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：
Figure PCTCN2018106100-appb-000001
Figure PCTCN2018106100-appb-000002
此外，本发明也提供一种猪萎缩性鼻炎免疫组合物。所述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(re-PmT)及/或一含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)。所述败血性巴氏杆菌毒素重组蛋白(re-PmT)包含如SEQ ID NOs:2、3以及4所示的抗原决定位。而所述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)包含如SEQ ID NOs:2、3以及4所示的抗原决定位以及一如SEQ ID NO:6所示的乙型肝炎病毒核心蛋白(HBc)，其中所述抗原定位插入或取代所述乙型肝炎病毒核心蛋白(HBc)的主要免疫显性区域(MIR)。
在某些实施例中，所述败血性巴氏杆菌毒素重组蛋白(re-PmT)具有如SEQ ID NOs:5、22、23、24、25或26所示的氨基酸序列。在一具体实施例中，所述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)具有如SEQ ID NO:9所示的氨基酸序列。在另一具体实施例中，所述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)具有如SEQ ID NO:10所示的氨基酸序列。在又一实施例中，所述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)具有如SEQ ID NOs:27、28、29、30、31、32、33、34、35、36所示的氨基酸序列之一。
本发明所提供的败血性巴氏杆菌毒素重组蛋白(re-PmT)以及含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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所述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(re-PmT)的核酸序列或编码含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的核酸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(re-PmT)的核酸序列的质粒或含有编码含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的核酸序列的核酸序列的质粒，再将所述质粒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。
所述表现载体系统包含，但不限于，pET载体系统以及pGEX载体系统等；所述生物表现系统(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)。
在一实施例中，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(B.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)。所述支气管败血性博德氏杆菌(B.bronchiseptica)的来源可为例如，但不限于，美国标准生物品收藏中心(American Type Culture Collection,ATCC)编号ATCC31437，所述败血性巴氏杆菌A型菌(PmA)的来源可为例如，但不限于，英国国家标准生物品收藏中心(National Collection of Type Cultures,NCTC)编号NCTC 12177，以及所述败血性巴氏杆菌D型菌(PmD)的来源可为例如，但不限于，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，所述病原抗原包含，但不限于：猪环状病毒第二型(PCV2)抗原、猪流感病毒(SIV)抗原、猪繁殖与呼吸综合症病毒(PRRSV)抗原、猪支原体(Mycoplasma)、猪小病毒(Parvovirus,PPV)、猪丹毒(Erysipelas)、伪狂犬病(Aujeszky's disease)，及/或猪胸膜肺炎放线杆菌(actinobacillus pleuropneumonia，APP)。
另外，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种或多种选自于下列药学上可接受的载体，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。
所述药学上可接受的载体包含一或多种选自于下列的试剂：溶剂(solvent)、乳化剂(emulsifier)、悬浮剂(suspending agent)、分解剂(decomposer)、黏结剂(binding agent)、赋形剂(excipient)、安定剂(stabilizing agent)、螯合剂(chelating agent)、稀释剂(diluent)、胶凝剂(gelling agent)、防腐剂(preservative)、润滑剂(lubricant)、界面活性剂(surfactant)、佐剂(adjuvant)，及其他类似或适用本发明的载体。
