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WO2016173504A1 - 猪胸膜肺炎放线杆菌重组毒素蛋白及其应用 - Google Patents

猪胸膜肺炎放线杆菌重组毒素蛋白及其应用 Download PDF

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WO2016173504A1
WO2016173504A1 PCT/CN2016/080469 CN2016080469W WO2016173504A1 WO 2016173504 A1 WO2016173504 A1 WO 2016173504A1 CN 2016080469 W CN2016080469 W CN 2016080469W WO 2016173504 A1 WO2016173504 A1 WO 2016173504A1
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pleuropneumoniae
toxin protein
recombinant
porcine
seq
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PCT/CN2016/080469
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English (en)
French (fr)
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杨滢臻
郭村勇
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金协国际实业有限公司
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Priority to EP16785935.4A priority Critical patent/EP3450461A4/en
Priority to CN201680024623.XA priority patent/CN107614534B/zh
Publication of WO2016173504A1 publication Critical patent/WO2016173504A1/zh
Priority to US16/171,802 priority patent/US20190048045A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the invention relates to the preparation and application of recombinant protein of Actinobacillus pleuropneumoniae, in particular to the use of recombinant toxin protein of Actinobacillus pleuropneumoniae to prevent pig infection of Actinobacillus pleuropneumoniae.
  • Porcine pleuropneumonia is a highly infectious swine respiratory disease caused by Actinobacillus pleuropneumoniae (App.). Its clinical symptoms include fever, cough, vomiting, and difficulty breathing. Depression. Infection with this disease may cause sudden death from acute pneumonia, or a chronic infection that causes asymptomatic presence. Pigs of various pig ages are infected with Actinobacillus pleuropneumonia pleuropneumonia, especially in piglets under 6 months of age.
  • Actinobacillus pleuropneumoniae belongs to Gram-negative bacilli and has two biotypes. Biotype 1 requires ⁇ -nicotinamide adenine dinucleotide ( ⁇ -NAD), while biotype 2 does not. Need ⁇ -NAD. In addition, according to the difference in capsular polysaccharide, 15 serotypes of Actinobacillus pleuropneumoniae have been confirmed, and all serotypes have pathogenic ability. Known virulence factors include capsules, lipopolysaccharides, outer membrane proteins, and most important cytotoxins (Apx).
  • Apx toxins are members of the repeats in structural toxin (RTX) family. A total of four different Apx toxins are produced by 15 serotype strains of Actinobacillus pleuropneumoniae. Strains of each serotype can produce up to three Apx toxins. ApxI is secreted by serotype strains 1, 5, 9, 10, 11, and 14; ApxII can be secreted except for serotypes other than 10 and 14 serotypes; ApxIII is composed of 2, 3, 4, 6, 8, and 15 sera. Type secreted; ApxIV is secreted by all serotypes, but only when infected.
  • RTX structural toxin
  • A. faecalis pleuropneumonia causes serious economic losses in the pig industry worldwide.
  • Treatment of Actinobacillus pleuropneumoniae infection includes the use of, for example, amoxicillin, ampicillin, ceftiofur, enrofloxacin, tiamulin, penicillin ( Penicillin) and antibiotics such as penicillin/streptomycin.
  • Penicillin penicillin
  • antibiotics such as penicillin/streptomycin.
  • Most commercially available Actinobacillus pleuropneumoniae vaccines are whole-bacterial vaccines that reduce pig mortality but do not prevent the original infection and the original state. Therefore, it is important to develop a more effective vaccine against Actinobacillus pleuropneumoniae.
  • the present invention provides a Recombinant Apx toxins of Actinobacillus pleuropneumoniae (Recombinant Apx toxins, re-Apx) is expressed by the following formula (I):
  • Each of these A represents an independent epitope of the A. pleuropneumoniae toxin protein
  • Each of the C3d fragments represents an amino acid sequence of an independent complement cleavage fragment C3d, and each C3d fragment is a group independently selected from the group consisting of SEQ ID NOs: 22, 23, 24, and 25;
  • n is an integer representing from 1 to about 30;
  • n represents an integer from 0 to about 10.
  • the invention provides a nucleotide sequence encoding the recombinant Avian porcine pleuropneumoniae recombinant toxin protein (re-Apx).
  • the present invention provides an immunological composition of Actinobacillus septicum pleuropneumonia comprising the recombinant Avian porcine pneumococcal recombinant toxin protein (re-Apx) and a pharmaceutically acceptable carrier.
  • Actinobacillus septicum pleuropneumonia comprising the recombinant Avian porcine pneumococcal recombinant toxin protein (re-Apx) and a pharmaceutically acceptable carrier.
  • the present invention provides a method for combating Pneumomicrocytogenes pleuropneumonia in an animal comprising administering an effective amount of the above immunological composition to administer an animal to enhance immunity of the animal against A. faecalis pleuropneumonia force.
  • the present invention provides an antibody against A. pleuropneumoniae toxin protein (Apx) which is prepared or derived from the recombinant toxin protein of Reovirus (A. pleuropneumoniae). .
  • the present invention provides a kit for detecting A. faecalis pleuropneumonia.
  • the test kit is for detecting whether the test sample contains the A. pleuropneumoniae toxin protein (Apx), or detecting whether the sample contains an anti-porcine Pleuropneumoniae toxin protein (Apx) antibody.
  • the immunocompetent composition of A. faecalis pleuropneumonia provided by the invention has the following advantages when compared with other conventional techniques:
  • the immunocompetent composition of Actinobacillus hominis pleuris pneumonia comprises a recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx).
  • the recombinant Avian porcine pleuropneumoniae recombinant toxin protein (re-Apx) comprises at least one epitope of the A. pleuropneumoniae toxin protein (Apx) and a partial amino acid sequence of the complement cleavage fragment C3d.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) is much shorter than the full-length amino acid sequence of the A. pleuropneumoniae toxin protein (Apx), is easier to express in the biological expression system, and produces recombinant protein. Higher rates can reduce the cost of manufacturing vaccines.
  • the recombinant Avian porcine pneumococcal recombinant toxin protein (re-Apx) contained in the immunocompetent composition of A. porcine pleuris pneumonia provided by the present invention has a partial amino acid sequence of the complement cleavage fragment C3d, thereby increasing specific immunity
  • the test results show that the immunological composition of Actinobacillus hominis pleuris pneumonia provided by the invention can improve the immunity of the animal against A. pleuropneumoniae infection, and significantly increase the survival rate of the infected animal.
  • Figure 2 is a diagram showing the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) of the present invention confirmed by Western blotting method according to Examples 1 to 4; M: protein molecular weight marker; first lane: porcine pleuropneumonia Recombinant toxin protein I (re-ApxI); lane 2: Actinobacillus pleuropneumoniae recombinant toxin protein II (re-ApxII); lane 3: Actinobacillus pleuropneumoniae recombinant toxin protein III (re-ApxIII Lane 4: Actinobacillus pleuropneumoniae recombinant toxin protein IV (re-ApxIV); primary antibody is alkaline phosphatase-conjugated mouse anti-His-labeled monoclonal antibody (Alkaline phosphatase-conjugated mouse anti-His)
  • Example 3 is a result of measuring the titer of an anti-ApxII antibody against Actinobacillus pleuropneumoniae by enzyme-linked immunoassay (ELISA) according to Example 9; the first group is a negative control group (PBS group).
  • the second group is the multi-valent vaccine of Actinobacillus pleuropneumoniae (App1, 2, 5) obtained in the sixth embodiment; the third group is the whole fungus and recombinant strain of Actinobacillus pleuropneumoniae obtained in the sixth embodiment.
  • Toxin protein mixed multivalent vaccine (App1, 2, 5+re-ApxI ⁇ III group); Group 4 is a commercially available A.
  • the present invention provides a recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) comprising at least one antigenic epitope (epitopes) of Actinobacillus pleuropneumoniae toxin protein (Apx) for inducing anti-porcine pleuropneumoniae
  • An antibody to the bacillus toxin protein (Apx) which may further comprise a full-length or partial amino acid sequence of the complement cleavage fragment C3d to increase the specific immune response.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) of the present invention is represented by the following formula (I):
  • Each of these A represents an independent epitope of the A. pleuropneumoniae toxin protein
  • Each of the C3d fragments represents the amino acid sequence of an independent complement cleavage fragment C3d
  • n is an integer representing from 1 to about 30;
  • n represents an integer from 0 to about 10.
  • 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: 25.
  • the partial amino acid sequence of the complement cleavage fragment C3d is the 211th amino acid to 238th amino acid fragment sequence (mC3d-p28) of the mouse complement cleavage fragment C3d, having SEQ ID NO: 24 The amino acid sequence shown.
  • 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:23.
  • 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, having SEQ ID NO: 22 The amino acid sequence shown.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) provided by the present invention has a full-length or partial amino acid sequence of the above-described complement cleavage fragment C3d of 0 to 10 unit repeats, and the full length of the complement cleavage fragment C3d Or partial amino acid sequence is preferred It is 1 to repeat 10 units, and more preferably 4 to 8 units are repeated.
  • each A is further joined by a linker, each linker being independently selected from Gly-Gly, Gly-Ser, and SEQ ID NOs: 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 and 36.
  • each C3d fragment is further joined by a linker, each of which is independently selected from Gly-Gly, Gly-Ser, and SEQ ID NOs: 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35 and 36.
  • a linker each of which is independently selected from Gly-Gly, Gly-Ser, and SEQ ID NOs: 26, 27, 28, 29, 30 , 31, 32, 33, 34, 35 and 36.
  • the A and C3d fragments are further joined by a linker selected from the group consisting of Gly-Gly, Gly-Ser, and SEQ ID NOs: 26, 27, 28, 29, 30, 31. , 32, 33, 34, 35 and 36.
  • a linker selected from the group consisting of Gly-Gly, Gly-Ser, and SEQ ID NOs: 26, 27, 28, 29, 30, 31. , 32, 33, 34, 35 and 36.
  • the porcine pleuropneumoniae toxin protein is A.
  • porcine pleuropneumoniae toxin protein I (ApxI)
  • the recombinant porcine pleuropneumoniae recombinant toxin protein is a recombinant toxin of Actinobacillus pleuropneumoniae Protein I (re-ApxI)
  • each A is independently selected from SEQ ID NOs: 37, 4, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 and The amino acid sequence shown in 51, the antigenic epitope of the A.
