TWI642681B - Recombinant protein of pasteurella multocida toxin, its virus like particle and application thereof - Google Patents

Abstract

The present invention relates to a recombinant protein of Pasteurella septicum toxin having an epitope of three S. septicum toxin proteins. The three epitopes have the sequences shown in SEQ ID NOs: 2, 3 and 4, respectively. The present invention also relates to a viroid-like particle containing the recombinant protein of Pasteurella septicum toxin, a recombinant protein encoding the S. septicum toxin or a nucleic acid sequence encoding the viral particle containing the recombinant protein of Pasteurella septicum toxin And a porcine atrophic rhinitis immunological composition comprising the recombinant protein of Pasteurella septicum and/or the viral particle containing the recombinant protein of Pasteurella septicum.

Description

Recombinant protein of Pasteurella septicum, its virion and its application

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 (AR) 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. In severe infections, 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 (AR) is caused by Bordetella bronchiseptica (Bb) and Pasteurella multocida type A (PmA) and D-type bacteria (PmD), especially Toxin produced by Pasteurella multocida toxin (PmD) of Pasteurella multocida (PmD). 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 of an S. septicum toxin protein having the amino acid sequence of SEQ ID NO: 2 (epitopes) An epitope of a S. septicum toxin protein having an amino acid sequence as shown in SEQ ID NO: 3, and a Pasteurella septicum having an amino acid sequence as shown in SEQ ID NO: The epitope of the toxin protein.

The invention provides a virus like particle (VLP) containing a recombinant protein of Pasteurella septicum toxin, comprising: a recombinant protein of Pasteurella septicum toxin and a hepatitis B virus core as described above Hepatitis B virus core protein (HB); wherein the recombinant protein of Pasteurella septicum toxin is inserted into the major immunodominant region (MIR) of the hepatitis B virus core protein.

The present invention provides, in a third part, a nucleic acid sequence encoding a recombinant protein of Pasteurella septicum toxin as described above.

In a fourth aspect, the invention provides a nucleic acid sequence encoding a viroid-like particle comprising a recombinant protein of Pasteurella septicum toxin as described above.

The present invention provides, in a fifth aspect, a porcine atrophic rhinitis immunological composition comprising at least one of the aforementioned S. septicum toxin recombinant protein and a viroid-like particle containing the S. septicum toxin recombinant protein as described above, and A pharmaceutically acceptable carrier.

In a sixth aspect, 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 a seventh aspect, an antibody against a septicum 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 selected from the group consisting of at least one of the following groups: a Pasteurella septicum a recombinant protein of toxin, a viroid-like particle containing the recombinant protein of Pasteurella multocida toxin as described above, an antibody prepared by the recombinant protein of Pasteurella septicum toxin as described above, and a septic-containing bar as described above An antibody prepared from a viroid-like particle of a recombinant protein of a toxin.

The present invention is exemplified by the following examples and the accompanying drawings, but the invention is not limited by the following examples.

The present invention provides a recombinant protein of septicum Pasteurella toxin (re-PmT) comprising three epitopes of P. septicum toxin protein (PmT) to induce an animal to produce a septic-resistant Pasteurella toxin Protein (PmT) antibody. The three epitopes are: epitope A: SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO: 2); epitope B: SISPDDPPREITD (SEQ ID NO: 3); and epitope C: LNSTPGTGRPMP (SEQ ID NO: 4) ).

In certain preferred embodiments, the amino acid sequences of each of the epitopes may be further linked by a linker comprising at least one glycine (Glycine, Gly), the linkage Subunits include, but are not limited to, Gly-Gly, Gly-Ser, SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. In one embodiment, the linker has an amino acid sequence as set forth in SEQ ID NO: 11. However, each of these epitope-determining amino acid sequences is not necessarily linked by a linker.

The recombinant S. septicum toxin (re-PmT) provided by the present invention can be represented by the following formula: (antigenic epitope 1) - (linker 1) _m - (antigenic epitope 2) - (linker) 2) _n - (epitope 3) Formula (I) This epitope 1, epitope 2, epitope 3, one of which has the amino acid sequence of SEQ ID NO: 2, the other two epitopes Each having an amino acid sequence as SEQ ID NO: 3 and SEQ ID NO: 4; 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; wherein m is an integer representing from 0 to about 10; wherein n is an integer from 0 to about 10.

In certain embodiments, the S. septicum toxin recombinant protein (re-PmT) provided by the present invention has at least the amino acid sequence shown in SEQ ID NOs: 5, 22, 23, 24, 25, and 26. One.

In some embodiments, 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. , 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%, Approximately 97%, approximately 98%, approximately 99% sequence homology.

The present invention also provides a viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella septicum toxin, which inserts or replaces an epitope of three S. septicum toxin proteins (PmT) into A major immunodominant region (MIR) of Hepatitis B virus core protein (HB) to form a viroid-like particle containing a recombinant protein of Pasteurella septicum (re-PmT VLP) ). The epitopes of the three P. septicum toxin proteins (PmT) are: epitope A: SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO: 2); epitope B: SISPDDPPREITD (SEQ ID NO: 3); Decision bit C: LNSTPGTGRPMP (SEQ ID NO: 4).

In certain preferred embodiments, the hepatitis B virus core protein (HBc) has an amino acid sequence as set forth in SEQ ID NO: 6.

In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located 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 located 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 located 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 located at the 78th to 79th amino acid positions of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at the 78th 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 located at the 78th to 82th amino acid positions of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at the 78th to 86th amino acid positions of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at the 78th to 89th amino acid positions of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at the 78th to 94th amino acid positions of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at positions 81-82 of the protein. In certain embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at positions 82-83 of the amino acid of the protein.

In certain preferred embodiments, the amino acid sequence 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 ligated by a linker (linker) a ligation comprising at least one glycine (Glycine, Gly), the linker comprising but not limited to: Gly-Gly, Gly-Ser, SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. In one embodiment, the linker has an amino acid sequence as set forth in SEQ ID NOs: 11 and/or 12. However, each of these epitope-determining amino acid sequences is not necessarily linked by a linker.

The viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella multocida toxin provided by the present invention can be represented by the following formula: (HBc-N terminal segment) - (linker 3) _p - (antigen determination) Bit 1)-(linker 1) _m - (antigenic epitope 2)- (linker 2) _n - (antigenic epitope 3) - (linker 4) _q - (HBc-C end segment) Formula (II) One of the epitope 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. The 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; wherein m is an integer representing from 0 to about 10; wherein n is an integer from 0 to about 10; wherein p is an integer from 0 to about 10; wherein q Is an integer representing from 0 to about 10.

In certain embodiments, the N-terminal portion of the hepatitis B virus core protein (HBc-N-terminal segment) has the sequence of the first to the 73 amino acids of the HBc protein, and the C-terminal portion of the hepatitis B virus core protein (HBc) -C-terminal segment) A sequence having 83 to 144 amino acids of HBc protein. In certain embodiments, the HBc-N terminus has a sequence of 1 to 75 amino acids of the HBc protein, and the HBc-C terminus has a sequence of 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 79 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 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. 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 87 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 90 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 95 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has a sequence of 1 to 81 amino acids of the HBc protein, and the HBc-C terminus has a sequence of 82 to 144 amino acids of the HBc protein. In certain embodiments, the HBc-N terminus has the sequence of the 1st to 82th amino acids of the HBc protein, and the HBc-C terminus has the sequence of the 83th to 144th amino acids of the HBc protein.

In certain embodiments, a viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella septicum 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 represented by 32, 33, 34, 35, and 36.

In some embodiments, 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 with the amino acid sequence represented by the above formula (II). Homology, preferably, has about 85% sequence homology, and 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 VLP) encoding the recombinant protein of Pasteurella multocida toxin of the present invention. Nucleic acid sequence. The S. septicum toxin recombinant protein (re-PmT) comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4. The viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4 and a B as shown in SEQ ID NO: 6. Hepatitis virus core protein (HBc), wherein the antigenic localization system inserts or replaces the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc).