所述药学上可接受的赋形剂可为适合在肠外、肠内或滴鼻施用的药学上可接受的有机或无机载体物质，且所述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、聚乙烯吡咯烷酮等。
所述药学上可接受的佐剂包含，但不限于，水性氢氧化铝胶、明矾、Freund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/O/W)；在一实施例中，所述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，包含将有效量的上述免疫组合物施予动物，以增强所述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，所述抗体是通过本发明所提供的败血性巴氏杆菌毒素重组蛋白(re-PmT)及/或含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)所制备或衍生而得；所述抗体包括，但不限于：单克隆抗体、多克隆抗体，以及经基因重组的抗体。在一实施例中，所述抗体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)施打在一动物体内而得到的多克隆抗体。
本发明并提供一种猪萎缩性鼻炎的检测试剂盒，所述检测试剂盒是用来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。所述检测试剂盒包含，但不限于：(1)一抗原，所述抗原为本发明所提供的败血性巴氏杆菌毒素重组蛋白(re-PmT)及/或含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)，在一实施例中，所述抗原置于一抗原盘上；及/或(2)一抗体，所述抗体是由本发明所提供的所述败血性巴氏杆菌毒素重组蛋白(re-PmT)及/或含有所述败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)所衍生、制备而得的单克隆抗体或多克隆抗体。
所述检测试剂盒的形式包含但不限于：酵素连结免疫分析(enzyme-linked immuNOsorbent assay,ELISA)试剂盒、微芯片检验试剂盒(Microchip kit)、免疫荧光分析法(immuNO fluorescent assay,IFA)检测试剂盒、或其他通过所述败血性巴氏杆菌毒素重组蛋白(re-PmT)及/或含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmTVLP)所制得的检测试剂盒。在一实施例中，所述检测试剂盒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(re-PmT)及/或含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的抗原盘，可用来检验样本中是否含有抗败血性巴氏杆菌毒素(PmT)的抗体。
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。
本文及所附的申请专利范围所使用的「约」、「大约」或「近乎」一词实质上代表所述的数值或范围位在20％以内，优选的为在10％以内，以及更佳者为在5％以内。在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。
除非上下文另有明确说明，本文及所附的权利要求所使用的「一」、「一个」，以及「所 述」的含义包括复数。此外，为了方便读者阅读，在本说明书中可能使用标题或副标题，这些对于本发明的范围没有影响。
以下实施例将更为具体地描述本发明，这些实施例仅为说明性的，因为对本领域技术人员而言，许多修改及变化将会变得显而易见。本发明的各种具体实施例将在下文中被详细地描述。
实施例一败血性巴氏杆菌毒素重组蛋白(re-PmT)的构筑
1.败血性巴氏杆菌毒素重组蛋白(re-PmT)氨基酸序列的设计
自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)选出三段抗原决定位(epitopes)，分别为：
抗原决定位A:SVGKEGAYYPDHDYGPEYNPVWGPNEQI(SEQ ID NO:2)；
抗原决定位B:SISPDDPPREITD(SEQ ID NO:3)；
抗原决定位C:LNSTPGTGRPMP(SEQ ID NO:4)。
分别在抗原决定位A(SEQ ID NO:2)、抗原决定位B(SEQ ID NO:3)，以及抗原决定位C(SEQ ID NO:4)之间各以一段连接子连接，所述连接子具有如SEQ ID NO:11所示的氨基酸序列。依照抗原决定位A、B、C自重组蛋白N端自C端排列顺序组合的不同，得到的败血性巴氏杆菌毒素重组蛋白(re-PmT)氨基酸序列分别如SEQ ID NOs:5、22、23、24、25，以及26所示，并以合成仪合成所述氨基酸序列或以基因选殖表现方式获得。
2.含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的氨基酸序列的设计
将上述构筑的败血性巴氏杆菌毒素重组蛋白(re-PmT)(SEQ ID NO:5)插入乙型肝炎病毒核心蛋白(HBc)(SEQ ID NO:6)的第78个与第79个氨基酸之间，并且分别在乙型肝炎病毒核心蛋白(HBc)N端段(第1～78个氨基酸；SEQ ID NO:7)与败血性巴氏杆菌毒素重组蛋白(re-PmT)(SEQ ID NO:5)之间，以及败血性巴氏杆菌毒素重组蛋白(re-PmT)(SEQ ID NO:5)与乙型肝炎病毒核心蛋白(HBc)C端段(第79～144个氨基酸；SEQ ID NO:8)之间，各以一段连接子连接，所述连接子具有如SEQ ID NO:12所示的氨基酸序列。得到含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的氨基酸序列如SEQ ID NO:9所示。所述氨基酸序列可以合成仪合成，或者先合成编码所述氨基酸序列的核酸序列，并将所述核酸序列选殖至表现载体中，在生物表现宿主中表现所述氨基酸序列并纯化。