  • pleuropneumoniae toxin protein I may be from 1 to about 30, and the sequence of each epitope amino acid sequence from the N-terminus to the C-terminus of the protein is It is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • the five epitopes of the epitope are each covered by an epitope of the above-mentioned A. porcine pleuropneumoniae toxin protein I (ApxI), the longer five epitopes.
  • the amino acid sequences are shown in SEQ ID NOs: 2, 3, 4, 5, 6, respectively.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein I (re-ApxI) of the present invention contains two or more of said longer epitope amino acid sequences, and each epitope amino acid sequence is derived from a protein.
  • the order of arrangement from the N-terminus to the C-terminus is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • the recombinant Avian porcine pleuropneumoniae recombinant toxin protein I (re-ApxI) comprises the amino acid sequence set forth in SEQ ID NO: 7 or 8.
  • the A. pleuropneumoniae toxin protein is Anopheles pneumoniae toxin protein II (ApxII), and the recombinant toxin of A. pleuropneumoniae is a recombinant toxin of A.
  • each A is independently selected from the group consisting of SEQ ID NOs: 14, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
  • the amino acid sequences shown in 65, 67 and 68, wherein the epitope of Actinobacillus pleuropneumoniae toxin protein II (ApxII) may be from 1 to about 30, and the amino acid sequence of each epitope is from the N-terminus of the protein to C.
  • the order of arrangement of the ends is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • the five epitopes of the epitope are each encompassing an epitope of the above-mentioned A. pleuropneumoniae toxin protein II (ApxII), the five longer epitopes.
  • the amino acid sequences are shown in SEQ ID NOs: 10, 11, 12, 13, and 14, respectively.
  • the recombinant Avian Streptococcus pneumoniae recombinant toxin protein II comprises two or more of said longer epitope amino acid sequences, and each epitope amino acid sequence is derived from a protein.
  • the order of arrangement from the N-terminus to the C-terminus is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • the A. pleuropneumoniae recombinant toxin protein II (re-ApxII) comprises the amino acid sequence set forth in SEQ ID NO: 15 or 16.
  • the A. pleuropneumoniae toxin protein is Anopheles pneumoniae toxin protein III (ApxIII), and the recombinant toxin of A. pleuropneumoniae is a recombinant toxin of A. pleuropneumoniae Protein III (re-ApxIII), and each A is independently selected from SEQ ID NOs: 69, 70, 71, 72, 73, 74, 75, 76, 77, The amino acid sequences shown in 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 and 88, the epitope of the A.
  • pleuropneumoniae toxin protein III may be from 1 to about 30, and the order of arrangement of the amino acid sequences of each epitope 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).
  • the antigenic determinant of the above-mentioned A. porcine pleuropneumoniae toxin protein III (ApxIII) is covered by two longer epitope epitopes, respectively, which are longer epitopes.
  • the amino acid sequences are shown in SEQ ID NOs: 18, 19, respectively.
  • the recombinant Avian porcine pleuropneumoniae recombinant toxin protein III comprises the two said longer epitope amino acid sequences, and each epitope amino acid sequence is self-determined.
  • the order of arrangement of 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).
  • the Actinobacillus pleuropneumoniae recombinant toxin protein III (re-ApxIII) comprises the amino acid sequence set forth in SEQ ID NO: 20 or 21.
  • the A. pleuropneumoniae toxin protein is Anopheles pneumoniae toxin protein IV (ApxIV), and the recombinant toxin of A. pleuropneumoniae is a recombinant toxin of A. pleuropneumoniae Protein IV (re-ApxIV), and each A is independently selected from the group consisting of SEQ ID NOs: 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, The amino acid sequences shown in 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 and 117, the epitope of the A.
  • pleuropneumoniae toxin protein IV may be from 1 to about 30, and the order of arrangement of the amino acid sequences of each epitope 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).
  • the three epitopes of the epitope are each covered by an epitope of the above-mentioned A. porcine pleuropneumoniae toxin protein IV (ApxIV), said three longer epitopes.
  • the amino acid sequences are shown in SEQ ID NOs: 66, 89, 90, respectively.
  • the recombinant Avian porcine pleuropneumoniae recombinant toxin protein IV comprises two or more of said longer epitope amino acid sequences, and each epitope amino acid sequence is derived from a protein.
  • the order of arrangement from the N-terminus to the C-terminus is not limited to being arranged in the order of the sequence identification number (SEQ ID NO).
  • the A. pleuropneumoniae recombinant toxin protein IV (re-ApxIV) comprises the amino acid sequence set forth in SEQ ID NO: 91 or 92.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) 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, having 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 present invention also provides a nucleotide sequence encoding a recombinant toxin protein (Re-Apx) of Actinobacillus pleuropneumoniae.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) comprises at least one antigenic epitope of the A. pleuropneumoniae toxin protein (Apx), and a portion of the complement cleavage fragment C3d of 0 to 10 units repeated Amino acid sequence.
  • the nucleotide sequence encoding the recombinant Avian porcine pleuropneumoniae recombinant toxin protein (re-Apx) of the present invention is derived from the amino acid sequence of the recombinant Avian porcine pleuropneumoniae recombinant toxin protein (re-Apx) of the present invention. .
  • the serine can be encoded by nucleotide sequences such as TCT, TCC, TCA, TCG, AGT, AGC.
  • the present invention provides an immunocompetitive composition of Actinobacillus avium pleuropneumoniae.
  • the A. porcine Aureus pleuropneumoniae immunological composition comprises the recombinant Avian porcine pleuropneumoniae recombinant toxin protein (re-Apx) and a pharmaceutically acceptable carrier.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein is a recombinant toxin protein I (re-ApxI) of Actinobacillus pleuropneumoniae, and recombinant toxin protein II of Actinobacillus pleuropneumoniae (re-ApxII), at least one of Actinobacillus pleuropneumoniae recombinant toxin protein III (re-ApxIII), and A. porcine pleuropneumoniae recombinant toxin protein IV (re-ApxIV).
  • the A is a recombinant toxin protein I (re-ApxI) of Actinobacillus pleuropneumoniae, and recombinant toxin protein II of Actinobacillus pleuropneumoniae (re-ApxIII), at least one of Actinobacillus pleuropneu
  • porcine Aureus pleuropneumoniae immunological composition comprises Recombinant Toxin Protein I (re-ApxI) of A. pleuropneumoniae, Recombinant Toxin Protein II of Resveratococcus pleuropneumoniae (re-ApxII) ), recombinant toxin protein III (re-ApxIII) with Actinobacillus pleuropneumoniae, and a pharmaceutically acceptable carrier.
  • the A. porcine Aureus pleuropneumoniae immunological composition comprises Recombinant Toxin Protein I (re-ApxI) of A.
  • pleuropneumoniae Recombinant Toxin Protein II of Resveratococcus pleuropneumoniae (re-ApxII) ), Actinobacillus pleuropneumoniae recombinant toxin protein III (re-ApxIII), and recombinant porcine pleuropneumoniae recombinant toxin protein IV (re-ApxIV), and a pharmaceutically acceptable carrier.
  • the A. anisopliae pleuropneumoniae immunological composition may further comprise at least one whole strain of Actinobacillus pleuropneumoniae serotype.
  • the serotypes of Actinobacillus pleuropneumoniae serotypes include, but are not limited to, Actinobacillus pleuropneumoniae serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15.
  • the A. faecalis pleuropneumoniae immunological composition further comprises a porcine Pleuropneumoniae serotype 1, 2, 5 phenotype.
  • the A. faecalis pleural pneumonia immunological composition provided by the present invention further comprises other pathogenic antigens, including, but not limited to, porcine circovirus type 2 (PCV2) antigen, pig Influenza virus (SIV) antigen, porcine reproductive and respiratory syndrome virus (PRRSV) antigen, Mycoplasma, Parvovirus (PPV), Erysipelas, Bordetella bronchiseptica , Pasteurella multocida, and Aujeszky's disease.
  • PCV2 porcine circovirus type 2
  • SIV pig Influenza virus
  • PRRSV porcine reproductive and respiratory syndrome virus
  • PSV Parvovirus
  • Erysipelas Bordetella bronchiseptica
  • Pasteurella multocida Pasteurella multocida
  • Aujeszky's disease including, but not limited to, porcine circovirus type 2 (PCV2) antigen, pig Influenza virus (SIV) antigen, porcine reproductive and respiratory
  • the A. porcine pleuris pneumonia immunological composition provided by the present invention 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, Excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, adjuvants, biotype carriers, and the like.
  • pharmaceutically acceptable carriers including: a solvent, an emulsifier, a suspending agent, a decomposing agent, a binder, Excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, adjuvants, biotype carriers, and the like.
  • 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 glycols, gelatin, amylose, lactose, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides, 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, Freund's incomplete adjuvant, oily An adjuvant, a water-soluble adjuvant, or a water-in-oil-in-water (W/O/W); in one embodiment, the adjuvant is aqueous hydroxide Aluminum glue.
  • the present invention provides a method for treating an animal against A. faecalis pleuropneumonia comprising administering an animal in an effective amount of the above immunological composition to enhance immunity of the animal against A. faecalis pleuropneumonia, Increase the survival rate after infection.
  • the present invention also provides an antibody against A. pleuropneumoniae toxin protein (Apx), which is prepared or derived by the recombinant avian pneumoniae recombinant toxin protein (re-Apx) provided by the present invention;
  • A. pleuropneumoniae toxin protein Apx
  • Such antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and recombinant antibodies.
  • the antibody is a plurality of antibodies obtained by administering the recombinant Avian porcine pleuropneumoniae recombinant toxin protein (re-Apx) provided in the present invention to an animal.
  • the present invention provides a kit for detecting A. faecalis pleuropneumonia.
  • the test kit is for detecting whether the test sample contains the A. pleuropneumoniae toxin protein (Apx), or detecting whether the sample contains an anti-porcine Pleuropneumoniae toxin protein (Apx) antibody.
  • the detection kit includes, but is not limited to: (1) an antigen, which is an A. pleuropneumoniae recombinant toxin protein (re-Apx) provided by the present invention.
  • the antigen is Is placed on an antigenic disk; and/or (2) an antibody obtained by the recombinant porcine pleuropneumoniae recombinant toxin protein (re-Apx) provided by the present invention. Single antibody or multiple antibodies.
  • the form of the detection kit includes, but is not limited to, an enzyme-linked immunosorbent assay (ELISA) kit, a microchip assay kit (Microchip kit), and an immunofluorescence assay (IFA).