Nucleotide sequence encoding the recombinant protein of Resin septicum toxin (re-PmT) of the present invention and the nucleic acid encoding the viroid-like particle (re-PmT VLP) of the recombinant protein of S. septicum toxin of the present invention The sequence is an amino acid sequence of the recombinant protein of recombinant Pasteurella toxin (re-PmT) of the present invention and an amine of the viroid-like particle (re-PmT VLP) of the recombinant protein of the present invention containing Pasteurella toxin The base acid sequence is derived. The amino acid sequence of the recombinant S. septicum toxin (re-PmT) amino acid sequence of the present invention and the viroid-like particle (re-PmT VLP) of the recombinant protein of S. septicum toxin of the present invention Each of the amino acids is replaced by a nucleotide sequence encoding the amino acid listed in the genetic code table (including various degenerate codons, or synonymous codons). The nucleotide sequence provided by the present invention can be obtained. For example, the septic Aspergillus toxin recombinant protein (re-PmT) of the present invention and the serine on the amino acid sequence of the viroid-like particle (re-PmT VLP) of the present invention containing the recombinant protein of Pasteurella toxin The serine can be encoded by a nucleotide sequence such as TCT, TCC, TCA, TCG, AGT, AGC or the like. The respective amino acids on 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 S. septicum toxin of the present invention may be The following nucleotide sequences are encoded:  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> The S. septicum toxin recombinant protein (rPMT) of the present invention and the septicemia of the present invention The amino acid of the amino acid sequence of the virus-like particle (re-PmT VLP) of the recombinant protein of the Pasteurella toxin </td><td> encodes the nucleotide sequence of the amino acid</td></ Tr><tr><td> Alanine </td><td> GCA, GCC, GCG, GCT </td></tr><tr><td> Cysteine </ Td><td> TGC, TGT </td></tr><tr><td> Aspartic acid </td><td> GAC, GAT </td></tr><tr ><td> Glutamic acid </td><td> GAA, GAG </td></tr><tr><td> Phenylalanine </td><td> TTC, TTT </td></tr><tr><td> Glycine </td><td> GGA, GGC, GGG, GGT </td></tr><tr><td> Histamine Acid (Histidine) </td><td> CAC, CAT </td></tr><tr><td> Isoleucine </td><td> ATA, ATC, ATT </td ></tr><tr><td> Lysine </td><td> AAA, AAG </td></tr><tr><td> Leucine </td ><td> CTA, CTC, CTG, CTT, TTA, TTG </td></tr><tr><td> Methylthioamide (Methionine) </td><td> ATG </td></tr><tr><td> Asparagine </td><td> AAC, AAT </td></tr> Tr><td> Proline </td><td> CCA, CCC, CCG, CCT </td></tr><tr><td> Glutamine </td> <td> CAA, CAG </td></tr><tr><td> Arginine </td><td> AGA, AGG, CGA, CGC, CGG, CGT </td></ Tr><tr><td> Serine </td><td> AGC, AGT, TCA, TCC, TCG, TCT </td></tr><tr><td> Hydroxybutyric acid (Threonine) </td><td> ACA, ACC, ACG, ACT </td></tr><tr><td> ursolic acid (Valine) </td><td> GTA, GTC, GTG , GTT </td></tr><tr><td> Tryptophan </td><td> TGG </td></tr><tr><td> Tyrosine </td><td> TAC, TAT </td></tr></TBODY></TABLE>

In addition, the present invention also provides a porcine atrophic rhinitis immune 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. The viroid-like particle (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin comprises an epitope as shown in SEQ ID NOs: 2, 3 and 4 and a B as shown in SEQ ID NO: 6. Hepatitis virus core protein (HBc), wherein the antigenic localization system inserts or replaces the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc).

In certain embodiments, the S. septicum toxin recombinant protein (re-PmT) has an amino acid sequence as set forth in SEQ ID NOs: 5, 22, 23, 24, 25 or 26. In a specific embodiment, the viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella multocida toxin has an amino acid sequence as set forth in SEQ ID NO: 9. In another specific embodiment, the viroid-like particle (re-PmT VLP) comprising a recombinant protein of Pasteurella septicum toxin has an amino acid sequence as set forth in SEQ ID NO: 10. In still another embodiment, 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, 35 One of the amino acid sequences shown at 36.

The septic Aspergillus 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, by genetic selection or by Synthesized by a peptide synthesizer; the method for obtaining the recombinant protein by gene selection may be, but not limited to, a nucleic acid sequence or coding containing a recombinant protein of recombinant Pasteurella toxin (re-PmT) The nucleic acid sequence of the viruvirus-like particle (re-PmT VLP) of 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 encoding the S. septicum toxin recombinant protein (re-PmT) a plastid containing a nucleic acid sequence encoding a nucleic acid sequence of a viroid-like particle (re-PmT VLP) containing a recombinant protein of Pasteurella septicum toxin, and then transferring the plastid into a biological expression host, after protein expression And the antigen protein obtained.

The expression vector system includes, but is not limited to, a pET vector system and a pGEX vector system, etc.; the biological expression system (host) includes, but is not limited to, a prokaryotic expression system (eg, E. coli ), a eukaryotic expression system (eg, : animal cells (insect cells or mammalian cells), plant cells).

In one embodiment, the porcine atrophic rhinitis immunological 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 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 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 bacteria (PmD) may be, for example, However, it is not limited to strains such as the British National Standards Collection of Biological Products (NCTC) number NCTC 12178, or strains derived from field isolation.

The porcine atrophic rhinitis immunological 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, porcine reproduction. Respiratory Syndrome Virus (PRRSV) antigen, Mycoplasma, Parvovirus (PPV), Erysipelas, Aujeszky's disease, and/or Actinobacillus pleuropneumonia , APP).

In addition, the porcine atrophic rhinitis 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, and a shape. Agents, 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, surfactant 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 which does not deleteriously react with the active composition. Suitable excipients include, but are not limited to, water, salt solutions, vegetable oils, polyethylene glycols, gelatin, amylose, lactose, magnesium stearate, talc, citric acid, viscous paraffins, 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 gum, alum, Freund's incomplete adjuvant, oily adjuvant, water soluble adjuvant, or water-in-oil-in-water two-phase adjuvant (water- In-oil-in-water, W/O/W); In one embodiment, the adjuvant is an aqueous aluminum hydroxide gel.

Further, 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 clinical symptoms. , survival rate, and weight gain trends.

The present invention also provides an antibody against P. septicum D-type toxin (PmT), which is a recombinant protein of septice septicum toxin (re-PmT) provided by the present invention and/or contains Pasteurella septicum The virus-like particle (re-PmT VLP) of the toxin recombinant protein is prepared or derived; the antibody includes, but is not limited to, a monoclonal antibody, a polyclonal antibody, and a recombinant antibody. In one embodiment, the anti-system is a plurality of antibodies obtained by administering the S. septicum toxin recombinant protein (rPMT) provided by the present invention to an animal.

The invention also provides a test kit for porcine atrophic rhinitis, which is used for detecting whether a sample contains a septicemia type D-toxin (PmT) or detecting whether a sample contains anti-septic An antibody to Pasteurella D-type toxin (PmT). The test kit comprises, but is not limited to: (1) an antigen which is a recombinant protein of septicum toxin of the septicum (re-PmT) and/or a recombinant protein containing Pasteurella septicum a virus-like particle (re-PmT VLP), in one embodiment, the antigen is placed on an antigenic disk; and/or (2) an antibody, the anti-system is provided by the present invention A monoclonal antibody or a plurality of antibodies derived from a recombinant protein of recombinant toxin (re-PmT) and/or a viroid-like particle (re-PmT VLP) containing a recombinant protein of Pasteurella multocida toxin.

The form of the test kit includes, but is not limited to, an enzyme-linked immu sorbent assay (ELISA) kit, a microchip test kit (Microchip kit), and an immunofluorescence assay (IMA) assay. Kit, or other test kit prepared by the recombinant protein of recombinant Pasteurella toxin (re-PmT) and/or virus-like particles (re-PmT VLP) containing recombinant protein of Pasteurella toxin . In one embodiment, the test kit comprises at least one virus-like particle containing the recombinant protein of Resin septicum (re-PmT) provided by the present invention and/or a recombinant protein containing Pasteurella septicum toxin (re An antigen disk of -PmT VLP) can be used to test whether the sample contains antibodies against P. septicum toxin (PmT).

All technical and scientific terms used in the specification, unless otherwise defined, are intended to be understood by those of ordinary skill in the art.

The words "about", "about" or "nearly" as used herein and the scope of the appended claims generally mean that the stated value or range is within 20%, preferably within 10%, and preferably. Within 5%. The amount of digitization provided herein is an approximation and is intended to be derived if the terms "about", "about" or "nearly" are not used.

The use of the terms "a", "an" and "the" Moreover, for ease of reading, headings or subtitles may be used in this specification, which have no effect on the scope of the invention.

The invention will be more specifically described in the following examples, which are intended to be illustrative, and many modifications and variations will be apparent to those skilled in the art. Various specific embodiments of the invention are described in detail below.

Embodiment 1 Septicemia Pasteurella toxin recombinant protein (re-PmT) Construction

1. Design of amino acid sequence of recombinant protein (re-PmT) of Pasteurella multocida toxin

The three-stage epitope (epitopes) is selected from the full-length amino acid sequence of the P. septicum toxin protein (PmT) (shown as SEQ ID NO: 1), respectively: 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 linker There is an amino acid sequence as shown in SEQ ID NO: 11. According to the difference of epitopes A, B, and C from the C-terminal arrangement of the recombinant protein N, the obtained recombinant septicum toxin recombinant protein (re-PmT) amino acid sequence is SEQ ID NOs: 5, respectively. 22, 23, 25, and 26 are shown, and the amino acid sequence is synthesized by a synthesizer or obtained by gene expression.