上述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)也可以使用基因选殖表现方式获得。以基因选殖方式构筑编码含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的核酸序列时，可以使用限制酶切位连接各个片段。例如，但不限于，将编码乙型肝炎病毒核心蛋白(HBc)N端段(第1～78个氨基酸；SEQ ID NO:7)的核酸序列、编码连接子(SEQ ID NO:12)的核酸序列、编码败血性巴氏杆菌毒素重组蛋白(re-PmT)(SEQ ID NO:5)的核酸序列、编码连接子(SEQ ID NO:12)的核酸序列，以及编 码乙型肝炎病毒核心蛋白(HBc)C端段(第79～144个氨基酸；SEQ ID NO:8)的核酸序列依序插入载体pET24的多种限制酶酵素切位(Multiple cloning site,MCS)内，并分别以SacI、NcoI、HindIII及KpnI等限制酶切位连接上述各核酸序列，以构筑形成编码含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的核酸序列，再将含有这段核酸序列的载体转殖到生物表现宿主中，经蛋白质表现后得到含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)，其氨基酸序列如SEQ ID NO:10所示。
3.含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)的观察
将上述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)纯化后，调整蛋白浓度至1mg/ml，再以0.22mm滤膜过滤后，以JEM-1400电子显微镜(JEOL公司，日本)进行负染色观察。首先制备300网目的镀碳铜网(carbon-coated copper grid)，接着将样品在每个铜网上滴8ml，静置3-5分钟后，以滤纸吸掉多余液体，接着以1％醋酸铀溶液(pH4.5)进行染色30秒，再以滤纸吸掉多余液体后进行显微镜观察。观察时电压设定为80kV。显微镜观察的结果如图1所示，本发明含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)呈现出类病毒颗粒状(如图1箭头所指)。
实施例二支气管败血性博德氏杆菌(Bordetella bronchiseptica)及败血性巴氏杆菌(Pasteurella multocida)的培养
1.支气管败血性博德氏杆菌(B.bronchiseptica)的培养
先将支气管败血性博德氏杆菌(B.bronchiseptica)接种在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)]，在37℃下培养隔夜后，挑选单一菌落接种在脑心浸出液(brain heart infusion,BHI)液体培养基内(BD公司，美国)，在37℃下震荡培养隔夜；接着取菌液再接种在BHI液体培养基内，在37℃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)，在室温下震荡24至36小时，对菌液进行灭活处理。
2.败血性巴氏杆菌(P.multocida)的培养
先将败血性巴氏杆菌A型菌(PmA)及具有产生毒素能力的败血性巴氏杆菌D型菌(PmD)分别接种在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)]，在37℃下培养隔夜后，挑选单一菌落接种在脑心浸出液(BHI)液体培养基内(BD公司，美国)，在37℃下震荡培养隔夜；接着取0.1％(v/v)菌液再接种在BHI液体培养基内，在37℃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灭活作用。
实施例三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(re-PmT)(SEQ ID NO:5)(最终浓度为250μg/ml)或含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)(SEQ ID  NO:10)(最终浓度为250μg/ml)与实施例二所得到的灭活的支气管败血性博德氏杆菌(B.bronchiseptica)(最终浓度为1x10 9CFU/ml)、灭活的败血性巴氏杆菌A型菌(PmA)(最终浓度为1x10 9CFU/ml)及灭活的败血性巴氏杆菌D型菌(PmD)(最终浓度为1x10 9CFU/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。
实施例四猪萎缩性鼻炎免疫组合物的免疫原性及中和抗体分析
1.小鼠免疫试验
将败血性巴氏杆菌抗体阴性3周龄健康的BALB/c小鼠(实验动物中心，台湾)随机分为5组；第1组为对照组，第2-5组为免疫试验组；各组每只小鼠分别以腹腔注射(intraperitoneal injection,ip.)注射0.2ml的以下物质：
第1组：含30％(v/v)铝胶的PBS缓冲溶液(负对照组)；