  • ELISA enzyme-linked immunosorbent assay
  • Microchip kit microchip assay kit
  • IFA immunofluorescence assay
  • the test kit comprises at least one antigen disk containing the recombinant Avian porcine pleuropneumoniae recombination toxin protein (re-Apx) provided by the present invention, which can be used to test whether the sample contains anti-porcine pleuropneumonia An antibody to the bacillus toxin protein (Apx).
  • re-Apx Avian porcine pleuropneumoniae recombination toxin protein
  • epitope epitopes were selected from the full-length amino acid sequence of Actinobacillus pleuropneumoniae toxin protein I (ApxI) (as shown in SEQ ID NO: 1), respectively:
  • epitopes include, but are not limited to, the following epitopes:
  • the epitope determinant ApxI-1, the epitope determinant ApxI-2, the epitope determinant ApxI-3, the epitope determinant ApxI-4, and the epitope determinant ApxI-5 are sequentially ligated from the N-terminus to the C-terminus, and Each of the epitope epitopes is ligated with more than one linker having the amino acid sequence set forth in SEQ ID NO: 26; and 6 at the C-terminus of the epitope epitope ApxI-5 pC3d-p31 bioadjuvant sequence (as shown in SEQ ID NO: 22), each pC3d-p31 bioadjuvant sequence linked by more than one linker (SEQ ID NO: 26); resulting porcine pleura
  • the amino acid sequence of the recombinant toxin protein I (re-ApxI) of Actinobacillus pneumoniae is set forth in SEQ ID NO: 7.
  • the amino acid sequence can be synthesized by a synthesizer.
  • a nucleic acid sequence encoding the amino acid sequence is first synthesized, and the nucleic acid sequence is selected into a expression vector, and the amino acid sequence is expressed in a biological expression host and purified.
  • a nucleic acid sequence encoding Recombinant Toxin Protein I (re-ApxI) of A. pleuropneumoniae can be constructed by gene selection to restriction the cleavage of the DNA fragment, and the nucleic acid sequence is selected into the expression vector. The amino acid sequence is expressed in a biological expression host and purified.
  • the DNA sequence encoding the epitope of the A. pleuropneumoniae toxin protein I and the DNA sequence encoding the pC3d-p31 bioadjuvant fragment are selected by HindIII restriction enzyme cleavage.
  • the amino acid sequence of the recombinant porcine pleuropneumoniae recombinant toxin protein I (re-ApxI) obtained after colonization is shown in SEQ ID NO: 8.
  • Recombinant toxin protein I (re-ApxI) of A. pleuropneumoniae purified by nickel affinity chromatography and ion exchange chromatography and polymerized with sodium lauryl sulfate The results were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The results are shown in Figure 1 (lane 1) and Figure 2 (lane 2).
  • the molecular weight of Actinobacillus pleuropneumoniae recombinant toxin protein I (re-ApxI) was as expected at about 46 kDa.
  • epitope epitopes were selected from the full-length amino acid sequence of Actinobacillus pleuropneumoniae toxin protein II (ApxII) (as shown in SEQ ID NO: 9), respectively:
  • epitopes include, but are not limited to, the following epitopes:
  • the epitope determinant ApxII-1, the epitope determinant ApxII-2, the epitope determinant ApxII-3, the epitope determinant ApxII-4, and the epitope determinant ApxII-5 are sequentially linked from the N-terminus to the C-terminus, and Each of the epitope epitopes is ligated with more than one linker having the amino acid sequence set forth in SEQ ID NO: 26; and 6 at the C-terminus of the epitope epitope ApxII-5 pC3d-p31 biological adjuvant sequence (as shown in SEQ ID NO: 22), each pC3d-p31 biological adjuvant sequence is linked by more than one linker (SEQ ID NO: 26); the resulting porcine pleuropneumonia lineup
  • the amino acid sequence of the recombinant recombinant toxin protein II (re-ApxII) is shown in SEQ ID NO: 15.
  • the amino acid sequence can be synthesized by a synthesizer.
  • a nucleic acid sequence encoding the amino acid sequence is first synthesized, and the nucleic acid sequence is selected into a expression vector, and the amino acid sequence is expressed in a biological expression host and purified.
  • the nucleic acid sequence encoding Recombinant Toxin Protein II (re-ApxII) of A. pleuropneumoniae can be constructed by gene selection to restriction the cleavage of the DNA fragment, and the nucleic acid sequence is selected into the expression vector. The amino acid sequence is expressed in a biological expression host and purified.
  • the DNA sequence encoding the epitope fragment of Actinobacillus pleuropneumoniae toxin protein II and the DNA sequence encoding the pC3d-p31 biological adjuvant fragment are ligated by HindIII restriction enzyme cleavage, and obtained after selection.
  • pleuropneumoniae is shown in SEQ ID NO: 16.
  • the molecular weight of Actinobacillus pleuropneumoniae recombinant toxin protein II (re-ApxII) was as expected at about 47 kDa.
  • Two epitope epitopes were selected from the full-length amino acid sequence of Actinobacillus pleuropneumoniae toxin protein III (ApxIII) (as shown in SEQ ID NO: 17), respectively:
  • epitopes include, but are not limited to, the following epitopes:
  • the epitope determinant ApxIII-1 and the epitope determinant ApxIII-2 are ligated sequentially from the N-terminus to the C-terminus, and are ligated with more than one linker between each epitope, respectively.
  • the adjuvant sequences are each ligated with more than one linker (SEQ ID NO: 26); the resulting amino acid sequence of A.
  • porcine pleuropneumoniae recombinant toxin protein III (re-ApxIII) is set forth in SEQ ID NO: 20.
  • the amino acid sequence can be synthesized by a synthesizer. Alternatively, a nucleic acid sequence encoding the amino acid sequence is first synthesized, and the nucleic acid sequence is selected into a expression vector, and the amino acid sequence is expressed in a biological expression host and purified.
  • a nucleic acid sequence encoding Recombinant Toxin Protein III (re-ApxIII) of A. pleuropneumoniae can be constructed by gene selection to restriction the cleavage of the DNA fragment, and the nucleic acid sequence is selected into a expression vector. The amino acid sequence is expressed in a biological expression host and purified.
  • the DNA sequence encoding the epitope fragment of Actinobacillus pleuropneumoniae toxin protein III and the DNA sequence encoding the pC3d-p31 bioadjuvant fragment are ligated with HindIII restriction enzyme cleavage, and obtained after selection.
  • the amino acid sequence of Actinobacillus pleuropneumoniae recombinant toxin protein III is shown in SEQ ID NO:21.
  • the molecular weight of Actinobacillus pleuropneumoniae recombinant toxin protein III (re-ApxIII) was as expected at about 49 kDa.
  • ApxIV Actinobacillus pleuropneumoniae toxin protein IV
  • epitopes include, but are not limited to, the following epitopes:
  • the epitope determinant ApxIV-1, the epitope determinant ApxIV-2, and the epitope determinant ApxIV-3 are sequentially ligated from the N-terminus to the C-terminus, and are ligated by more than one linker between each epitope epitope.
  • the linker has the amino acid sequence set forth in SEQ ID NO: 26; and the C-terminus of the epitope epitope ApxIV-3 is added with six pC3d-p31 biological adjuvant sequences (as set forth in SEQ ID NO: 22) , each pC3d-p31 biological adjuvant sequence is ligated with more than one linker (SEQ ID NO: 26); the resulting recombinant Avian porcine pleuropneumoniae recombinant toxin protein IV (re-ApxIV) amino acid sequence is SEQ ID NO :91 is shown.
  • the amino acid sequence can be synthesized by a synthesizer. Alternatively, a nucleic acid sequence encoding the amino acid sequence is first synthesized, and the nucleic acid sequence is selected into a expression vector, and the amino acid sequence is expressed in a biological expression host and purified.
  • a nucleic acid sequence encoding Recombinant Toxin Protein IV (re-ApxIV) of A. pleuropneumoniae can be constructed by gene selection to restriction the cleavage of the DNA fragment, and the nucleic acid sequence is selected into the expression vector. The amino acid sequence is expressed in a biological expression host and purified.
  • the DNA sequence encoding the epitope fragment of Actinobacillus pleuropneumoniae toxin protein IV and the DNA sequence encoding the pC3d-p31 biological adjuvant fragment are ligated with a HindIII restriction enzyme cleavage, and obtained after colonization.
  • the amino acid sequence of Actinobacillus pleuropneumoniae recombinant toxin protein IV is set forth in SEQ ID NO:92.
  • Recombinant toxin protein IV (re-ApxIV) of A. pleuropneumoniae purified by nickel affinity chromatography and ion exchange chromatography and polymerized with sodium lauryl sulfate Acrylamide gel electrophoresis (SDS-PAGE) and Western blotting confirmed the results as shown in Figure 1 (Track 4) and Figure 2 (Track 5).
  • the molecular weight of Actinobacillus pleuropneumoniae recombinant toxin protein IV (re-ApxIV) was as expected at about 50 kDa.
  • the serotype of Actinobacillus pleuropneumoniae serotype 1 (Taiwan wild isolate, secreting ApxI, ApxII, ApxIV toxin) and type 2 (Taiwan wild isolate, secreting ApxII, ApxIII, ApxIV toxin) Bacterial inoculation of brain heart extract (BHI) liquid medium containing 0.01% (v/v) nicotinamide adenine dinucleotide ( ⁇ -NAD) and 10% (v/v) horse serum Internal (BD company, USA), cultured overnight at 37 ° C, 5% CO 2 .
  • BHI brain heart extract
  • ⁇ -NAD nicotinamide adenine dinucleotide
  • horse serum Internal (BD company, USA)
  • the above-mentioned bacteria of the porcine Pleuropneumoniae serotype type 1 and 2 were treated with an ultrasonic oscillator (SONOPULS, Bandelin, Germany) to crush the bacteria, and then centrifuged in a high-speed centrifuge (KUBOTA, Japan). After the supernatant was filtered through a 0.22 ⁇ m pore size filtration membrane (Millipore, USA), the filtrate was a crude extract ApxI-IV of the genus Actinobacillus pleuropneumoniae, and stored after packaging. Stand by at -80 °C.
  • the recombinant porcine pleuropneumoniae recombinant toxin protein re-ApxI (SEQ ID NO: 8) (final concentration 500 ⁇ g/ml) and re-ApxII (SEQ ID NO: 16) obtained in Example 1 to Example 4, respectively.
