Recombinant protein of Pasteurella multocida toxin, its virus-like particles and their applications

Abstract

This invention provides a recombinant Pasteurella multocida toxin protein having three antigenic determinants of the Pasteurella multocida toxin protein. The three antigenic determinants have sequences as shown in SEQ ID NOs:2, 3, and 4, respectively. This invention also provides a virus-like particle containing the aforementioned recombinant Pasteurella multocida toxin protein, a nucleic acid sequence encoding the aforementioned recombinant Pasteurella multocida toxin protein or encoding the virus-like particle containing the aforementioned recombinant Pasteurella multocida toxin protein, and a porcine atrophic rhinitis immune composition containing the aforementioned recombinant Pasteurella multocida toxin protein and / or the virus-like particle containing the aforementioned recombinant Pasteurella multocida toxin protein.

Description

Technical Field

[0001] This invention relates to recombinant proteins of Pasteurella multocida toxin, and more particularly to recombinant proteins containing Pasteurella multocida toxin antigenic determinants, and virus-like particles containing said recombinant proteins. Background Technology

[0002] Atrophic rhinitis (AR) is one of the three major infectious diseases of the respiratory system in pigs. It causes facial bone deformities and chronic purulent rhinitis in infected pigs, leading to atrophy of the nasal turbinate bones. In severe infections, lesions may also occur in the nasal cavity, jawbone, and maxilla. The downstream fossa of the inferior turbinate is most commonly infected. The superior turbinate, inferior turbinate, nasal septum, and ethmoid bone can all be infected.

[0003] Porcine atrophic rhinitis (AR) is caused by *Bordetella bronchiseptica* (Bb) and *Pasteurella multocida* type A (PmA) and type D (PmD), especially by the toxin (PMT) produced by *Pasteurella multocida* type D (PmD). Inoculation of four-week-old piglets with PMT via intramuscular, intraperitoneal, and intranasal routes can induce nasal turbinate bone atrophy. Furthermore, it can affect systemic skeletal development, causing growth retardation. High doses can even lead to liver damage, resulting in jaundice and death in pigs.

[0004] Atrophic rhinitis (AR) in pigs is prevalent in pig-farming regions worldwide. Affected pigs experience slow growth and reduced feed utilization efficiency. While the mortality rate is low when infected alone, the contamination rate is high, and it easily induces other complications or infections with other pathogens, leading to high mortality and increased production costs. In pig farms severely affected by AR, economic losses range from 15% to 38%, and severely infected farms exhibit significant growth retardation, with average daily weight gain being 5-8% lower than that of healthy pigs. Therefore, developing an effective vaccine for atrophic rhinitis in pigs to prevent AR is both urgent and extremely important. Summary of the Invention

[0005] The present invention provides a recombinant Pasteurella multocida toxin protein in its first part, comprising: an epitope of a Pasteurella multocida toxin protein having the amino acid sequence shown in SEQ ID NO:2, an epitope of a Pasteurella multocida toxin protein having the amino acid sequence shown in SEQ ID NO:3, and an epitope of a Pasteurella multocida toxin protein having the amino acid sequence shown in SEQ ID NO:4.

[0006] In its second part, the present invention provides a virus-like particle (VLP) containing a recombinant Pasteurella multocida toxin protein, comprising: a recombinant Pasteurella multocida toxin protein as described above and a hepatitis B virus core protein (HBc); wherein the recombinant Pasteurella multocida toxin protein is inserted into the major immunodominant region (MIR) of the hepatitis B virus core protein.

[0007] In its third part, the present invention provides a nucleic acid sequence encoding a recombinant protein of the septicemic pasteurellosis toxin as described above.

[0008] In its fourth part, this invention provides a nucleic acid sequence encoding a virus-like particle containing a recombinant protein of Pasteurella multocida toxin as described above.

[0009] In its fifth part, the present invention provides an immune composition for porcine atrophic rhinitis comprising at least one of the recombinant Pasteurella multocida toxin protein as described above and the virus-like particles containing the recombinant Pasteurella multocida toxin protein as described above, and a pharmaceutically acceptable carrier.