第2组：实施例二所得到的支气管败血性博德氏杆菌(B.bronchiseptica)(1x10 9CFU/ml)、败血性巴氏杆菌A型菌(PmA)(1x10 9CFU/ml)以及败血性巴氏杆菌D型菌(PmD)(1x10 9CFU/ml)的免疫组合物(含30％(v/v)铝胶佐剂)(B.b+PmA+PmD组)；
第3组：实施例一所得到的败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(SEQ ID NO:10)(浓度为250μg/ml)(re-PmT VLP组)；
第4组：实施例三所得到的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(SEQ ID NO:10)的猪萎缩性鼻炎免疫组合物(B.b+PmA+PmD+re-PmT VLP组)；以及
第5组：市售猪萎缩性鼻炎疫苗(市售疫苗组)；每剂量(2ml)含有：猪博德氏杆菌Bb-1型NO.12-1株灭活菌(灭活前)≥1x109CFU、猪败血性巴氏杆菌A型菌Pm-A-8株灭活菌(灭活前)≥1x10 9CFU、猪败血性巴氏杆菌D型菌Pm-D-8株灭活菌(灭活前)≥1x10 9CFU、猪败血性巴氏杆菌毒素基因Tox1转型大肠杆菌E.coli BL21/rsPMT/Tox1株重组毒素(猪败血性巴氏杆菌毒素第1-487个氨基酸残基)≥20μg、猪败血性巴氏杆菌毒素基因Tox2转型大肠杆菌E.coli BL21/rsPMT/Tox2株重组毒素(猪败血性巴氏杆菌毒素第485-987个氨基酸残基)≥20μg、猪败血性巴氏杆菌毒素基因Tox7转型大肠杆菌E.coliBL21/rsPMT/Tox7株重组毒素(猪败血性巴氏杆菌毒素第986-1282个氨基酸残基)≥20μg、甲醛≤0.004ml，以及氢氧化铝胶佐剂6mg。
每只小鼠分别在初次免疫前24小时(第0天)采血保存，初次免疫(第1天)后，在第13天再进行采血，在第14天以相同免疫剂量再进行二次免疫，并在第24天再次采血。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)与中和抗体试验。
2.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)(Abcam公司，美国)作为抗原，并将抗原涂布(coating)在ELISA用96孔盘(Thermo公司，美国)，在4℃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％BSA的wash buffer)，在室温下静置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，在室温下静置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)，所述二级抗体先以阻隔缓冲液稀释5,000倍后再加入96孔盘(100μl/孔)，在室温下静置1小时后，去除二级抗体，并以清洗缓冲液清洗后，每孔加入100μl 3,3’,5,5’-四甲基联苯氨二盐酸(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(
Figure PCTCN2018106100-appb-000003
M2/M2ELISA Reader，Molecular Devices公司，美国)读取波长650nm的吸光值。
酵素连结免疫分析结果如图2所示。二次免疫后，免疫含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(第4组，即B.b+PmA+PmD+re-PmT VLP组)的小鼠血清中含有的抗败血性巴氏杆菌毒素(PMT)抗体效价最高，其次为免疫实施例一所得的败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(第3组，即re-PmT VLP组)的小鼠血清；而免疫市售猪萎缩性鼻炎疫苗(第5组，即市售疫苗组)的小鼠血清中所含有的抗败血性巴氏杆菌毒素(PMT)抗体效价，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(第2组，即B.b+PmA+PmD组)的小鼠血清中的抗体效价则无显著差异。而各免疫试验组(第2-5组)的小鼠血清中含有的抗败血性巴氏杆菌毒素(PMT)抗体效价皆比第1组(负对照组)的小鼠血清中的抗体效价高，并且具有显著差异(p<0.05或p<0.01)。由此可知，本发明所提供的败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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，而且免疫效果比市售猪萎缩性鼻炎疫苗好。
3.中和抗体效价试验
以绿猴肾脏细胞(Vero cell)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类病毒颗粒的猪萎缩性鼻炎免疫组合物(第4组，即B.b+PmA+PmD+re-PmT VLP组)的小鼠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。进行中和抗体效价测试之前，先进行Vero细胞的最小毒性剂量(minimum toxin dose，MTD)测定。
(a)Vero细胞的最小毒性剂量(MTD)测定
将Vero细胞接种在96孔培养盘中，待细胞长成单层后，去除培养液，并加入使用不含血清的DMEM培养液(GIBCO公司，美国)稀释的败血性巴氏杆菌毒素(PMT，Abcam公司)(0、20、50、100ng)进行处理。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) 的最低败血性巴氏杆菌毒素(PMT)浓度为最小毒性剂量(MTD)。试验结果显示，Vero细胞的败血性巴氏杆菌毒素(PMT)的最小毒性剂量为50ng。