  • the inactivated porcine Pleuropneumoniae serotype type 1, 2, and 5 whole bacteria (the final concentration of each strain was 1 ⁇ 10 9 cfu/ml) and the phosphate buffer solution (PBS) were uniformly mixed.
  • An aluminum gel [final concentration of 30% (v/v)] was added as an adjuvant to prepare a multivalent vaccine against Actinobacillus pleuropneumoniae (App1, 2, 5).
  • the recombinant porcine pleuropneumoniae recombinant toxin protein re-ApxI (SEQ ID NO: 8) obtained from Example 1 to Example 3 (final concentration 20 ⁇ g/ml), re-ApxII (SEQ ID NO: 16) (final Inactivated porcine Pleuropneumoniae serotype 1st, 2nd, and 5th strains obtained in Example 5 at a concentration of 20 ⁇ g/ml, re-ApxIII (SEQ ID NO: 21) (final concentration of 20 ⁇ g/ml) (The final concentration of each strain is 1x 10 9 cfu/ml) and phosphate buffer solution (PBS) is mixed evenly, and aluminum glue [final concentration of 30% (v/v)] is added as an adjuvant to prepare into porcine pleura.
  • a multivalent vaccine of Actinobacillus pneumoniae and recombinant toxin protein (App1, 2, 5+re-ApxI-III).
  • mice 60 healthy ICR mice (National Experimental Animal Center, Taiwan) with negative antibody to Actinobacillus pleuropneumoniae were randomly divided into 6 groups, 10 in each group; the first group was negative control group, the first group Groups 2 to 6 were immunoassay groups; each group was injected intraperitoneally (ip.) with 0.2 ml of the following substances:
  • Group 1 PBS buffer solution (PBS group) containing 30% (v/v) aluminum gel;
  • Group 2 a subunit vaccine (re-ApxI group) containing Recombinant toxin protein I (re-ApxI) (SEQ ID NO: 8) of P. aeruginosa pneumoniae obtained in Example 1;
  • Group 3 sub-single of recombinant porcine pleuropneumoniae recombinant toxin protein II (re-ApxII) (SEQ ID NO: 16) obtained in Example 2.
  • Vaccine (re-ApxII group);
  • Group 4 subunit vaccine (re-ApxIII group) containing Recombinant toxin protein III (re-ApxIII) (SEQ ID NO: 21) of P. aeruginosa pneumoniae obtained in Example 3;
  • Group 5 a subunit vaccine (re-ApxIV group) comprising the recombinant Avian Streptococcus pneumoniae recombinant toxin protein IV (re-ApxIV) (SEQ ID NO: 92) obtained in Example 4;
  • Group 6 The live multivalent vaccine of Actinobacillus pleuropneumoniae (App1, 2, 5) obtained in Example 6.
  • Each mouse was immunized a second time with the same immunization dose on the 14th day after the first immunization. On the 10th day after the second immunization, each mouse was injected with 0.1 ml of the porcine pleuropneumoniae obtained in Example 5.
  • the LD 90 dose of the whole bacterial crude extract toxin protein extracting APP1 6.5 ⁇ 10 9 cfu/ml and APP2 bacterial amount 9.65 ⁇ 10 10 cfu/ml was used for challenge, and the number of mouse deaths was recorded after ten days to calculate each Group survival rate.
  • Mouse survival was counted by Kaplan-Meier Survival Analysis (Log-Rank test) and was considered statistically significant difference at p ⁇ 0.05.
  • mice challenge test results of the mouse challenge test are shown in Table 1.
  • Each of the subunit vaccines containing the recombinant toxins of the porcine Pleuropneumoniae recombinant proteins re-ApxI, re-ApxII, re-ApxIII, and re-ApxIV ie, groups 2 to 5
  • group 1 survival rate 0%
  • it can induce the protective effect of mice, and is resistant to the challenge of Actinobacillus pleuropneumoniae crude extract toxin protein ApxI ⁇ IV, and improve the survival rate.
  • mice Survival rate Group 1 10 0% Group 2 (re-ApxI group) 10 50%* Group 3 (re-ApxII group) 10 50%* Group 4 (re-ApxIII group) 10 50%** Group 5 (re-ApxIV group) 10 30%* Group 6 (App1, 2, 5 groups) 10 20%
  • Example 8 Analysis of protective efficacy of Actinobacillus pleuropneumoniae multivalent vaccine against serotype 2 strain (App2) of Actinobacillus pleuropneumoniae serotype
  • Group 1 PBS buffer solution (PBS group) containing 30% (v/v) aluminum gel;
  • Group 2 The porcine pleuropneumoniae whole-cell multivalent vaccine obtained in Example 6 (App1, 2, 5 groups);
  • Group 3 the live multi-valent vaccine of Actinobacillus pleuropneumoniae and recombinant toxin protein obtained in Example 6 (App1, 2, 5+re-ApxI-III group);
  • Group 4 Commercially available vaccine against Actinobacillus pleuropneumonia pleuropneumonia, containing the serotypes of Actinobacillus pleuropneumoniae serotype 1, 2, 3, 4, 5, 7 (App1, 2, 3, 4, 5 , 7 groups).
  • mice were given a second immunization with the same immunization dose on the 14th day after the first immunization, and each mouse was injected with 0.1 ml of LD 90 dose (7.5 x 10 8 cfu/ on the 10th day after the second immunization).
  • LD 90 dose 7.5 x 10 8 cfu/ on the 10th day after the second immunization.
  • App2 porcine Pleuropneumoniae serotype type 2 whole bacteria
  • mice challenge test The results of the mouse challenge test are shown in Table 2. Compared with the negative control group (the first group, the survival rate was 20%), the survival rate of each immunoassay group was significantly increased and statistically different.
  • the multivalent vaccine App1, 2, 5+re-ApxI-III group
  • the recombinant toxin protein re-ApxI-III of the present invention had the best protective effect on mice and the highest survival rate.
  • mice Survival rate Group 1 10 20% Group 2 (App1, 2, 5 groups) 10 70%** Group 3 (App1, 2, 5+re-ApxI to III) 10 80%** Group 4 (commercial App1, 2, 3, 4, 5, 7 groups) 10 60%*
  • mice National Experimental Animal Center, Taiwan
  • Actinobacillus pleuropneumoniae 50 healthy ICR mice (National Experimental Animal Center, Taiwan) with negative anti-bacterial activity of Actinobacillus pleuropneumoniae were randomly divided into 4 groups; the first group was negative control group, the second group was 4 to 4 Immunoassay group; each group was injected intraperitoneally (ip.) with 0.2 ml of the following substances:
  • Each mouse was collected for blood collection 24 hours before the first immunization, and 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 the blood samples were separated. Serum for enzyme-linked immunoassay (ELISA) of toxin antibodies; after blood collection, each mouse was injected with 0.1 ml of LD 90 dose (7.32 x 10 8 cfu/ml) of A. pleuropneumoniae serotype 5 The whole type of bacteria (App5) was used for challenge, and the mice were observed for 10 days and the number of deaths was recorded to calculate the survival rate of each group.
  • ELISA enzyme-linked immunoassay
  • Actinobacillus pleuropneumoniae recombinant toxin protein II (re-ApxII, SEQ ID NO: 16) was used as an antigen, and the antigen was coated on a 96-well plate (Thermo, USA) (100 ng/well) for ELISA. , allowed to stand at 4 ° C for 16 hours. 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 wash 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 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 calibrated with Horseradish peroxidase (HRP).
  • HRP Horseradish peroxidase
  • Goat anti-mouse conjugated HRP Gene Tex, USA
  • the secondary antibody was diluted 5,000 times in blocking buffer and then added to a 96-well plate (100 ⁇ l/well). After standing at room temperature for 1 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 was added to each well.
  • mice challenge test The results of the mouse challenge test are shown in Table 3. Compared with the negative control group (the first group, the survival rate was 6.7%), the survival rate of each immunoassay group was significantly increased and statistically different.
  • the multivalent vaccine App1, 2, 5+re-ApxI-III group
  • the recombinant toxin protein re-ApxI-III of the present invention had the best protective effect on mice and the highest survival rate.
  • mice Survival rate Group 1 PBS group 15 6.7%
  • Group 2 App1, 2, 5 groups
  • Group 3 App1, 2, 5+re-ApxI to III
  • Group 4 commercial App1, 2, 3, 4, 5, 7 groups
  • faecalis pleuropneumonia inactivated vaccine Group 4, ie Antibody titers of commercially available App1, 2, 3, 4, 5, 7 groups, p ⁇ 0.01. It can be seen that the whole bacterium of the porcine pleuropneumoniae and the recombinant protein mixed multivalent vaccine can effectively induce the anti-porcine porcine pleuropneumoniae toxin protein antibody in the animal, and has immunogenicity and effect. Significantly better than the conventional vaccine.
  • the recombinant proteins recombinant re-ApxI (SEQ ID NO: 8), re-ApxII (SEQ ID NO: 16), and re-ApxIII (SEQ ID NO: 21) obtained from Examples 1 to 4, respectively.