2. Design of amino acid sequence of viroid-like particles (re-PmT VLP) containing recombinant protein of Pasteurella multocida toxin

The above-described constructed S. septicum toxin recombinant protein (re-PmT) (SEQ ID NO: 5) was inserted into the 78th and 79th amines of hepatitis B virus core protein (HBc) (SEQ ID NO: 6). Between the base acids and the N-terminal segment of the hepatitis B virus core protein (HBc) (1st to 78th amino acids; SEQ ID NO: 7) and the recombinant protein of septicum Pasteurella toxin (re-PmT) (SEQ ID NO: 5), and the S. septicum toxin recombinant protein (re-PmT) (SEQ ID NO: 5) and the hepatitis B virus core protein (HBc) C-terminal segment (79-144) Amino acids; SEQ ID NO: 8), each linked by a linker having an amino acid sequence as 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, and the amino acid sequence can be expressed and purified in a biological expression host.

The above-mentioned virus-like particles (re-PmT VLP) containing the recombinant protein of Pasteurella multocida toxin can also be obtained by gene colonization expression. When 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 restricted by cleavage. The nucleic acid sequence encoding the N-terminal portion of the hepatitis B virus core protein (HBc) (1 to 78 amino acids; SEQ ID NO: 7), the nucleic acid sequence encoding the linker (SEQ ID NO: 12), and the coding The nucleic acid sequence of the recombinant Pasteurella toxin (re-PmT) (SEQ ID NO: 5), the nucleic acid sequence encoding the linker (SEQ ID NO: 12), and the C-terminal encoding the hepatitis B virus core protein (HBc) The nucleic acid sequence of the fragment (79-144 amino acid; SEQ ID NO: 8) was sequentially inserted into various restriction enzyme enzyme cloning sites (MCS) of vector pET24, and Sac I, Nco I, respectively. A restriction enzyme cleavage site such as Hind III and Kpn I 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 the nucleic acid sequence is contained. 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.

3. Observation of viroid-like particles (re-PmT VLP) containing recombinant protein of Pasteurella multocida toxin

After purifying the above-mentioned virus-like particles (re-PmT VLP) containing the recombinant protein of Pasteurella septicum toxin, 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). The company, Japan) conducted negative staining observations. First, 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 1% acetic acid. The uranium solution (pH 4.5) was dyed for 30 seconds, and the excess liquid was sucked off with a filter paper and observed under a microscope. The voltage was set to 80 kV during observation. The results of microscopic observation are shown in Fig. 1. 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).

Embodiment 2 B. bronchiseptica ( Bordetella bronchiseptica ) Pasteurella septicum ( Pasteurella multocida ) Cultivation

1. Culture of B. bronchiseptica

B. bronchiseptica was first 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) Incubate overnight at 37 ° C; then inoculate the solution in BHI liquid medium, shake overnight at 37 ° C, and calculate the colony forming unit (CFU) value; finally add formaldehyde (formaldehyde ), shaking at room temperature for 24 to 36 hours, inactivation of the bacterial solution.

2. Culture of P. multocida

First, the Pasteurella multocida type A (PmA) and the bacterium of the bacterium S. septicum (PmD) having the ability to produce toxin were inoculated on the TSB solid medium [containing 5% (v/v) yeast extract, 10% (v/v) serum, soy protein-degraded protein casein medium (TSB, BD, USA), after overnight incubation at 37 ° C, single colonies were selected for inoculation into brain heart extract (BHI) liquid medium (BD) Company, 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.

Embodiment 3 Preparation of porcine atrophic rhinitis immune composition

The recombinant S. septicum toxin recombinant protein (re-PmT) (SEQ ID NO: 5) obtained in Example 1 (final concentration of 250 μg/ml) or viroid-like granule containing recombinant protein of S. septicum toxin (re-PmT VLP) (SEQ ID NO: 10) (final concentration 250 μg/ml) and the inactivated B. bronchiseptica obtained in Example 2 (final concentration 1x10 ⁹ CFU/ml), inactivated Pasteurella septicum 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.

Embodiment 4 Analysis of immunogenicity and neutralizing antibody of porcine atrophic rhinitis immune composition

Mouse immunoassay

BALB/c mice (National Experimental Animal Center, Taiwan), which were negative for 3 weeks old with Pasteurella septicum antibody, were randomly divided into 5 groups; the first group was the control group, and the second group was the immunoassay group; Each mouse in each group 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 (1 × 10 ⁹ CFU/ml), Pasteurella multocida type A (PmA) (1×10 ⁹ CFU/ml) and Pasteurella septicum obtained in Example 2 Immunoassay of type D bacteria (PmD) (1x10 ⁹ CFU/ml) (containing 30% (v/v) aluminum gel adjuvant) (Bb + PmA + PmD group); Group 3: defeat of Example 1 Virion-like particles of the recombinant protein of Pasteurella multocida (SEQ ID NO: 10) (concentration: 250 μg/ml) (re-PmT VLP group); Group 4: The septic-containing Pasteurella toxin obtained in Example 3 Porcine atrophic rhinitis immunocompetent composition of recombinant protein-like virulent particle (SEQ ID NO: 10) (Bb + PmA + PmD + re-PmT VLP group); and Group 5: commercial porcine atrophic rhinitis vaccine (commercially available) Vaccine group).

Each mouse was collected for blood collection 24 hours before the first immunization (Day 0). After the first immunization (Day 1), blood was collected again on the 13th day, and then the second dose was performed on the 14th day with the same immunization dose. Immunize and collect blood again on the 24th day. The serum in the blood sample is separated for Enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody assay.

2. Enzyme-linked immunoassay (ELISA) for toxin antibodies

Commercially available S. septicum toxin (PMT) (Abcam, USA) was used as an antigen, and the antigen was coated on a 96-well plate (Thermo, USA) for ELISA, and allowed to stand at 4 ° C. 16 hours. After removing excess antigen, add washing buffer (wash buffer; 0.9% NaCl; 0.1% Tween20), wash 3 times, and then dry. Then, blocking buffer (wash buffer containing 1% BSA) was added, and after standing at room temperature for 1 hour, it was washed with washing buffer, and then serum samples collected from the above groups of mice were used as PBS buffer solution. After dilution, the diluted mouse serum was added to each well, and after standing at room temperature for 1 hour, the serum sample was removed, washed with a washing buffer, and then added with horseradish peroxidase (HRP). Mouse secondary antibody (goat anti-mouse conjugated HRP, Gene Tex, USA), this secondary antibody is diluted 5,000 times in blocking buffer and then added to a 96-well plate (100 ml / well) at room temperature After standing for 1 hour, the secondary antibody was removed and washed with washing buffer, and 100 ml of 3,3',5,5'-tetramethylbenzidine dihydrochloride (3, 3', 5, 5 was added to each well. '-tetramethylbenzidine, TMB, KPL, USA) solution was protected from light for 10 minutes and read at 650 nm with an enzyme-linked immunoassay reader (SpectraMax® M2/M2 ELISA Reader, Molecular Devices, USA) value.

The results of the enzyme-linked immunoassay are shown in Figure 2. After the second immunization, the mouse serum of the porcine atrophic rhinitis immunocomplex (Group 4, ie, Bb + PmA + PmD + re-PmT VLP group) immunized with viroid-like particles of the recombinant protein of Pasteurella toxin The anti-Septica Pasteurin toxin (PMT) antibody contained in the drug is the most expensive, followed by the viroid-like particle of the recombinant protein of the Pasteurella septicum toxin obtained in the first embodiment (Group 3, ie, the re-PmT VLP group) Mouse sera; and immunosuppressed porcine atrophic rhinitis vaccine (Group 5, the commercially available vaccine group) of mouse serum containing anti-Septic Pasteurin toxin (PMT) antibody titer, and immunization Antibody abilities in mouse sera of B. bronchiseptica, P. septicum type A and S. septicum D-type bacteria (Group 2, ie Bb + PmA + PmD group) obtained in Example 2 There is no significant difference in the price. The anti-Septic Pasteurin toxin (PMT) antibody titers contained in the serum of each immunoassay group (Groups 2-5) were higher than those in the serum of the first group (negative control group). The price was high and there was a significant difference ( p < 0.05 or p < 0.01). It can be seen that the 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.

3. Neutralizing antibody strength test

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 Bb + PmA + PmD + re- Whether the anti-Septic Pasteurin toxin (PMT) antibody contained in the mouse serum of the PmT VLP group is a neutralizing antibody capable of neutralizing PMT toxicity. The minimum toxin dose (MTD) measurement of Vero cells was performed prior to the neutralization antibody titer test.