(b)中和抗体效价试验
首先，分别将免疫含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(第4组，即B.b+PmA+PmD+re-PmT VLP组)的小鼠血清，以及免疫市售猪萎缩性鼻炎疫苗(第5组，即市售疫苗组)的小鼠血清稀释10倍后，再进行连续稀释(40、80、120、160、200、240倍)。在96孔培养盘中，分别加入上述稀释的小鼠血清，并在各孔中加入含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℃中反应1小时。接着将上述反应液加入培养在96孔盘的Vero细胞中，在37℃、5％CO 2培养箱中培养后，观察所述血清是否能够抑制Vero细胞产生细胞病变(CPE)。
结果如图3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3B所示，可以看到细胞呈现典型的结节样(如图3B箭头所示)。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3A所示；而以免疫含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(第4组，即B.b+PmA+PmD+re-PmT VLP组)的小鼠血清稀释160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3C所示。而以免疫市售猪萎缩性鼻炎疫苗(第5组，即市售疫苗组)的小鼠血清稀释160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3D所示。图3A与图3C所示的细胞皆未出现如图3B所示的典型的结节样，图3D尚有少许结节样的细胞形态，显示免疫含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(第4组，即B.b+PmA+PmD+re-PmT VLP组)的小鼠血清，以及免疫市售猪萎缩性鼻炎疫苗(第5组，即市售疫苗组)的小鼠血清中皆含有抗败血性巴氏杆菌毒素(PMT)的中和抗体，而且前者的中和抗体含量高于后者。这些结果表示，相较于市售猪萎缩性鼻炎疫苗，本发明提供的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物可以引起受试动物体产生较多的中和抗体。
4.统计方法
所有实验数据利用SigmaState软件与单因子变异数分析(One-Way ANOVA)进行统计分析，实验结果以平均值±标准偏差(mean±SEM)表示。并使用学生氏-纽曼-柯尔斯检验(Student Newman-keuls test)统计方法进行各组间的比较；符号*与**代表与第1组(负对照组)相比具有显著差异(分别表示p<0.05与p<0.01)。符号#与##代表与第2组(B.b+PmA+PmD组)相比具有显著差异(分别表示p<0.05与p<0.01)。符号++代表与第5组(市售疫苗组)相比具有显著差异(表示p<0.01)。
实施例五含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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，将败血性巴氏杆菌抗体阴性3周龄BALB/c小鼠(实验动物中心，台湾)随机分为3组，第1组为对照组，第2组为B.b+PmA+PmD+re-PmT VLP免疫试验组，第3组为市售疫苗免疫试验组；每只小鼠分别以腹腔注射(ip.)注射0.5ml的10倍稀释待测物，各组分别为：
第1组：含30％(v/v)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的类病毒颗粒(SEQ ID NO:10)的猪萎缩性鼻炎免疫组合物(B.b+PmA+PmD+re-PmT VLP组)；以及
第3组：市售猪萎缩性鼻炎疫苗(市售疫苗组)，成分如实施例四所述。
免疫试验组(第2组及第3组)在免疫后第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)1x10 6CFU/ml、1x10 7CFU/ml、1x10 8CFU/ml三种浓度活菌液0.1ml腹腔注射。同时对照组(第1组)小鼠也分为三小组，依组序分别以巴氏杆菌强毒菌株1x10 5CFU/ml、1x10 6CFU/ml、1x10 7CFU/ml三种浓度活菌液0.1ml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LD 50，且各免疫试验组的防御指数须高于对照组1x10 0.5以上。贝卡二氏法防御指数计算法如下：
LD 50＝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1所示，含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的猪萎缩性鼻炎免疫组合物(第2组，即B.b+PmA+PmD+re-PmT VLP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，且其防御指数大于1x10 2.8，高于台湾检验标准(1x10 0.5)以及市售猪萎缩性鼻炎疫苗(市售疫苗组)的防御指数(1x10 2.5)。
表1含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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 PCTCN2018106100-appb-000004