  • re-ApxIV SEQ ID NO: 92
  • FCA Freund's complete adjuvant
  • FCA Freund's complete adjuvant

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Abstract

本发明提供了一种猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)重组毒素蛋白，包括至少一个猪胸膜肺炎放线杆菌毒素蛋白的抗原决定位；当所述抗原决定位为复数个时，各抗原决定位之间可有连接子。所述重组蛋白可包含至少一个补体裂解片段C3d的氨基酸序列，且所述抗原决定位与C3d的氨基酸序列之间可有连接子。本发明还提供了编码所述蛋白的核苷酸序列，以及含有所述蛋白的免疫组合物。

Description

猪胸膜肺炎放线杆菌重组毒素蛋白及其应用 技术领域
本发明是关于猪胸膜肺炎放线杆菌重组蛋白的制备与应用，特别是关于使用猪胸膜肺炎放线杆菌重组毒素蛋白以预防猪只感染猪胸膜肺炎放线杆菌。
背景技术
猪放线杆菌胸膜肺炎(porcine pleuropneumonia)是一种由猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,App.)所造成的高感染性猪只呼吸疾病，其临床症状包括发烧、咳嗽、呕吐、呼吸困难、抑郁。感染本病后可能会引起急性肺炎造成骤死，或是慢性感染造成无症状出现的带原者。各种猪龄的猪只均会感染猪放线杆菌胸膜肺炎，尤其是6月龄以下的小猪发病率和死亡率最高。
猪胸膜肺炎放线杆菌属于革兰氏阴性杆菌，具有二种生物型，生物型1需要β-烟碱酰胺腺二核苷酸(β-nicotinamide adenine dinucleotide,β-NAD)，生物型2则不需要β-NAD。此外，根据荚膜多醣的差异，已有15种血清型的猪胸膜肺炎放线杆菌被确认，且所有的血清型皆具有致病能力。已知的毒力因子包括荚膜、脂多醣、外膜蛋白，以及最重要的细胞毒素(Apx)。
Apx毒素属于结构重复毒素家族(repeats in structural toxin,RTX)的成员。猪胸膜肺炎放线杆菌的15种血清型菌株共可产生四种不同的Apx毒素。每种血清型的菌株最多可产生3种Apx毒素。ApxI由1、5、9、10、11、14血清型菌株所分泌；ApxII除了第10、14血清型以外的其他血清型都可分泌；ApxIII由2、3、4、6、8、15血清型所分泌；ApxIV由所有的血清型所分泌，但只在感染时才会表现。
猪放线杆菌胸膜肺炎在全世界的养猪业造成严重的经济损失。猪胸膜肺炎放线杆菌感染的治疗包括使用如阿莫西林(amoxicillin)、氨芐青霉素(ampicillin)、头孢噻呋(ceftiofur)、恩诺沙星(enrofloxacin)、泰妙菌素(tiamulin)、青霉素(penicillin)以及青霉素/链霉素(penicillin/streptomycin)等的抗生素。然而，因为对抗生素产生抗性的问题日益严重且消费者对食物安全的需求日益增加，以疫苗预防猪胸膜肺炎放线杆菌感染已变得重要。大部分市售猪胸膜肺炎放线杆菌疫苗为全菌疫苗，这些疫苗可降低猪只死亡率，但却无法预防最初的感染及带原状态的发生。因此，发展更有效的猪胸膜肺炎放线杆菌疫苗是重要的。
发明内容
于一方面，本发明提供一种猪胸膜肺炎放线杆菌重组毒素蛋白(recombinant Apx toxins, re-Apx)，是以下列式(I)表示：
(A)m-(C3d片段)n；                    式(I)；
其中每一个A代表一个独立的猪胸膜肺炎放线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，每一个C3d片段是各自独立选自于SEQ ID NOs:22、23、24及25所组成的群组；
其中m是代表从1至约30的整数；以及
其中n是代表从0至约10的整数。
于另一方面，本发明提供一种编码所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)的核苷酸序列。
于另一方面，本发明提供一种猪放线杆菌胸膜肺炎免疫组合物，包含所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)以及一药学上可接受的载体。
于又一方面，本发明提供一种动物对抗猪放线杆菌胸膜肺炎的方法，包含使用有效量的上述免疫组合物以施予一动物，以增强所述动物对抗猪放线杆菌胸膜肺炎的免疫力。
于再一方面，本发明提供一种抗猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗体，是藉由所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)所制备或衍生而得。
于另一方面，本发明提供一种猪放线杆菌胸膜肺炎的检测试剂盒。所述检测试剂盒是用以侦测检验样本中是否含有猪胸膜肺炎放线杆菌毒素蛋白(Apx)，或侦测检验样本内是否含有抗猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗体。
本发明所提供的猪放线杆菌胸膜肺炎免疫组合物，与其他习用技术相互比较时，更具有下列的优点：
本发明所提供的猪放线杆菌胸膜肺炎免疫组合物含有一猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)。所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)包含至少一个猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗原决定位(epitopes)以及补体裂解片段C3d的部份氨基酸序列。所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)远比猪胸膜肺炎放线杆菌毒素蛋白(Apx)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，本发明所提供的猪放线杆菌胸膜肺炎免疫组合物所含的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)具有补体裂解片段C3d的部份氨基酸序列，因此可以增加特异性免疫反应，经试验结果显示，本发明所提供的猪放线杆菌胸膜肺炎免疫组合物可提高动物对抗猪胸膜肺炎放线杆菌感染的免疫性，显著增加受感染动物的存活率。
本发明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所示为根据实施例一至四，以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)确认纯化的本发明猪胸膜肺炎放线杆菌重组毒素蛋 白(re-Apx)；M：蛋白质分子量标记；第1道：猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)；第2道：猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)；第3道：猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)；第4道：猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)。
图2所示为根据实施例一至四，以西方墨点法确认纯化的本发明猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)；M：蛋白质分子量标记；第1道：猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)；第2道：猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)；第3道：猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)；第4道：猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)；一级抗体为碱性磷酸酶缀合的小鼠抗His标记单株抗体(Alkaline phosphatase-conjugated mouse anti-His，Invitrogen)。
图3为根据实施例九，以酵素连结免疫分析(ELISA)测定抗猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)抗体效价的结果；第1组为负对照组(PBS组)；第2组为含有实施例六所得的猪胸膜肺炎放线杆菌全菌多价疫苗(App1,2,5组)；第3组为实施例六所得的猪胸膜肺炎放线杆菌全菌及重组毒素蛋白混合多价疫苗(App1,2,5+re-ApxI～III组)；第4组为市售猪放线杆菌胸膜肺炎不活化疫苗，含有猪胸膜肺炎放线杆菌血清型第1,2,3,4,5,7型全菌(App1,2,3,4,5,7组)。符号**代表所述组与负对照组之间具有显著差异(p<0.01)；符号##代表两组之间具有显著差异(p<0.01)。
具体实施方式
本发明提供一种猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)，包含至少一个猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗原决定位(epitopes)，以诱导动物产生抗猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗体，并可进一步含补体裂解片段C3d的全长或部份氨基酸序列，以增加特异性免疫反应。本发明的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)是以下列式(I)表示：
(A)m-(C3d片段)n；                式(I)；
其中每一个A代表一个独立的猪胸膜肺炎放线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；以及
其中n是代表从0至约10的整数。
于一实施例中，所述补体裂解片段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:25所示的氨基酸序列。于一较佳实施例中，所述补体裂解片段C3d的部份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:24所示的氨基酸序列。于另一实施例中，所述补体裂解片段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:23所示的氨基酸序列。于另一较佳实施例中，所述补体裂解片段C3d的部份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:22所示的氨基酸序列。本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)具有0至10个单元重复的上述补体裂解片段C3d的全长或部份氨基酸序列，所述补体裂解片段C3d的全长或部份氨基酸序列较佳 为1至重复10个单元，更佳为重复4至8个单元。
于一实施例中，每一个A之间进一步以一个连接子连接，每一个连接子是各自独立选自于Gly-Gly、Gly-Ser及SEQ ID NOs:26、27、28、29、30、31、32、33、34、35及36。
于一实施例中，每一个C3d片段之间进一步以一个连接子连接，每一个连接子是各自独立选自于Gly-Gly、Gly-Ser及SEQ ID NOs:26、27、28、29、30、31、32、33、34、35及36。
于一实施例中，A与C3d片段之间进一步以一连接子连接，所述连接子是选自于Gly-Gly、Gly-Ser及SEQ ID NOs:26、27、28、29、30、31、32、33、34、35及36。
于一实施例中，所述猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白I(ApxI)，所述猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)，且每一个A是各自独立选自于SEQ ID NOs:37、4、39、40、41、42、43、44、45、46、47、48、49、50及51所示的氨基酸序列，所述猪胸膜肺炎放线杆菌毒素蛋白I(ApxI)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，分别以五段较长的抗原决定位氨基酸序列涵盖部分上述猪胸膜肺炎放线杆菌毒素蛋白I(ApxI)的抗原决定位，所述五段较长的抗原决定位氨基酸序列是分别如SEQ ID NOs:2、3、4、5、6所示。于一较佳实施例中，本发明的猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)含有二个以上所述较长的抗原决定位氨基酸序列，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，所述猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)包含如SEQ ID NO:7或8所示的氨基酸序列。
于一实施例中，所述猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白II(ApxII)，所述猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)，且每一个A是各自独立选自于SEQ ID NOs:14、52、53、54、55、56、57、58、59、60、61、62、63、64、65、67及68所示的氨基酸序列，所述猪胸膜肺炎放线杆菌毒素蛋白II(ApxII)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，分别以五段较长的抗原决定位氨基酸序列涵盖部分上述猪胸膜肺炎放线杆菌毒素蛋白II(ApxII)的抗原决定位，所述五段较长的抗原决定位氨基酸序列是分别如SEQ ID NOs:10、11、12、13、14所示。于一较佳实施例中，本发明的猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)含有二个以上所述较长的抗原决定位氨基酸序列，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，所述猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)包含如SEQ ID NO:15或16所示的氨基酸序列。