(a) Determination of the minimum toxic dose (MTD) of Vero cells

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 (PMT, diluted with serum-free DMEM medium (GIBCO, USA) was added. Abcam) (0, 20, 50, 100 ng) processing. 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 trial showed that the minimal toxic dose of the septic Aspergillus toxin (PMT) of Vero cells was 50 ng.

(b) Neutralizing antibody titer test

First, mouse sera of a porcine atrophic rhinitis immunological composition (Group 4, ie, Bb + PmA + PmD + re-PmT VLP group) immunized with viroid-like particles containing a recombinant protein of Pasteurella septicum toxin, respectively, and The mouse serum of the commercially available porcine 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 a 4-fold minimum toxic dose (MTD) was added to each well, followed by a reaction at 37 ° C. 1 hour. Subsequently, the above reaction solution was added to Vero cells cultured in a 96-well plate, and cultured at 37 ° C in a 5% CO ₂ incubator, and it was observed whether the serum could inhibit Vero cell-producing cytopathic effect (CPE).

The result is shown in Figure 3. 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, and the cells showed typical nodule (Fig. 3B arrow) Shown). In contrast, Vero cells were cultured in DMEM containing fetal bovine serum (FBS) as a negative control group, and the cell morphology was as shown in Fig. 3A; and the virus-like particles containing the recombinant protein of Pasteurella septicum toxin were immunized. The porcine atrophic rhinitis immune composition (Group 4, ie Bb + PmA + PmD + re-PmT VLP group) was diluted 160-fold in serum with 4 times the minimum toxic dose (MTD) of S. septicum toxin After neutralization of (PMT), the morphology of 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). Thereafter, 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 typical nodules as shown in Fig. 3B, and Fig. 3D still had a few nodular cell morphology, showing the immunization of viroid-like particles containing the recombinant protein of Pasteurella toxin. Mouse sera of porcine atrophic rhinitis immune composition (Group 4, ie Bb + PmA + PmD + re-PmT VLP group), and immunologically marketed porcine atrophic rhinitis vaccine (Group 5, commercially available vaccine group) The mouse serum contains anti-Septic Pasteurin toxin (PMT) neutralizing antibodies, and the former has a higher neutralizing antibody content than the latter. These results indicate that the porcine atrophic rhinitis immunological composition of the present invention containing the virulent granules of the recombinant protein of septicum Pasteurella toxin can cause the body of the test animal to produce more than the commercially available porcine atrophic rhinitis vaccine. And antibodies.

4. Statistical methods

All experimental data were statistically analyzed using SigmaState software and One-Way ANOVA, and the results were expressed as mean±SEM. The Student Newman-keuls test statistical method was used to compare the groups; the symbols * and ** were significantly different 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 (Bb + 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).

Embodiment 5 Viral particle containing recombinant protein of Pasteurella multocida toxin (re-PmT VLP) Analysis of immunogenicity and protective efficacy of immune composition of porcine atrophic rhinitis 1- Taiwan pig atrophic rhinitis vaccine test standard ( Pasteurella efficacy test )

According to the test criteria for Taiwan atrophic rhinitis bacterin, the BALB/c mice (National Experimental Animal Center, Taiwan) with negative antibody to Pasteurella septicum were randomly divided into 3 groups. The first group was the control group, the second group. The group was Bb + PmA + PmD + re-PmT VLP immunoassay 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 was: Group 1: PBS buffer solution containing 30% (v/v) aluminum gel (control group); Group 2: virus-like particles containing recombinant protein of Pasteurella septicum toxin obtained in Example 3 Porcine atrophic rhinitis immunological composition (SEQ ID NO: 10) (Bb + PmA + PmD + re-PmT VLP group); and Group 3: commercial porcine atrophic rhinitis vaccine (commercially available vaccine group).

The immunoassay group (Group 2 and Group 3) was divided into 3 groups on the 14th day after immunization, and the virulent strains of Pasteurella pneumoniae type D (PmD) with the ability to produce toxin were used in the order. Second ^{embodiment] 1x10 6 CFU / ml, 1x10} 7 CFU / ml, 1x10 8 CFU / ml of live bacteria at three concentrations 0.1 ml intraperitoneal injection. At the same time, the control group (Group 1) 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 Mm was injected intraperitoneally. After 10 days of observation, 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. The Beka's two-defense index is calculated as follows: LD ₅₀ = minimum dilution factor for the challenge dose - [(sum of mortality for each group / 100) - 0.5] x 1 defense index = {minimum dilution of control dose Multiples - [(sum of mortality in each group / 100) - 0.5] x 1} - {Minimum dilution factor for the dose of the immunization group - [(sum of deaths per group / 100) - 0.5] x 1}.

As a result, as shown in Table 1, the porcine atrophic rhinitis immunological composition (group 2, Bb + PmA + PmD + re-PmT VLP group) containing the viroid-like particles of the recombinant protein of Pasteurella septicum was indeed able to induce small The rat has protective effects and is resistant to the virulent strain of Pasteurella multocida D-type bacteria (PmD), and its defense index is greater than 1x10 ^2.8 , higher than the Taiwan test standard (1x10 ^0.5 ) and the commercial atrophic rhinitis of pigs. The defense index of the vaccine (commercial vaccine group) (1x10 ^2.5 ).

Table 1 Immunogenicity and protective efficacy of porcine atrophic rhinitis immunocompetent composition containing viroid-like particles (re-PmT VLP) of recombinant protein of Pasteurella toxin  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Groups</td><td> Number of attacking bacteria (CFU/ml) </td ><td> Mortality (%) </td><td> LD₅₀ (CFU/ml) </td><td> Defense Index</td></tr><tr> <td> Group 1 (control group) </td><td> 1x10⁵</td><td> 36 </td><td> 1x10^5.2 </td><td> -- </td></tr><tr><td> 1x10⁶</td><td> 88 </td></tr><tr ><td> 1x10⁷</td><td> 100 </td></tr><tr><td> Group 2 (Bb + PmA + PmD + re-PmT VLP group) ) </td><td> 1x10⁶</td><td> 0 </td><td> > 1x10⁸</td><td> 1x10 ^2.8</td></tr><tr><td> 1x10⁷</td><td> 0 </td></tr><tr> <td> 1x10⁸</td><td> 0 </td></tr><tr><td> Group 3 (commercial vaccine group) </td><td> 1x10⁶</td><td> 0 </td><td> 1x10^7.7</td><td> 1x10^2.5< /td></tr><tr><td> 1x10⁷</td><td> 16.7 </td></tr><tr><td> 1x10<sup>8< /sup></td><td> 60 </td></tr></TBODY></TABLE>

Embodiment 6 Viral particle containing recombinant protein of Pasteurella multocida toxin (re-PmT VLP) Analysis of immunogenicity and protective efficacy of immune composition of porcine atrophic rhinitis 2

BALB/c mice (National Experimental 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 1: 0.2 ml PBS buffer solution (control group) per mouse; Group 2-1: Each One mouse was injected with 0.2 ml of the porcine atrophic rhinitis immunological composition stock solution (Bb + PmA + PmD + re-PmT) containing the viroid-like particle of the S. septicum toxin recombinant protein (SEQ ID NO: 10) obtained in Example 3. VLP group); Group 2-2: Each mouse was injected with 0.2 ml of 5-fold diluted porcine atrophy of the viroid-like particle containing septicum Pasteurella toxin recombinant protein (SEQ ID NO: 10) obtained in Example 3. Rhinitis immune composition (1/5 Bb + PmA + PmD + re-PmT VLP group); Group 2-3: Each mouse was injected with 0.2 ml of 25-fold diluted Example 3 containing Pasteurella septicum Porcine atrophic rhinitis immunocompetent composition of the virus-like particle of the recombinant protein (SEQ ID NO: 10) (1/25 Bb + PmA + PmD + re-PmT VLP group); Group 3-1: 0.2 injection per mouse Ml commercially available porcine atrophic rhinitis vaccine stock (commercial vaccine group); Group 3-2: 0.2 ml diluted 5 times of commercial pig atrophy per mouse Polio vaccine (a commercial vaccine group 1/5); Group 3-3: mice were injected with 0.2 ml per 25-fold dilution of a commercially available porcine atrophic rhinitis vaccine (1/25 commercial vaccine group).

The immunization test group (groups 2 and 3) was immunized twice on the 14th day after the first immunization, and the dose was the same as the first immunization; the control group (group 1) was injected with 0.2 ml of PBS buffer solution again; after the second immunization 10 days of challenge test, each mouse was injected intraperitoneally with 0.2 ml of a virulent strain of Pasteurella multocida type D (PmD) with the ability to produce toxin [same as in the second example] 100 LD ₅₀ live bacteria, The survival rate was recorded for 10 days. 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 (group 2-2, group 3-2) must be higher than 50%, 25 times. The survival rate of the diluted immunized group (Groups 2-3, 3-3) must be higher than 20%, and the control group must all die.