Figure PCTCN2018106100-appb-000005
实施例六含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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
将败血性巴氏杆菌抗体阴性、体重15～20g的BALB/c小鼠(实验动物中心，台湾)随机分为3组，第1组为对照组，第2、3组为免疫试验组。分别将各免疫试验再细分为3小组；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0.2ml PBS缓冲溶液(对照组)；
第2-1组：每只小鼠注射0.2ml实施例三所得的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(SEQ ID NO:10)的猪萎缩性鼻炎免疫组合物原液(B.b+PmA+PmD+re-PmT VLP组)；
第2-2组：每只小鼠注射0.2ml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的类病毒颗粒(SEQ ID NO:10)的猪萎缩性鼻炎免疫组合物(1/5B.b+PmA+PmD+re-PmT VLP组)；
第2-3组：每只小鼠注射0.2ml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的类病毒颗粒(SEQ ID NO:10)的猪萎缩性鼻炎免疫组合物(1/25B.b+PmA+PmD+re-PmT VLP组)；
第3-1组：每只小鼠注射0.2ml市售猪萎缩性鼻炎疫苗原液，成分如实施例四所述(市售疫苗组)；
第3-2组：每只小鼠注射0.2ml稀释5倍的市售猪萎缩性鼻炎疫苗，成分如实施例四所述(1/5市售疫苗组)；
第3-3组：每只小鼠注射0.2ml稀释25倍的市售猪萎缩性鼻炎疫苗，成分如实施例四所述(1/25市售疫苗组)。
免疫试验组(第2、3组)在初次免疫后第14日进行二次免疫，剂量同初次免疫；对照组(第1组)小鼠则再次注射0.2ml 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0.2ml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]100LD 50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2所示，含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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、免疫稀释5倍疫苗的小鼠，以及免疫稀释25倍疫苗的小鼠的存活率皆为100％，全部符合上述存活率的标准。
表2含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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
Figure PCTCN2018106100-appb-000006
实施例七抗败血性巴氏杆菌毒素(PmT)抗体的制备
1.抗败血性巴氏杆菌毒素(PmT)的多克隆抗体
将实施例一所得到的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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(PmT)的多克隆抗体。
其中所述抗败血性巴氏杆菌毒素(PmT)的多克隆抗体，可视需要与显色剂或荧光结合。
其中所述动物经施予初级免疫及二次免疫后，可视需要增加免疫次数，以提高抗体效价。
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(PmT)的单克隆抗体
将实施例一所得到的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒(re-PmT VLP)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。从所述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(PmT)的单克隆抗体的 融合细胞株。
经上述制备所得的抗体，可用在免疫检测试剂、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之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 (21)

  1. 一种败血性巴氏杆菌Pasteurella multocida毒素重组蛋白，其特征在于，包含：
    一具有如SEQ ID NO:2所示氨基酸序列的败血性巴氏杆菌毒素蛋白的抗原决定位；
    一具有如SEQ ID NO:3所示氨基酸序列的败血性巴氏杆菌毒素蛋白的抗原决定位；
    以及
    一具有如SEQ ID NO:4所示氨基酸序列的败血性巴氏杆菌毒素蛋白的抗原决定位。
  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，所述各抗原决定位之间由连接子连接。
  3. 如权利要求2所述的败血性巴氏杆菌毒素重组蛋白，其特征在于，所述连接子为各自独立选自于由Gly-Gly、Gly-Ser及SEQ ID NOs:11、12、13、14、15、16、17、18、19、20、21所组成的群组。