于一实施例中，所述猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白III(ApxIII)，所述猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)，且每一个A是各自独立选自于SEQ ID NOs:69、70、71、72、73、74、75、76、77、 78、79、80、81、82、83、84、85、86、87及88所示的氨基酸序列，所述猪胸膜肺炎放线杆菌毒素蛋白III(ApxIII)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，分别以二段较长的抗原决定位氨基酸序列涵盖部分上述猪胸膜肺炎放线杆菌毒素蛋白III(ApxIII)的抗原决定位，所述二段较长的抗原决定位氨基酸序列是分别如SEQ ID NOs:18、19所示。于一较佳实施例中，本发明的猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)含有所述二个所述较长的抗原决定位氨基酸序列，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，所述猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)包含如SEQ ID NO:20或21所示的氨基酸序列。
于一实施例中，所述猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白IV(ApxIV)，所述猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)，且每一个A是各自独立选自于SEQ ID NOs:93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116及117所示的氨基酸序列，所述猪胸膜肺炎放线杆菌毒素蛋白IV(ApxIV)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猪胸膜肺炎放线杆菌毒素蛋白IV(ApxIV)的抗原决定位，所述三段较长的抗原决定位氨基酸序列是分别如SEQ ID NOs:66、89、90所示。于一较佳实施例中，本发明的猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)含有二个以上所述较长的抗原决定位氨基酸序列，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，所述猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)包含如SEQ ID NO:91或92所示的氨基酸序列。
于部分实施态样中，本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)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。
本发明亦提供一种编码猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)的核苷酸序列。所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)包含至少一个猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗原决定位，以及重复0至10个单元的补体裂解片段C3d的部份氨基酸序列。所述编码本发明的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)的核苷酸序列，是由本发明的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)的氨基酸序列衍生而来。将本发明的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)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，本发明的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)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。
此外，本发明提供一种猪放线杆菌胸膜肺炎免疫组合物。所述猪放线杆菌胸膜肺炎免疫组合物包含本发明的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)以及一药学上可接受的载体。于一实施例中，所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)为猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)、猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)、猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)、猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)至少其中一种。于一较佳实施例中，所述猪放线杆菌胸膜肺炎免疫组合物包含猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)、猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)，与猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)，以及一药学上可接受的载体。于一较佳实施例中，所述猪放线杆菌胸膜肺炎免疫组合物包含猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)、猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)、猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)，与猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)，以及一药学上可接受的载体。
于一实施例中，所述猪放线杆菌胸膜肺炎免疫组合物可进一步包含至少一个猪胸膜肺炎放线杆菌血清型全菌。所述猪胸膜肺炎放线杆菌血清型全菌包括，但不限于，猪胸膜肺炎放线杆菌血清型1、2、3、4、5、6、7、8、9、10、11、12、13、14、15。于一较佳实施例中，所述猪放线杆菌胸膜肺炎免疫组合物进一步包含猪胸膜肺炎放线杆菌血清型1、2、5三型。
于一实施例中，本发明所提供的猪放线杆菌胸膜肺炎免疫组合物，进一步包含其他病原抗原，所述病原抗原包括，但不限于，猪环状病毒第二型(PCV2)抗原、猪流感病毒(SIV)抗原、猪繁殖与呼吸症候群病毒(PRRSV)抗原、猪支原体(Mycoplasma)、猪小病毒(Parvovirus,PPV)、猪丹毒(Erysipelas)、支气管败血性博德氏杆菌(Bordetella bronchiseptica)、败血性巴氏杆菌(Pasteurella multocida)，以及伪狂犬病(Aujeszky's disease)。
另，本发明所提供的猪放线杆菌胸膜肺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)；于一实施例中，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放线杆菌胸膜肺炎的方法，包含使用有效量的上述免疫组合物以施予一动物，以增强所述动物对抗猪放线杆菌胸膜肺炎的免疫力，增加感染后的存活率。
本发明并提供一种抗猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗体，是藉由本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)所制备或衍生而得；所述抗体包括但不限于：单株抗体、多株抗体，以及经基因重组的抗体。于一实施例中，所述抗体是为经由将本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)施打于一动物体内而得到的多株抗体。
于另一方面，本发明提供一种猪放线杆菌胸膜肺炎的检测试剂盒。所述检测试剂盒是用以侦测检验样本中是否含有猪胸膜肺炎放线杆菌毒素蛋白(Apx)，或侦测检验样本内是否含有抗猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗体。所述检测试剂盒包含但不限于：(1)一抗原，所述抗原是为本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)，于一实施例中，所述抗原是置于一抗原盘上；及/或(2)一抗体，所述抗体是由所述本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)所衍生、制备而得的单株抗体或多株抗体。
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、微晶片检验试剂盒(Microchip kit)、免疫萤光分析法(immuno fluorescent assay,IFA)检测试剂盒、或其他藉由所述猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)所制得的检测试剂盒。于一实施例中，所述检测试剂盒至少包含一含有本发明所提供的猪胸膜肺炎放线杆菌重组毒素蛋白(re-Apx)的抗原盘，可用以检验样本中是否含有抗猪胸膜肺炎放线杆菌毒素蛋白(Apx)的抗体。
于本发明中所使用的单数形式「一」、及「所述」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，包含复数个所述等样本及对所述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，皆为所述所属领域具有通常技艺者可共同了解的意义。
本发明是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。
实施例一猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)的构筑
自猪胸膜肺炎放线杆菌毒素蛋白I(ApxI)全长氨基酸序列(如SEQ ID NO:1所示)选出5个抗原决定位(epitopes)片段，分别为：
抗原决定位片段ApxI-1:
Figure PCTCN2016080469-appb-000001
抗原决定位片段ApxI-2:
Figure PCTCN2016080469-appb-000002
抗原决定位片段ApxI-3:
Figure PCTCN2016080469-appb-000003
抗原决定位片段ApxI-4:
Figure PCTCN2016080469-appb-000004
抗原决定位片段ApxI-5:
Figure PCTCN2016080469-appb-000005
这些抗原决定位片段中包含，但不限于，以下抗原决定位：
Figure PCTCN2016080469-appb-000006
由N端至C端依序连接抗原决定位片段ApxI-1、抗原决定位片段ApxI-2、抗原决定位片段ApxI-3、抗原决定位片段ApxI-4、抗原决定位片段ApxI-5，且分别在各抗原决定位片段之间以一个以上的连接子连接，所述连接子具有如SEQ ID NO:26所示的氨基酸序列；并且在抗原决定位片段ApxI-5的C端加上6个pC3d-p31生物佐剂(bioadjuvant)序列(如SEQ ID NO:22所示)，每个pC3d-p31生物佐剂序列皆以一个以上的连接子(SEQ ID NO:26)连接；得到的猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)氨基酸序列如SEQ ID NO:7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，于生物表现宿主中表现所述氨基酸序列并纯化。
此外，亦可以基因选殖方式构筑编码猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)的核酸序列，以限制酶切位连接DNA片段，并将所述核酸序列选殖至表现载体中，于生物表现宿主中表现所述氨基酸序列并纯化。于本实施例中，以HindIII限制酶切位连接编码猪胸膜肺炎放线杆菌毒素蛋白I的抗原决定位片段的DNA序列以及编码pC3d-p31生物佐剂片段的DNA序列，经过选 殖后得到的猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)的氨基酸序列如SEQ ID NO:8所示。以镍亲和管柱(nickel affinity chromatography)以及离子交换层析管柱(ion exchange chromatography)纯化后的猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)再以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)以及西方墨点法(western blot)确认，结果分别如图1(第1道)及图2(第2道)所示。猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)的分子量如预期的为约46kDa。
实施例二猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)的构筑
自猪胸膜肺炎放线杆菌毒素蛋白II(ApxII)全长氨基酸序列(如SEQ ID NO:9所示)选出5个抗原决定位片段，分别为：
抗原决定位片段ApxII-1:
Figure PCTCN2016080469-appb-000007
抗原决定位片段ApxII-2:
Figure PCTCN2016080469-appb-000008
抗原决定位片段ApxII-3:
Figure PCTCN2016080469-appb-000009
Figure PCTCN2016080469-appb-000010
抗原决定位片段ApxII-4:
Figure PCTCN2016080469-appb-000011
抗原决定位片段ApxII-5:
Figure PCTCN2016080469-appb-000012
这些抗原决定位片段中包含，但不限于，以下抗原决定位：
Figure PCTCN2016080469-appb-000013
Figure PCTCN2016080469-appb-000014