The results are shown in Table 2, porcine atrophic rhinitis immunocomplexes containing viroid-like particles of the recombinant protein of Pasteurella toxin (re-PmT VLP) (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.

Table 2 Immunogenicity and protective efficacy analysis of porcine atrophic rhinitis immunocompetent composition containing viroid-like particles (re-PmT VLP) of recombinant protein of Pasteurella toxin  <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Groups</td><td> Number of dead mice/number of challenged mice</ Td><td> Survival rate (%) </td></tr><tr><td> Group 1 (control group) </td><td> 5/5 </td><td> 0 < /td></tr><tr><td> Group 2-1 (B.b+PmA+ PmD+re-PmT VLP group) </td><td> 0/10 </td><td> 100 </td></tr><tr><td> Group 2-2 (1/5 B.b+PmA+ PmD+re-PmT VLP group) </td><td> 0/10 </td> <td> 100 </td></tr><tr><td> Group 2-3 (1/25 B.b+PmA+PmD+re-PmT VLP group) </td><td> 0/ 10 </td><td> 100 </td></tr><tr><td> Group 3-1 (commercial vaccine group) </td><td> 0/10 </td><td > 100 </td></tr><tr><td> Group 3-2 (1/5 Commercial Vaccine Group) </td><td> 0/10 </td><td> 100 </ Td></tr><tr><td> Group 3-3 (1/25 Commercial Vaccine Group) </td><td> 4/10 </td><td> 60 </td></ Tr></TBODY></TABLE>

Example 7 Anti-Septic Pasteurella toxin (PmT) Antibody preparation

1. Anti-septic Pasteurella toxin (PmT) Multiple antibodies

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 (such as 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), can be a second immunization as needed. After appropriate time intervals (eg, 2 to 3 weeks), the serum of immunized animals (eg, mice, rats, pigs, goats, rabbits) is collected to obtain the most resistant to Pasteurella toxin (PmT). Strain antibody.

The multi-strain antibody of the anti-Septic bacillus toxin (PmT) can be combined with a developer or fluorescent light as needed.

After the primary immunization and the second immunization, the animal may increase the number of immunizations as needed to increase the antibody titer.

The animal to be administered includes, but is not limited to, mice, rats, rabbits, poultry (eggs), pigs, goats, cattle, and aquatic animals.

2. Anti-septic Pasteurella (PmT) Individual antibody

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 (such as 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), can be a second immunization as needed. After appropriate time intervals (eg, 2 to 3 weeks), serum from immunized animals (eg, mice) is collected to assess mice suitable for collecting spleen cells. The spleen cells and the myeloma cells (e.g., the FO cell strain, the NS cell strain) are collected from the suitable mouse for cell fusion with PEG (Polyethylene Glycol, such as PEG 1500). A fusion cell strain suitable for producing a monoclonal antibody against P. septicum (PmT) can be obtained by screening a fusion cell having a secretory ability and singulating it.

The antibody obtained by the above preparation can be used for an immunoassay reagent, a therapeutic agent, or added to a food or a feed to make the consumer immune.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, the technical features disclosed in this case have fully complied with the statutory invention patent requirements of novelty and progressiveness. If you apply in accordance with the law, you are requested to approve the application for this invention patent to encourage invention.

Fig. 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 type of viral particle of the invention. Scale bar: 50 μm.

Figure 2 shows the results of measuring the valence of anti-Septic bacillus 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 Immunological composition of B. bronchiseptica (Bb), Pasteurella multocida type A (PmA) and Pasteurella multocida type D (PmD) obtained in Example 2 (Bb + PmA + PmD group) The third group is the viroid-like particle containing the recombinant protein of Pasteurella septicum toxin obtained in the first embodiment (re-PmT VLP group); the fourth group is the recombinant protein containing the Pasteurella septicum toxin obtained in the third embodiment The porcine atrophic rhinitis immunocompetent composition (Bb + PmA + PmD + re-PmT VLP group); the fifth group 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 (Bb + 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).

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 medium containing fetal bovine serum (FBS) (negative control group); Figure 3B shows the Pasteurella septicum containing 4 times the minimum toxic dose (MTD). Toxin (PMT) treatment of the cell morphology of Vero cells (positive control group), the cells showed typical nodular (as indicated by the arrow); Figure 3C shows the virus with recombinant protein containing Pasteurella toxin Granules of porcine atrophic rhinitis immune composition (Bb + PmA + PmD + re-PmT VLP group) immunized mice serum diluted 160-fold with 4 times the minimum toxic dose (MTD) of septicemia pasteurin toxin (PMT) After neutralization, the cell morphology co-cultured with Vero cells was added; Figure 3D shows the dilution of the serum of the mice immunized with the commercially available porcine atrophic rhinitis vaccine (commercial vaccine group) 160 times and the 4 times minimum toxic dose (MTD). After neutralization of the S. septicum toxin (PMT), the morphology of the cells co-cultured with Vero cells was observed, and the cells still showed typical nodule (as indicated by the arrows).