  4. 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，所述败血性巴氏杆菌毒素重组蛋白具有如SEQ ID NOs:5、22、23、24、25以及26所示序列之一。
  5. 一种含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，包含：
    一如权利要求1所述的败血性巴氏杆菌毒素重组蛋白；以及
    一乙型肝炎病毒核心蛋白；
    其中如权利要求1所述的败血性巴氏杆菌毒素重组蛋白插入所述乙型肝炎病毒核心蛋白的主要免疫显性区域。
  6. 如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，所述乙型肝炎病毒核心蛋白具有如SEQ ID NO:6所示的氨基酸序列，以及所述乙型肝炎病毒核心蛋白的主要免疫显性区域为选自于由所述乙型肝炎病毒核心蛋白的第73～94个氨基酸、第73～82个氨基酸、第75～81个氨基酸、第78～79个氨基酸、第78～81个氨基酸、第78～82个氨基酸、第78～86个氨基酸、第78～89个氨基酸、第78～94个氨基酸、第81～82个氨基酸，以及第82～83个氨基酸所组成的群组。
  7. 如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，如权利要求1所述的败血性巴氏杆菌毒素重组蛋白取代所述乙型肝炎病毒核心蛋白的主要免疫显性区域。
  8. 如权利要求7所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，所述乙型肝炎病毒核心蛋白具有如SEQ ID NO:6所示的氨基酸序列，以及所述乙型肝炎病毒核心蛋白的主要免疫显性区域为选自于由所述乙型肝炎病毒核心蛋白的第73～94个氨基酸、第73～82个氨基酸、第75～81个氨基酸、第78～79个氨基酸、第78～81个氨基酸、第78～82个氨基酸、第78～86个氨基酸、第78～89个氨基酸、第78～94个氨基酸、第81～82个氨基酸，以及第82～83个氨基酸所组成的群组。
  9. 如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，如权利要求1所述的败血性巴氏杆菌毒素重组蛋白与所述乙型肝炎病毒核心蛋白之间由连接子连接。
  10. 如权利要求9所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，所述连接子为各自独立选自于由Gly-Gly、Gly-Ser及SEQ ID NOs:11、12、13、14、15、16、17、18、19、20、21所组成的群组。
  11. 如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒，其特征在于，所述含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒具有如SEQ ID NOs:9、10、27、28、29、30、31、32、33、34、35、36所示的氨基酸序列之一。
  12. 一种编码如权利要求1所述的败血性巴氏杆菌毒素重组蛋白的核酸序列。
  13. 一种编码如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒的核酸序列。
  14. 一种猪萎缩性鼻炎免疫组合物，其特征在于，包含如权利要求1所述的败血性巴氏杆菌毒素重组蛋白以及如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒至少其中一种，以及一药学上可接受的载体。
  15. 如权利要求14所述的猪萎缩性鼻炎免疫组合物，其特征在于，进一步包含一支气管败血性博德氏杆菌Bordetella bronchiseptica、一败血性巴氏杆菌A型菌Pasteurella multocida Type A以及一败血性巴氏杆菌D型菌Pasteurella multocida Type D。
  16. 如权利要求14所述的猪萎缩性鼻炎免疫组合物，其特征在于，进一步包含其他病原抗原，所述病原抗原为选自由下列群组所组成者：猪环状病毒第二型抗原、猪流感病毒抗原、猪繁殖与呼吸综合症病毒抗原、猪支原体、猪小病毒、猪丹毒、猪胸膜肺炎放线杆菌，以及伪狂犬病。
  17. 一种如权利要求14所述的猪萎缩性鼻炎免疫组合物在制备动物对抗猪萎缩性鼻炎的药物的应用。
  18. 一种抗败血性巴氏杆菌D型菌毒素的抗体，其特征在于，通过如权利要求1所述的败血性巴氏杆菌毒素重组蛋白或如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒所制备而得。
  19. 如权利要求18所述的抗体，其特征在于，所述抗体包含至少下列其中一种：一单克隆抗体、一多克隆抗体，以及一经基因重组的抗体。
  20. 一种猪萎缩性鼻炎的检测试剂盒，其特征在于，包含一侦测单元，所述侦测单元为选自于下列群组所组成中至少一者：一如权利要求1所述的败血性巴氏杆菌毒素重组蛋白、一如权利要求5所述的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒、一如权利要求1所述的败血性巴氏杆菌毒素重组蛋白所制备的抗体，以及一如权利要求5所述 的含有败血性巴氏杆菌毒素重组蛋白的类病毒颗粒所制备的抗体。
  21. 如权利要求20所述的检测试剂盒，其特征在于，所述抗体包含至少下列其中一种：一单克隆抗体、一多克隆抗体，以及一经基因重组的抗体。
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