由N端至C端依序连接抗原决定位片段ApxII-1、抗原决定位片段ApxII-2、抗原决定位片段ApxII-3、抗原决定位片段ApxII-4、抗原决定位片段ApxII-5，且分别在各抗原决定位片段之间以一个以上的连接子连接，所述连接子具有如SEQ ID NO:26所示的氨基酸序列；并且在抗原决定位片段ApxII-5的C端加上6个pC3d-p31生物佐剂序列(如SEQ ID NO:22所示)，每个pC3d-p31生物佐剂序列皆以一个以上的连接子(SEQ ID NO:26)连接；得到的猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)氨基酸序列如SEQ ID NO:15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，于生物表现宿主中表现所述氨基酸序列并纯化。
此外，亦可以基因选殖方式构筑编码猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)的核酸序列，以限制酶切位连接DNA片段，并将所述核酸序列选殖至表现载体中，于生物表现宿主中表现所述氨基酸序列并纯化。于本实施例中，以HindIII限制酶切位连接编码猪胸膜肺炎放线杆菌毒素蛋白II的抗原决定位片段的DNA序列以及编码pC3d-p31生物佐剂片段的DNA序列，经过选殖后得到的猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxII)的氨基酸序列如SEQ ID NO:16所示。以镍亲和管柱(nickel affinity chromatography)以及离子交换层析管柱(ion exchange chromatography)纯化后的猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)再以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)以及西方墨点法(western blot)确认，结果分别如图1(第2道)及图2(第3道)所示。猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)的分子量如预期的为约47kDa。
实施例三猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)的构筑
自猪胸膜肺炎放线杆菌毒素蛋白III(ApxIII)全长氨基酸序列(如SEQ ID NO:17所示)选出2个抗原决定位片段，分别为：
抗原决定位片段ApxIII-1:
Figure PCTCN2016080469-appb-000015
抗原决定位片段ApxIII-2:
Figure PCTCN2016080469-appb-000016
这些抗原决定位片段中包含，但不限于，以下抗原决定位：
Figure PCTCN2016080469-appb-000017
Figure PCTCN2016080469-appb-000018
由N端至C端依序连接抗原决定位片段ApxIII-1与抗原决定位片段ApxIII-2，且分别在各抗原决定位片段之间以一个以上的连接子连接，所述连接子具有如SEQ ID NO:26所示的氨基酸序列；并且在抗原决定位片段ApxIII-2的C端加上6个pC3d-p31生物佐剂序列(如SEQ ID NO:22所示)，每个pC3d-p31生物佐剂序列皆以一个以上的连接子(SEQ ID NO:26)连接；得到的猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)氨基酸序列如SEQ ID NO:20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，于生物表现宿主中表现所述氨基酸序列并纯化。
此外，亦可以基因选殖方式构筑编码猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)的核酸序列，以限制酶切位连接DNA片段，并将所述核酸序列选殖至表现载体中，于生物表现宿主中表现所述氨基酸序列并纯化。于本实施例中，以HindIII限制酶切位连接编码猪胸膜肺炎放线杆菌毒素蛋白III的抗原决定位片段的DNA序列以及编码pC3d-p31生物佐剂片段的DNA序列，经过选殖后得到的猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)的氨基酸序列如SEQ ID NO:21所示。以镍亲和管柱(nickel affinity chromatography)以及离子交换层析管柱(ion exchange chromatography)纯化后的猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)再以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)以及西方墨点法(western blot)确认，结果分别如图1(第3道)及图2(第4道)所示。猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)的分子量如预期的为约49kDa。
实施例四猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)的构筑
自猪胸膜肺炎放线杆菌毒素蛋白IV(ApxIV)全长氨基酸序列(如SEQ ID NO:38所示)选出3个抗原决定位片段，分别为：
抗原决定位片段ApxIV-1:
Figure PCTCN2016080469-appb-000019
抗原决定位片段ApxIV-2:
Figure PCTCN2016080469-appb-000020
抗原决定位片段ApxIV-3:
Figure PCTCN2016080469-appb-000021
这些抗原决定位片段中包含，但不限于，以下抗原决定位：
Figure PCTCN2016080469-appb-000022
Figure PCTCN2016080469-appb-000023
由N端至C端依序连接抗原决定位片段ApxIV-1、抗原决定位片段ApxIV-2、抗原决定位片段ApxIV-3，且分别在各抗原决定位片段之间以一个以上的连接子连接，所述连接子具有如SEQ ID NO:26所示的氨基酸序列；并且在抗原决定位片段ApxIV-3的C端加上6个pC3d-p31生物佐剂序列(如SEQ ID NO:22所示)，每个pC3d-p31生物佐剂序列皆以一个以上的连接子(SEQ ID NO:26)连接；得到的猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)氨基酸序列如SEQ ID NO:91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，于生物表现宿主中表现所述氨基酸序列并纯化。
此外，亦可以基因选殖方式构筑编码猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)的核酸序列，以限制酶切位连接DNA片段，并将所述核酸序列选殖至表现载体中，于生物表现宿主中表现所述氨基酸序列并纯化。于本实施例中，以HindIII限制酶切位连接编码猪胸膜肺炎放线杆菌毒素蛋白IV的抗原决定位片段的DNA序列以及编码pC3d-p31生物佐剂片段的DNA序列，经过选殖后得到的猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)的氨基酸序列如SEQ ID NO:92所示。以镍亲和管柱(nickel affinity chromatography)以及离子交换层析管柱(ion exchange chromatography)纯化后的猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)再以十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)以及西方墨点法(western blot)确认，结果分别如图1(第4道)及图2(第5道)所示。猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)的分子量如预期的为约50kDa。
实施例五猪胸膜肺炎放线杆菌毒素萃取
1.猪胸膜肺炎放线杆菌的培养
将具有生产毒素能力的猪胸膜肺炎放线杆菌血清型第1型(台湾野外分离株，分泌ApxI、ApxII、ApxIV毒素)、第2型(台湾野外分离株，分泌ApxII、ApxIII、ApxIV毒素)全菌接种于含有0.01％(v/v)烟酰胺腺嘌呤二核苷酸(β-Nicotinamide adenine dinucleotide,β-NAD)以及10％(v/v)马血清的脑心浸出液(BHI)液体培养基内(BD公司，美国)，于37℃、5％CO2下培养隔夜。
2.猪胸膜肺炎放线杆菌毒素的制备
将上述猪胸膜肺炎放线杆菌血清型第1、2型菌液以超音波振荡器(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0.22μm孔径过滤膜(Millipore公司，美国)过滤后，此过滤液即为猪胸膜肺炎放线杆菌粗萃毒素蛋白ApxI～IV(crude extracted ApxI～IV)，分装后保存于-80℃备用。
3.不活化猪胸膜肺炎放线杆菌的制备
将具有生产毒素能力的猪胸膜肺炎放线杆菌血清型第1型(台湾野外分离株)、第2型(台湾野外分离株)、第5型(台湾野外分离株)全菌接种于含有0.01％(v/v)烟酰胺腺嘌呤二核苷酸(β-NAD)以及10％(v/v)马血清的脑心浸出液(BHI)液体培养基内(BD公司，美国)，于37℃、5％CO2下培养至少隔夜，接着加入甲醛以进行灭活作用，得到灭活猪胸膜肺炎放线杆菌。
实施例六猪胸膜肺炎放线杆菌疫苗的配制
1.猪胸膜肺炎放线杆菌重组毒素蛋白亚单位疫苗(re-ApxI、re-ApxII、re-ApxIII、re-ApxIV)的配制
分别将实施例一至实施例四所得到的猪胸膜肺炎放线杆菌重组毒素蛋白re-ApxI(SEQ ID NO:8)(最终浓度为500μg/ml)、re-ApxII(SEQ ID NO:16)(最终浓度为500μg/ml)、re-ApxIII(SEQ ID NO:21)(最终浓度为500μg/ml)、re-ApxIV(SEQ ID NO:92)(最终浓度为500μg/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，以配制成为猪胸膜肺炎放线杆菌重组蛋白亚单位疫苗。
2.猪胸膜肺炎放线杆菌全菌多价疫苗(App1,2,5)的配制
将实施例五所得到的灭活猪胸膜肺炎放线杆菌血清型第1,2,5型全菌(各菌株最终浓度皆为1x 109cfu/ml)以及磷酸盐缓冲溶液(PBS)混和均匀，并加入铝胶[最终浓度为30％(v/v)]作为佐剂，以配制成为猪胸膜肺炎放线杆菌全菌多价疫苗(App1,2,5)。
3.猪胸膜肺炎放线杆菌全菌及重组毒素蛋白混合多价疫苗(App1,2,5+re-ApxI～III)的配制
将实施例一至实施例三所得到的猪胸膜肺炎放线杆菌重组毒素蛋白re-ApxI(SEQ ID NO:8)(最终浓度为20μg/ml)、re-ApxII(SEQ ID NO:16)(最终浓度为20μg/ml)、re-ApxIII(SEQ ID NO:21)(最终浓度为20μg/ml)、实施例五所得到的灭活猪胸膜肺炎放线杆菌血清型第1,2,5型全菌(各菌株最终浓度皆为1x 109cfu/ml)以及磷酸盐缓冲溶液(PBS)混和均匀，并加入铝胶[最终浓度为30％(v/v)]作为佐剂，以配制成为猪胸膜肺炎放线杆菌全菌及重组毒素蛋白混合多价疫苗(App1,2,5+re-ApxI～III)。
实施例七猪胸膜肺炎放线杆菌重组毒素蛋白疫苗有效性分析
1.小鼠免疫及以猪胸膜肺炎放线杆菌粗萃毒素蛋白攻毒试验
取猪胸膜肺炎放线杆菌抗体阴性的3～4周龄健康ICR小鼠(国家实验动物中心，台湾)60只，随机分为6组，每组10只；第1组为负对照组，第2～6组为免疫试验组；各组每只小鼠分别以腹腔注射(ip.)0.2ml的以下物质：
第1组：含30％(v/v)铝胶的PBS缓冲溶液(PBS组)；
第2组：含实施例一所得的猪胸膜肺炎放线杆菌重组毒素蛋白I(re-ApxI)(SEQ ID NO:8)的亚单位疫苗(re-ApxI组)；
第3组：含实施例二所得的猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII)(SEQ ID NO:16)的亚单 位疫苗(re-ApxII组)；
第4组：含实施例三所得的猪胸膜肺炎放线杆菌重组毒素蛋白III(re-ApxIII)(SEQ ID NO:21)的亚单位疫苗(re-ApxIII组)；
第5组：含实施例四所得的猪胸膜肺炎放线杆菌重组毒素蛋白IV(re-ApxIV)(SEQ ID NO:92)的亚单位疫苗(re-ApxIV组)；以及
第6组：含实施例六所得的猪胸膜肺炎放线杆菌全菌多价疫苗(App1,2,5组)。
每只小鼠第一次免疫后第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天再对每只小鼠注射0.1ml的实施例五所得的猪胸膜肺炎放线杆菌粗萃毒素蛋白ApxI～IV以进行攻毒。以所述全菌粗萃毒素蛋白的LD90剂量(萃取APP1菌量6.5x109cfu/ml及APP2菌量9.65x1010cfu/ml)进行攻毒，十天后纪录小鼠死亡数，以计算各组存活率。
2.统计方法
小鼠存活率以Kaplan-Meier存活分析(Kaplan-Meier Survival Analysis:Log-Rank test)进行统计，并以p<0.05视为具有统计上的显著差異。
3.结果
小鼠攻毒试验结果如表1所示，各别含有猪胸膜肺炎放线杆菌重组毒素蛋白re-ApxI、re-ApxII、re-ApxIII、re-ApxIV的亚单位疫苗(即第2～5组)相较于负对照组(第1组，存活率0％)确实能诱发小鼠产生保护效力，并耐过猪胸膜肺炎放线杆菌粗萃毒素蛋白ApxI～IV的攻毒，提高存活率并且具有统计上的显著差异。
表1小鼠攻毒试验存活率
试验组别 小鼠数目 存活率
第1组(PBS组) 10 0％
第2组(re-ApxI组) 10 50％*
第3组(re-ApxII组) 10 50％*
第4组(re-ApxIII组) 10 50％**
第5组(re-ApxIV组) 10 30％*
第6组(App1,2,5组) 10 20％
*表示相对于负对照组有统计上的显著差异(p<0.05)。
**表示相对于负对照组有统计上的显著差异(p<0.01)。
实施例八猪胸膜肺炎放线杆菌多价疫苗对猪胸膜肺炎放线杆菌血清型第2型菌株(App2)的保护效力的分析
1.小鼠免疫及以猪胸膜肺炎放线杆菌血清型第2型菌株(App2)攻毒试验
取猪胸膜肺炎放线杆菌抗体阴性3～4周龄健康的ICR小鼠(国家实验动物中心，台湾)40只，随机分为4组，每组10只；第1组为负对照组，第2～4组为免疫试验组；各组每只小鼠分别以腹腔注射(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(PBS组)；