<110> Jinxie International Industrial Co., Ltd. <120> Recombinant protein of Pasteurella septicum, its virion and its application <130> P17-0221 <160> 36 <170> PatentIn version 3.5 <210> 1 <211 > 1281 <212> PRT <213> Pasteurella multocida <300><308> GenBank: ABR23002 <309> 2007-08-13 <313> (2)..(1282) <400> 1 Lys Thr Lys His Phe Phe Asn Ser Asp Phe Thr Val Lys Gly Lys Ser 1 5 10 15 Ala Asp Glu Ile Phe Arg Arg Leu Cys Thr Asp His Pro Asp Lys Gln 20 25 30 Leu Asn Asn Val Lys Trp Lys Glu Val Phe Ile Asn Arg Phe Gly Gln 35 40 45 Met Met Leu Asp Thr Pro Asn Pro Arg Lys Ile Val Glu Lys Ile Ile 50 55 60 Asn Glu Gly Leu Glu Lys Gln Gly Leu Lys Asn Ile Asp Pro Glu Thr 65 70 75 80 Thr Tyr Phe Asn Ile Phe Ser Ser Ser Asp Ser Ser Asp Gly Asn Val 85 90 95 Phe His Tyr Asn Ser Leu Ser Glu Ser Tyr Arg Val Thr Asp Ala Cys 100 105 110 Leu Met Asn Ile Phe Val Glu Arg Tyr Phe Asp Asp Trp Asp Leu Leu 115 120 125 Asn Ser Leu Ala Ser Asn Gly Il e Tyr Ser Val Gly Lys Glu Gly Ala 130 135 140 Tyr Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly 145 150 155 160 Pro Asn Glu Gln Ile Tyr His Ser Arg Val Ile Ala Asp Ile Leu Tyr 165 170 175 Ala Arg Ser Val Trp Asp Glu Phe Lys Lys Tyr Phe Met Glu Tyr Trp 180 185 190 Gln Lys Tyr Ala Gln Leu Tyr Thr Glu Met Leu Ser Asp Thr Phe Leu 195 200 205 Ala Met Ala Ile Gln Gln Tyr Thr Arg Gln Thr Leu Thr Asp Glu Gly 210 215 220 Phe Leu Met Val Cys Asn Thr Tyr Tyr Gly Asn Lys Glu Glu Val Gln 225 230 235 240 Ile Thr Leu Leu Asp Ile Tyr Gly Tyr Pro Ser Thr Asp Ile Ile Cys 245 250 255 Ile Glu Gln Lys Gly Leu Pro Thr Pro Lys Val Ile Leu Tyr Ile Pro 260 265 270 Gly Gly Thr Gln Pro Phe Val Glu Phe Leu Asn Thr Asp Asp Leu Lys 275 280 285 Gln Trp Ile Ala Trp His Leu Lys Asp Asn Lys His Met Val Ala Phe 290 295 300 Arg Lys His Phe Ser Leu Lys Gln Arg Gln Glu Gly Glu Thr Phe Thr 305 310 315 320 Gly Ile Asp Lys Ala Leu Gln Tyr Ile Ala Glu Glu Ser Pro Glu Trp 325 330 335 Pro Ala Asn Lys Tyr Ile Leu Ty r Asn Pro Thr His Leu Glu Thr Glu 340 345 350 Asn Leu Phe Asn Ile Met Met Lys Arg Thr Glu Gln Arg Met Leu Glu 355 360 365 Asp Ser Asp Val Gln Ile Arg Ser Asn Ser Glu Ala Thr Arg Asp Tyr 370 375 380 Ala Leu Ser Leu Leu Glu Thr Phe Ile Ser Gln Leu Ser Ala Ile Asp 385 390 395 400 Met Leu Val Pro Ala Val Gly Ile Pro Ile Asn Phe Ala Leu Ser Ala 405 410 415 Thr Ala Leu Gly Leu Ser Ser Asp Ile Val Val Asn Gly Asp Ser Tyr 420 425 430 Glu Lys Arg Lys Tyr Gly Ile Gly Ser Leu Val Gln Ser Ala Leu Phe 435 440 445 Thr Gly Ile Asn Leu Ile Pro Val Ile Ser Glu Thr Ala Glu Ile Leu 450 455 460 Ser Ser Phe Ser Arg Thr Glu Glu Asp Ile Pro Ala Phe Phe Thr Glu 465 470 475 480 Glu Gln Ala Leu Ala Gln Arg Phe Glu Ile Val Glu Glu Leu His 485 490 495 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp Glu Asn Leu 500 505 510 His Lys Ile Arg Leu Val Arg Leu Asn Asn Glu Asn Gln Pro Leu Val 515 520 525 Val Leu Arg Arg Leu Gly Gly Asn Lys Phe Ile Arg Ile Glu Pro Ile 530 535 540 Thr Phe Gln Glu Ile Lys Gly Ser Le u Val Ser Glu Val Ile Asn Pro 545 550 555 560 Val Thr Asn Lys Thr Tyr Tyr Val Ser Asn Ala Lys Leu Leu Gly Gly 565 570 575 Ser Pro Tyr Ser Pro Phe Arg Ile Gly Leu Glu Gly Val Trp Thr Pro 580 585 590 Glu Val Leu Lys Ala Arg Ala Ser Val Ile Gly Lys Pro Ile Gly Glu 595 600 605 Ser Tyr Lys Arg Ile Leu Ala Lys Leu Gln Arg Ile His Asn Ser Asn 610 615 620 Ile Leu Asp Glu Arg Gln Gly Leu Met His Glu Leu Met Glu Leu Ile 625 630 635 640 Asp Leu Tyr Glu Glu Ser Gln Pro Ser Ser Glu Arg Leu Asn Ala Phe 645 650 655 Arg Glu Leu Arg Thr Gln Leu Glu Lys Ala Leu Tyr Leu Pro Glu Met 660 665 670 Glu Ala Leu Lys Lys Gln Ile Leu Gln Ile Pro Asn Lys Gly Ser Gly 675 680 685 Ala Ala Arg Phe Leu Leu Arg Thr Ala Met Asn Glu Met Ala Gly Glu 690 695 700 Thr Ser Glu Ser Thr Ala Asp Leu Ile Arg Phe Ala Leu Gln Asp Thr 705 710 715 720 Val Ile Ser Ala Pro Phe Arg Gly Tyr Ala Gly Ala Ile Pro Glu Ala 725 730 735 Ile Asp Phe Pro Val Lys Tyr Val Ile Glu Asp Ile Ser Val Phe Asp 740 745 750 Lys Ile Gln Thr Asn Tyr Trp Glu Le u Pro Ala Tyr Glu Ser Trp Asn 755 760 765 Glu Gly Ser Asn Ser Ala Leu Leu Pro Gly Leu Leu Arg Glu Ser Gln 770 775 780 Ser Lys Gly Met Leu Ser Lys Cys Arg Ile Ile Glu Asn Ser Leu Tyr 785 790 795 800 Ile Gly His Ser Tyr Glu Glu Met Phe Tyr Ser Ile Ser Pro Tyr Ser 805 810 815 Asn Gln Val Gly Gly Pro Tyr Glu Leu Tyr Pro Phe Thr Phe Phe Ser 820 825 830 Met Leu Gln Glu Val Gln Gly Asp Leu Gly Phe Glu Gln Ala Phe Ala 835 840 845 Thr Arg Asn Phe Phe Asn Thr Leu Val Ser Asp Arg Leu Ser Leu Met 850 855 860 Glu Asn Thr Met Leu Leu Thr Glu Ser Phe Asp Tyr Thr Pro Trp Asp 865 870 875 880 Ala Ile Tyr Gly Asp Ile Asn Tyr Asp Glu Gln Phe Ala Ala Met Ser 885 890 895 Ile Asn Glu Arg Ile Glu Lys Cys Met Asn Thr Tyr Arg Gly Val Ala 900 905 910 Phe Gln Asn Ser Ser Lys Ser Ile Asp Phe Phe Leu Asn Asn Leu Thr 915 920 925 Thr Phe Ile Asp Asn Gly Leu Thr Glu Ile Ala Ile Ser Asp Leu Pro 930 935 940 Tyr Asp Ile Val Gln Gln Glu Ile Ser Gln Phe Leu Gln Gly Ser Asn 945 950 955 960 Glu Trp Lys Thr Leu Asp Ala Met Le u Phe Asn Leu Asp Lys Gly Asp 965 970 975 Ile Asn Gly Ala Phe Arg Lys Leu Leu Gln Ser Ala Lys Asp Asn Asn 980 985 990 Ile Lys Phe Arg Ala Ile Gly His Ser Asp Asn Ser Val Pro Pro Phe 995 1000 1005 Asn Asn Pro Tyr Lys Ser Leu Tyr Tyr Lys Gly Asn Ile Ile Ala 1010 1015 1020 Glu Ala Ile Glu Lys Leu Asp Arg Glu Gly Gln Lys Phe Val Val 1025 1030 1035 Phe Ala Asp Ser Ser Leu Leu Asn Ser Thr Pro Gly Thr Gly Arg 1040 1045 1050 Pro Met Pro Gly Leu Val Gln Tyr Leu Lys Ile Pro Ala Thr Val 1055 1060 1065 Val Asp Ser Asp Gly Ala Trp Gln Phe Leu Pro Asp Val Ala Ser 1070 1075 1080 Ser Arg Val Pro Ile Glu Val Thr Glu Leu Glu Asn Trp Gln Val 1085 1090 1095 Leu Thr Pro Pro Gln Gly Lys Ile Leu Gly Leu Lys Gln Phe Lys 1100 1105 1110 Leu Thr Ala Gly Phe Pro Thr Glu Gln Ser Arg Leu Pro Leu Leu 1115 1120 1125 Glu Asn Ser Val Ser Glu Asp Leu Arg Glu Glu Leu Met Gln Lys 1130 1135 1140 Ile Asp Ala Ile Lys Asn Asp Val Lys Met Asn Ser Leu Val Cys 1145 1150 1155 Met Glu Ala Gly Ser Cys Asp Ser Val Ser Pro Lys Val Al a Ala 1160 1165 1170 Arg Leu Lys Asp Met Gly Leu Glu Ala Gly Met Gly Ala Ser Ile 1175 1180 1185 Thr Trp Trp Arg Arg Glu Gly Gly Met Glu Phe Ser His Gln Met 1190 1195 1200 His Thr Thr Ala Ser Phe Lys Phe Ala Gly Lys Glu Phe Ala Val 1205 1210 1215 Asp Ala Ser His Leu Gln Phe Val His Asp Gln Leu Asp Thr Thr 1220 1225 1230 Ile Leu Ile Leu Pro Val Asp Asp Trp Ala Leu Glu Ile Ala Gln 1235 1240 1245 Arg Asn Arg Ala Ile Asn Pro Phe Val Glu Tyr Val Ser Lys Thr 1250 1255 1260 Gly Asn Met Leu Ala Leu Phe Met Pro Pro Leu Phe Thr Lys Pro 1265 1270 1275 Arg Leu Thr 1280 <210> 2 <211> 28 <212> PRT <213> Pasteurella multocida <400> 2 Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro 1 5 10 15 Glu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile 20 25 <210> 3 <211> 13 <212> PRT <213> Pasteurella multocida <400> 3 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp 1 5 10 <210> 4 <211> 12 <212> PRT <213> Pasteurella multocida <400> 4 Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro 1 5 10 <210> 5 <211> 61 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of recombinant prasemic toxin recombinant protein (re-PmT) <400> 5 Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro 1 5 10 15 Glu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr Ala 20 25 30 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr 35 40 45 Ala Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro 50 55 60 <210> 6 <211> 144 <212> PRT <213> Hepatitis B <400> 6 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Pro Ala 65 70 75 80 Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys 85 90 95 Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg 100 105 110 Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr 115 120 125 Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 130 135 140 <210> 7 <211> 78 <212> PRT <213> Hepatitis B <400> 7 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp 65 70 75 <210> 8 <211> 66 <212> PRT <213> Hepatitis B <400> 8 Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly 1 5 10 15 Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe 20 25 30 Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile 35 40 45 Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr 50 55 60 Leu Pro 65 <210> 9 <211> 223 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of a viroid-like particle (re-PmT VLP) containing a recombinant protein of Pasteurella septicum <400> 9 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Ser Val Gly Lys Glu Gly Ala Tyr Tyr 85 90 95 Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly Pro Asn 100 105 110 Glu Gln Ile Gly Ser Thr Ala Ser Ile Ser Pro Asp Asp Pro Pro Arg 115 120 125 Glu Ile Thr Asp Gly Ser Thr Ala Leu Asn Ser Thr Pro Gly Thr Gly 130 135 140 Arg Pro Met Pro Gly Gly Gly G Ly Ser Gly Gly Gly Gly Pro Ala Ser 145 150 155 160 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 165 170 175 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 180 185 190 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 195 200 205 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 210 215 220 <210> 10 <211> 231 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of viroid-like particle (re-PmT VLP) containing recombinant protein of Pasteurella septicum <400> 10 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Glu Leu 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Trp Ser Val Gly Lys Glu 85 90 95 Gly Ala Tyr Tyr Pr o Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val 100 105 110 Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr Ala Ser Ile Ser Pro Asp 115 120 125 Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr Ala Leu Asn Ser Thr 130 135 140 Pro Gly Thr Gly Arg Pro Met Pro Lys Leu Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Gly Thr Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val 165 170 175 Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile 180 185 190 Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 195 200 205 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 210 215 220 Pro Ile Leu Ser Thr Leu Pro 225 230 <210> 11 <211> 4 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of GSTA linker <400> 11 Gly Ser Thr Ala 1 <210 > 12 <211> 9 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of GS linker <400> 12 Gly Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 13 <211 > 14 <212> PRT <213> Artificial sequence 220><223> Amino acid sequence of GSGGG linker <400> 13 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220><223> (GS)3 Linker amino acid sequence <400> 14 Gly Ser Gly Ser Gly Ser 1 5 <210> 15 <211> 15 <212> PRT <213> Artificial sequence <220><223> (GGGGS)3 Amino Acid Sequence of Linker <400> 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 16 <211> 4 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of GGSG linker <400> 16 Gly Gly Ser Gly 1 <210> 17 <211> 8 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of (GGSG)2 linker <400> 17 Gly Gly Ser Gly Gly Gly Ser Gly 1 5 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220><223> (GGSG ) 3 linker amino acid sequence <400> 18 Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 <210> 19 <211> 10 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of GENLYFQSGG linker <400> 19 Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly 1 5 10 <210> 20 <211> 36 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of GAST linker <400> 20 Gly Gly Ser Ala Gly Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Ser 1 5 10 15 Gly Ala Ser Gly Thr Gly Thr Ala Gly Gly Thr Gly Ser Gly Ser Gly 20 25 30 Thr Gly Ser Gly 35 <210> 21 <211> 36 <212> PRT <213> Artificial Sequence <220><223> Amino Acid Sequence of SEG Linker <400> 21 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Ser Glu Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser 35 <210> 22 <211> 61 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of recombinant