第2组：实施例六所得的猪胸膜肺炎放线杆菌全菌多价疫苗(App1,2,5组)；
第3组：实施例六所得的猪胸膜肺炎放线杆菌全菌及重组毒素蛋白混合多价疫苗(App1,2,5+re-ApxI～III组)；以及
第4组：市售猪放线杆菌胸膜肺炎灭活疫苗，含有猪胸膜肺炎放线杆菌血清型第1,2,3,4,5,7型全菌(App1,2,3,4,5,7组)。
每只小鼠第一次免疫后第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天再对每只小鼠注射0.1ml的LD90剂量(7.5x108cfu/ml)的猪胸膜肺炎放线杆菌血清型第2型全菌(App2)以进行攻毒，观察小鼠10天并纪录死亡数，以计算各组存活率。
2.统计方法同实施例七所述。
3.结果
小鼠攻毒试验结果如表2所示，相较于负对照组(第1组，存活率为20％)，各免疫试验组的存活率皆显著增加，并具有统计上的差异。尤其是含有本发明重组毒素蛋白re-ApxI～III的多价疫苗(App1,2,5+re-ApxI～III组)对小鼠的保护效果最好，存活率最高。
表2以猪胸膜肺炎放线杆菌血清型第2型菌株(App2)对小鼠攻毒试验的存活率
试验组别 小鼠数目 存活率
第1组(PBS组) 10 20％
第2组(App1,2,5组) 10 70％**
第3组(App1,2,5+re-ApxI～III组) 10 80％**
第4组(市售App1,2,3,4,5,7组) 10 60％*
*表示相对于负对照组有统计上的显著差异(p<0.05)。
**表示相对于负对照组有统计上的显著差异(p<0.01)。
实施例九猪胸膜肺炎放线杆菌多价疫苗对猪胸膜肺炎放线杆菌血清型第5型菌株(App5) 的保护效力的分析
1.小鼠免疫及以猪胸膜肺炎放线杆菌血清型第5型菌株(App5)攻毒试验
取猪胸膜肺炎放线杆菌抗体阴性3～4周龄健康的ICR小鼠(国家实验动物中心，台湾)50只，随机分为4组；第1组为负对照组，第2～4组为免疫试验组；各组每只小鼠分别以腹腔注射(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(PBS组)(n＝15)；
第2组：实施例六所得的猪胸膜肺炎放线杆菌全菌多价疫苗(App1,2,5组)(n＝15)；
第3组：实施例六所得的猪胸膜肺炎放线杆菌全菌及重组毒素蛋白混合多价疫苗(App1,2,5+re-ApxI～III组)(n＝10)；以及
第4组：市售猪放线杆菌胸膜肺炎灭活疫苗，含有猪胸膜肺炎放线杆菌血清型第1,2,3,4,5,7型全菌(App1,2,3,4,5,7组)(n＝10)。
每只小鼠分别于第一次免疫前24小时采血保存，第一次免疫后第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(ELISA)；采血后，对每只小鼠注射0.1ml的LD90剂量(7.32x108cfu/ml)的猪胸膜肺炎放线杆菌血清型第5型全菌(App5)以进行攻毒，观察小鼠10天并纪录死亡数，以计算各组存活率。
2.毒素抗体的酵素连结免疫分析(ELISA)
以猪胸膜肺炎放线杆菌重组毒素蛋白II(re-ApxII，SEQ ID NO:16)作为抗原，并将抗原涂布(coating)于ELISA用96孔盘(Thermo公司，美国)(100ng/孔)，于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)，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(1:100)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)，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)，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100μl3,3’,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(
Figure PCTCN2016080469-appb-000024
M2/M2 ELISA Reader，Molecular Devices公司，美国)读取波长650nm的吸光值(OD650nm)。
3.统计方法
小鼠存活率以Kaplan-Meier存活分析(Kaplan-Meier Survival Analysis:Log-Rank test)进行统计，并以p<0.05视为具有统计上的显著差異。酵素连结免疫分析(ELISA)结果以Student Newman-Keuls Method方法进行统计，并以p<0.05视为具有统计上的显著差異。
4.结果
小鼠攻毒试验结果如表3所示，相较于负对照组(第1组，存活率为6.7％)，各免疫试验组的存活率皆显著增加，并具有统计上的差异。尤其是含有本发明重组毒素蛋白re-ApxI～III的多价疫苗(App1,2,5+re-ApxI～III组)对小鼠的保护效果最好，存活率最高。
表3以猪胸膜肺炎放线杆菌血清型第5型菌株(App5)对小鼠攻毒试验的存活率
试验组别 小鼠数目 存活率
第1组(PBS组) 15 6.7％
第2组(App1,2,5组) 15 26.7％*
第3组(App1,2,5+re-ApxI～III组) 10 70％**
第4组(市售App1,2,3,4,5,7组) 10 60％*
*表示相对于负对照组有统计上的显著差异(p<0.05)。
**表示相对于负对照组有统计上的显著差异(p<0.01)。
酵素连结免疫分析(ELISA)结果如图3所示，免疫含有本发明的猪胸膜肺炎放线杆菌全菌及重组蛋白混合多价疫苗的小鼠(第3组，即App1,2,5+ApxI～III组)，在第二次免疫第10天后，其血清中的抗猪胸膜肺炎放线杆菌毒素蛋白抗体效价显著高于负对照组小鼠(第1组，即PBS组，p<0.01)、免疫猪胸膜肺炎放线杆菌全菌多价疫苗(第2组，即App1,2,5组，p<0.01)、免疫市售猪放线杆菌胸膜肺炎灭活疫苗(第4组，即市售App1,2,3,4,5,7组，p<0.01)的小鼠的抗体效价。由此可知，本发明所提供的猪胸膜肺炎放线杆菌全菌及重组蛋白混合多价疫苗可以在动物体内有效地诱导出抗猪胸膜肺炎放线杆菌毒素蛋白抗体，具免疫原性，且效果显著优于习知疫苗。
实施例十抗猪胸膜肺炎放线杆菌毒素I～IV(ApxI～IV)多株抗体的制备
分别将实施例一至实施例四所得到的猪胸膜肺炎放线杆菌重组蛋白re-ApxI(SEQ ID NO:8)、re-ApxII(SEQ ID NO:16)、re-ApxIII(SEQ ID NO:21)、re-ApxIV(SEQ ID NO:92)与佛氏完全佐剂(Freund’s complete adjuvant,FCA，Sigma)充分混合乳化后，再以腹腔注射方式施与Balb/c小鼠(0.2ml/只)以进行初级免疫，3周后以相同方式进行第二次免疫，隔周采集免疫小鼠的血清，即制得抗猪胸膜肺炎放线杆菌毒素I～IV(ApxI～IV)的多株抗体，所述多株抗体是用于西方墨点法进行抗原分析。
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 (22)

  1. 一种猪胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae)重组毒素蛋白，其特征在于以下列式(I)表示：
    (A)m-(C3d片段)n；  式(I)；
    其中每一个A代表一个独立的猪胸膜肺炎放线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，所述每一个C3d片段是各自独立选自于由SEQ ID NOs:22、23、24及25所组成的群组；
    其中m是代表从1至约30的整数；以及
    其中n是代表从0至约10的整数。
  2. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于每一个A之间以一个连接子连接，每一个连接子是各自独立选自于由Gly-Gly、Gly-Ser及SEQ ID NOs:26、27、28、29、30、31、32、33、34、35及36所组成的群组。
  3. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于每一个C3d片段之间以一个连接子连接，每一个连接子是各自独立选自于由Gly-Gly、Gly-Ser及SEQ ID NOs:26、27、28、29、30、31、32、33、34、35及36所组成的群组。
  4. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于A与C3d片段之间以一连接子连接，所述连接子是选自于由Gly-Gly、Gly-Ser及SEQ ID NOs:26、27、28、29、30、31、32、33、34、35及36所组成的群组。
  5. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白I，所述的猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白I，且每一个A是各自独立选自于由SEQ ID NOs:2、3、4、5、6、37、39、40、41、42、43、44、45、46、47、48、49、50及51所组成的群组。
  6. 根据权利要求5所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌重组毒素蛋白I包含如SEQ ID NO:7或8所示的氨基酸序列。
  7. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白II，所述的猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白II，且每一个A是各自独立选自于由SEQ ID NOs:10、11、12、13、14、52、53、54、55、56、57、58、59、60、61、62、63、64、65、67及68所组成的群组。
  8. 根据权利要求7所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌重组毒素蛋白II包含如SEQ ID NO:15或16所示的氨基酸序列。
  9. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白III，所述的猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白III，且每一个A是各自独立选自于由SEQ ID NOs:18、19、69、 70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87及88所组成的群组。
  10. 根据权利要求9所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌重组毒素蛋白III包含如SEQ ID NO:20或21所示的氨基酸序列。
  11. 根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌毒素蛋白为猪胸膜肺炎放线杆菌毒素蛋白IV，所述的猪胸膜肺炎放线杆菌重组毒素蛋白为猪胸膜肺炎放线杆菌重组毒素蛋白IV，且每一个A是各自独立选自于由SEQ ID NOs:66、89、90、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116及117所组成的群组。
  12. 根据权利要求11所述的猪胸膜肺炎放线杆菌重组毒素蛋白，其特征在于所述的猪胸膜肺炎放线杆菌重组毒素蛋白IV包含如SEQ ID NO:91或92所示的氨基酸序列。
  13. 一种编码根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白的核苷酸序列。
  14. 一种猪放线杆菌胸膜肺炎免疫组合物，其特征在于包含一根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白以及一药学上可接受的载体。
  15. 根据权利要求14所述的猪放线杆菌胸膜肺炎免疫组合物，其特征在于所述的猪胸膜肺炎放线杆菌重组毒素蛋白是选自由下列群组所组成的至少一者：一根据权利要求5所述的猪胸膜肺炎放线杆菌重组毒素蛋白I、一根据权利要求7所述的猪胸膜肺炎放线杆菌重组毒素蛋白II、一根据权利要求9所述的猪胸膜肺炎放线杆菌重组毒素蛋白III，以及一根据权利要求11所述的猪胸膜肺炎放线杆菌重组毒素蛋白IV。
  16. 根据权利要求14所述的猪放线杆菌胸膜肺炎免疫组合物，其特征在于进一步包含至少一个猪胸膜肺炎放线杆菌血清型全菌。
  17. 根据权利要求14所述的猪放线杆菌胸膜肺炎免疫组合物，其特征在于进一步包含其他病原抗原，所述病原抗原是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、猪支原体(Mycoplasma)、猪小病毒(Parvovirus,PPV)、猪丹毒(Erysipelas)、支气管败血性博德氏杆菌(Bordetella bronchiseptica)、败血性巴氏杆菌(Pasteurella multocida)，以及伪狂犬病(Aujeszky's disease)。
  18. 一种动物对抗猪放线杆菌胸膜肺炎的方法，其特征在于包含使用根据权利要求14所述的免疫组合物以施予一动物，以增强所述动物对抗猪放线杆菌胸膜肺炎的免疫力。
  19. 一种抗猪胸膜肺炎放线杆菌毒素蛋白的抗体，其特征在于藉由根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白所制备而得。
  20. 根据权利要求19所述的抗体，其特征在于所述的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。
  21. 一种猪放线杆菌胸膜肺炎的检测试剂盒，其特征在于包含一侦测单元，所述的侦测单元是选自于下列群组所组成中至少一者：一根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白，以及一藉由根据权利要求1所述的猪胸膜肺炎放线杆菌重组毒素蛋白所制备的抗体。
  22. 根据权利要求21所述的检测试剂盒，其特征在于所述的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/CN2016/080469 2015-04-28 2016-04-28 猪胸膜肺炎放线杆菌重组毒素蛋白及其应用 WO2016173504A1 (zh)

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