protein (re-PmT) of Pasteurella septicum <400> 22 Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro 1 5 10 15 Glu Tyr Asn Pro Val Trp Gly Pro A Sn Glu Gln Ile Gly Ser Thr Ala 20 25 30 Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala 35 40 45 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp 50 55 60 <210> 23 <211> 61 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of recombinant prase aspergillus toxin (re-PmT) <400> 23 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr 1 5 10 15 Ala Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly 20 25 30 Pro Glu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr 35 40 45 Ala Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro 50 55 60 <210> 24 <211> 61 <212> PRT <213> Artificial sequence <220><223> S. septicum toxin recombinant protein (re-PmT) Amino acid sequence <400> 24 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr 1 5 10 15 Ala Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro Gly Ser Thr 20 25 30 Ala Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly 35 40 45 Pro G Lu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile 50 55 60 <210> 25 <211> 61 <212> PRT <213> Artificial sequence <220><223> S. septicum toxin recombinant protein (re-PmT) Amino acid sequence <400> 25 Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala 1 5 10 15 Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro 20 25 30 Glu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr Ala 35 40 45 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp 50 55 60 <210> 26 <211> 61 <212> PRT <213> Artificial Sequence <220><223> Amino acid sequence of Recombinant Protein of Pasteurella toxin (re-PmT) <400> 26 Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala 1 5 10 15 Ser Ile Ser Pro Asp Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr 20 25 30 Ala Ser Val Gly Lys Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly 35 40 45 Pro Glu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile 50 55 60 <210> 27 <211> 223 <212> PRT <213> Human Procedure <220><223> Amino acid sequence of viroid-like particles (re-PmT VLP) containing recombinant protein of Pasteurella septicum <400> 27 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Ser Val Gly Lys Glu Gly Ala Tyr Tyr 85 90 95 Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly Pro Asn 100 105 110 Glu Gln Ile Gly Ser Thr Ala Leu Asn Ser Thr Pro Gly Thr Gly Arg 115 120 125 Pro Met Pro Gly Ser Thr Ala Ser Ile Ser Pro Asp Asp Pro Pro Arg 130 135 140 Glu Ile Thr Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Ala Ser 145 150 155 160 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 165 170 175 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 180 185 190 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 195 200 205 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 210 215 220 <210> 28 <211 > 231 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of viroid-like particle (re-PmT VLP) containing recombinant protein of Pasteurella septicum <400> 28 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Glu Leu 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Trp Ser Val Gly Lys Glu 85 90 95 Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val 100 105 110 Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr Ala Leu Asn Ser Thr Pro 115 120 125 Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala Ser Ile Ser Pro Asp 130 135 140 Asp Pro Pro Arg Glu Ile Thr Asp Lys Leu Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Gly Thr Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val 165 170 175 Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile 180 185 190 Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 195 200 205 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 210 215 220 Pro Ile Leu Ser Thr Leu Pro 225 230 <210> 29 <211> 223 <212> PRT <213> Artificial sequence <220><223> Virion particles containing recombinant protein of Pasteurella septicum toxin Amino acid sequence of (re-PmT VLP) <400> 29 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Gly Gl y 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Ser Ile Ser Pro Asp Asp Pro Pro Arg 85 90 95 Glu Ile Thr Asp Gly Ser Thr Ala Ser Val Gly Lys Glu Gly Ala Tyr 100 105 110 Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly Pro 115 120 125 Asn Glu Gln Ile Gly Ser Thr Ala Leu Asn Ser Thr Pro Gly Thr Gly 130 135 140 Arg Pro Met Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Ala Ser 145 150 155 160 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 165 170 175 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 180 185 190 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 195 200 205 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 210 215 220 <210> 30 <211> 231 <212> PRT <213> Artificial sequence <220><223> Contains Amino acid sequence of virus-like particles (re-PmT VLP) of the recombinant protein of Pasteurella multocida toxin <400> 30 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Va l Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Glu Leu 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Trp Ser Ile Ser Pro Asp 85 90 95 Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr Ala Ser Val Gly Lys 100 105 110 Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro 115 120 125 Val Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr Ala Leu Asn Ser Thr 130 135 140 Pro Gly Thr Gly Arg Pro Met Pro Lys Leu Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Gly Thr Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val 165 170 175 Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile 180 185 190 Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 195 200 205 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 210 215 220 Pro Ile Leu Ser Thr Leu Pro 225 230 <210 > 31 <211> 223 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of viroid-like particle (re-PmT VLP) containing recombinant protein of Pasteurella septicum <400> 31 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Ser Ile Ser Pro Asp Asp Pro Pro Arg 85 90 95 Glu Ile Thr Asp Gly Ser Thr Ala Leu Asn Ser Thr Pro Gly Thr Gly 100 105 110 Arg Pro Met Pro Gly Ser Thr Ala Ser Val Gly Lys Glu Gly Ala Tyr 115 120 125 Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly Pro 130 135 140 Asn Glu Gln Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Ala Ser 145 150 155 160 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 165 170 175 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 180 185 190 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 195 200 205 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 210 215 220 <210> 32 <211> 231 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of viroid-like particle (re-PmT VLP) containing recombinant protein of Pasteurella septicum toxin <400> 32 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Glu Leu 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Trp Ser Ile Ser Pro Asp 85 90 95 Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr Ala Leu Asn Ser Thr 100 105 110 Pro Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala Ser Val Gly Lys 115 120 125 Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro 130 135 140 Val Trp Gly Pro Asn Glu Gln Ile Lys Leu Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Gly Thr Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val 165 170 175 Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile 180 185 190 Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 195 200 205 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 210 215 220 Pro Ile Leu Ser Thr Leu Pro 225 230 <210> 33 <211> 223 <212> PRT <213> Artificial sequence <220><223> Contains septic Amino acid sequence of the viroid-like particle (re-PmT VLP) of the recombinant protein of the bacillus toxin <400> 33 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Tr p Val Gly Asn Asn Leu Glu Asp Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Leu Asn Ser Thr Pro Gly Thr Gly Arg 85 90 95 Pro Met Pro Gly Ser Thr Ala Ser Val Gly Lys Glu Gly Ala Tyr Tyr 100 105 110 Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly Pro Asn 115 120 125 Glu Gln Ile Gly Ser Thr Ala Ser Ile Ser Pro Asp Asp Pro Pro Arg 130 135 140 Glu Ile Thr Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Ala Ser 145 150 155 160 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 165 170 175 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 180 185 190 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 195 200 205 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 210 215 220 <210> 34 <211> 231 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of viroid-like particle (re-PmT VLP) containing recombinant protein of Pasteurella septicum <400> 34 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Ph e Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Glu Leu 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Trp Leu Asn Ser Thr Pro 85 90 95 Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala Ser Val Gly Lys Glu 100 105 110 Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val 115 120 125 Trp Gly Pro Asn Glu Gln Ile Gly Ser Thr Ala Ser Ile Ser Pro Asp 130 135 140 Asp Pro Pro Arg Glu Ile Thr Asp Lys Leu Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Gly Thr Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val 165 170 175 Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile 180 185 190 Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 195 200 205 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 210 215 220 Pro Ile Leu Ser Thr Leu Pr o 225 230 <210> 35 <211> 223 <212> PRT <213> Artificial sequence <220><223> Amino acid sequence of viroid-like particle (re-PmT VLP) containing recombinant protein of Pasteurella septicum toxin <400> 35 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Gly Gly 65 70 75 80 Gly Gly Ser Gly Gly Gly Gly Leu Asn Ser Thr Pro Gly Thr Gly Arg 85 90 95 Pro Met Pro Gly Ser Thr Ala Ser Ile Ser Pro Asp Asp Pro Pro Arg 100 105 110 Glu Ile Thr Asp Gly Ser Thr Ala Ser Val Gly Lys Glu Gly Ala Tyr 115 120 125 Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro Val Trp Gly Pro 130 135 140 Asn Glu Gln Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Ala Ser 145 150 155 160 Arg Asp Leu Val Val Asn Tyr Val Asn Thr Asn Met Gly Leu Lys Ile 165 170 175 Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg Glu 180 185 190 Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr Pro 195 200 205 Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro 210 215 220 <210> 36 <211> 231 <212> PRT <213> Artificial sequence <220><223> Viroid-like particles containing the recombinant protein of Pasteurella septicum toxin (re -PmT VLP) Amino acid sequence <400> 36 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15 Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Val Arg Asp Leu Leu Asp 20 25 30 Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys 35 40 45 Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu 50 55 60 Leu Met Thr Leu Ala Thr Trp Val Gly Asn Asn Leu Glu Asp Glu Leu 65 70 75 80 Gly Gly Gly Gly Ser Gly Gly Gly Gly Pro Trp Leu Asn Ser Thr Pro 85 90 95 Gly Thr Gly Arg Pro Met Pro Gly Ser Thr Ala Ser Ile Ser Pro Asp 100 105 110 Asp Pro Pro Arg Glu Ile Thr Asp Gly Ser Thr Ala Ser Val Gly Lys 115 120 125 Glu Gly Ala Tyr Tyr Pro Asp His Asp Tyr Gly Pro Glu Tyr Asn Pro 130 135 140 Val Trp Gly Pro Asn Glu Gln Ile Lys Leu Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Gly Thr Pro Ala Ser Arg Asp Leu Val Val Asn Tyr Val 165 170 175 Asn Thr Asn Met Gly Leu Lys Ile Arg Gln Leu Leu Trp Phe His Ile 180 185 190 Ser Cys Leu Thr Phe Gly Arg Glu Thr Val Leu Glu Tyr Leu Val Ser 195 200 205 Phe Gly Val Trp Ile Arg Thr Pro Pro Ala Tyr Arg Pro Pro Asn Ala 210 215 220 Pro Ile Leu Ser Thr Leu Pro 225 230

Claims (16)

A Pasteurella multocida toxin recombinant protein consisting of an epitope (epitopes) having a septic Aspergillus toxin protein having the amino acid sequence of SEQ ID NO: 2; An epitope of a S. septicum toxin protein having an amino acid sequence as set forth in SEQ ID NO: 3; and a S. septicum toxin protein having an amino acid sequence as set forth in SEQ ID NO: An epitope; wherein each of the epitopes is optionally joined by a linker, each of which is independently selected from Gly-Gly, Gly-Ser, and SEQ ID NOs: 11, 12, 13, and 14. a group consisting of 15, 16, 17, 18, 19, 20, and 21. A virus like particle (VLP) containing a recombinant protein of Pasteurella septicum toxin, comprising: a recombinant protein of Pasteurella septicum as described in claim 1; and a hepatitis B virus Hepatitis B virus core protein (HBc); wherein the recombinant protein of Pasteurella septicum as described in claim 1 is inserted into the major immunodominant region of the hepatitis B virus core protein (Major immunodominant) Region, MIR). The viroid-like particle containing the recombinant protein of Pasteurella septicum as described in claim 2, wherein the hepatitis B virus core protein has an amino acid sequence as shown in SEQ ID NO: 6, and the B The main immunodominant region (MIR) of the hepatitis B virus core protein is selected from the 73th to 94th amino acids, 73th to 82th amino acids, 75th to 81th amino acids, and the first 78~79 amine groups Acid, 78-81 amino acid, 78-82 amino acid, 78-86 amino acid, 78-89 amino acid, 78-94 amino acid, 81# A group consisting of 82 amino acids and 82 to 83 amino acids. The viroid-like particle containing the recombinant protein of Pasteurella septicum as described in claim 2, wherein the recombinant protein of Pasteurella septicum as described in claim 1 of the patent scope replaces the hepatitis B The main immunodominant region of the viral core protein. The viroid-like particle containing the recombinant protein of Pasteurella septicum as described in claim 4, wherein the hepatitis B virus core protein has an amino acid sequence as shown in SEQ ID NO: 6, and the B The main immunodominant region of the hepatitis C virus core protein is selected from the 73th to 94th amino acids of the protein, the 73th to 82th amino acids, the 75th to 81th amino acids, and the 78th to 79th Amino acid, 78-81 amino acid, 78-82 amino acid, 78-86 amino acid, 78-89 amino acid, 78-94 amino acid, A group consisting of 81 to 82 amino acids and 82 to 83 amino acids. The viroid-like particle containing the recombinant protein of Pasteurella septicum as described in claim 2, wherein the recombinant protein of Pasteurella septicum as described in claim 1 and the hepatitis B virus The core proteins are linked by a linker. The viroid-like particle containing the recombinant protein of Pasteurella septicum as described in claim 6, wherein the linker is each independently selected from Gly-Gly, Gly-Ser and SEQ ID NOs: 11, 12 Groups of 13, 14, 15, 16, 17, 18, 19, 20, and 21. The viroid-like particle containing the recombinant protein of Pasteurella septicum as described in claim 2, wherein the viroid-like particle containing the recombinant protein of Pasteurella septicum has SEQ ID NOs: 9, 10, 27 One of the amino acid sequences shown in 28, 29, 30, 31, 32, 33, 34, 35, and 36. A nucleic acid molecule encoding a recombinant protein of Pasteurella septicum as described in claim 1. A nucleic acid molecule encoding a viroid-like particle containing a recombinant protein of Pasteurella septicum as described in claim 2 of the patent application. A porcine atrophic rhinitis immunological composition comprising the recombinant protein of Pasteurella septicum as described in claim 1 and the recombinant protein containing Pasteurella septicum as described in claim 2 At least one of the viral particles, and a pharmaceutically acceptable carrier. The porcine atrophic rhinitis immunological composition according to claim 11, further comprising a Bordetella bronchiseptica , a Pasteurella multocida Type A, and a Pasteurella multocida Type D. The porcine atrophic rhinitis immunological composition according to claim 11, further comprising another pathogenic antigen selected from the group consisting of porcine circovirus type 2 (PCV2) antigen, pig Influenza virus (SIV) antigen, porcine reproductive and respiratory syndrome virus (PRRSV) antigen, Mycoplasma, Parvovirus (PPV), Erysipelas, Actinobacillus pleuropneumonia (actinobacillus pleuropneumonia, APP), as well as Aujeszky's disease. An application of the porcine atrophic rhinitis immunological composition according to claim 11 for the preparation of an animal for treating atrophic rhinitis in pigs. A multi-strain antibody against a bacterium of the genus Pasteurella D-type toxin, which is obtained by the recombinant protein of Pasteurella septicum as described in claim 1 or as claimed in claim 2 A virus-like particle of a recombinant protein of Pasteurella multocida toxin is prepared. A detection kit for porcine atrophic rhinitis, comprising a detecting unit selected from at least one of the following groups: a Pasteurella septicum as described in claim 1 a recombinant protein of toxin, a viroid-like particle containing a recombinant protein of Pasteurella septicum as described in claim 2, as prepared by a recombinant protein of Pasteurella septicum as described in claim 1. A plurality of antibodies, and a plurality of antibodies prepared by the same type of virus-like particles containing the recombinant protein of Pasteurella septicum as described in claim 2 of the patent application.