[0010] Part VI of this invention provides the use of a porcine atrophic rhinitis immune composition for the preparation of a medicament for animals to combat porcine atrophic rhinitis.

[0011] In Part VII, this invention provides an antibody against Pasteurella multocida type D toxin, which is prepared by recombinant Pasteurella multocida toxin as described above or by virus-like particles containing recombinant Pasteurella multocida toxin as mentioned above.

[0012] In its eighth part, this invention provides a detection kit for porcine atrophic rhinitis, comprising a detection unit selected from at least one of the following groups: recombinant Pasteurella multocida toxin as described above, virus-like particles containing recombinant Pasteurella multocida toxin as described above, antibodies prepared from recombinant Pasteurella multocida toxin as described above, and antibodies prepared from virus-like particles containing recombinant Pasteurella multocida toxin as described above.

[0013] The present invention is illustrated by the following embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Attached Figure Description

[0014] Figure 1 The image shown is an electron microscope image of a virus-like particle (VLP) containing the recombinant Pasteurella multocida toxin protein of the present invention, in one embodiment. The arrow points to a virus-like particle of the present invention. Scale bar: 50 μm.

[0015] Figure 2 The image shows the results of determining the antibody titer against Pasteurella multocida toxin (PMT) using enzyme-linked immunosorbent assay (ELISA) in one embodiment. Group 1 is the negative control group; Group 2 is the immune composition containing *B. b.* (Bb), *Pasteurella multocida* type A (PmA), and *Pasteurella multocida* type D (PmD) obtained in Example 2 (B. b + PmA + PmD group); Group 3 is the virus-like particles containing recombinant *Pasteurella multocida* toxin protein obtained in Example 1 (re-PmT VLP group); Group 4 is the porcine atrophic rhinitis immune composition containing virus-like particles containing recombinant *Pasteurella multocida* toxin protein obtained in Example 3 (B. b + PmA + PmD + re-PmT VLP group); Group 5 is a commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group). The symbols * and ** represent significant differences compared to Group 1 (negative control group) (p<0.05 and p<0.01, respectively). The symbols # and ## represent significant differences compared to Group 2 (B.b+PmA+PmD group) (p<0.05 and p<0.01, respectively). The symbol ++ represents significant differences compared to Group 5 (commercially available vaccine group) (p<0.01).

[0016] Figures 3A to 3C The image shows the results of a neutralizing antibody assay in one embodiment. Figure 3A The image shows the cell morphology of Vero cells cultured in DMEM medium containing fetal bovine serum (FBS) (negative control group); Figure 3BThe image shows the cell morphology of Vero cells treated with 4 times the minimum toxic dose (MTD) of Pasteurella multocida toxin (PMT) (positive control group). The cells exhibit a typical nodular appearance (as indicated by the arrows). Figure 3C The image shows the cell morphology of mice immunized with a porcine atrophic rhinitis immunization composition (B.b+PmA+PmD+re-PmTVLP group) containing recombinant Pasteurella multocida toxin protein, diluted 160 times, neutralized with 4 times the minimum toxic dose (MTD) of Pasteurella multocida toxin (PMT), and then co-cultured with Vero cells. Figure 3D The image shows the cell morphology of mice immunized with commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group) diluted 160 times and neutralized with 4 times the minimum toxic dose (MTD) of septicemic pasteurellosis toxin (PMT), then co-cultured with Vero cells. The cells still exhibit typical nodular appearance (as shown by the arrow). Detailed Implementation

[0017] This invention provides a recombinant Pasteurella multocida toxin protein (re-PmT) containing three epitopes of the Pasteurella multocida toxin protein (PmT) to induce animals to produce antibodies against the Pasteurella multocida toxin protein (PmT). The three epitopes are:

[0018] Antigenic determinant A: SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO:2);

[0019] Antigenic determinant B: SISPDDPPREITD (SEQ ID NO:3); and

[0020] Antigenic determinant C: LNSTPGTGRPMP (SEQ ID NO:4).

[0021] In some preferred embodiments, the amino acid sequences of each of the stated antigenic determinants may be further linked by linkers containing at least one glycine (Gly) residue, including but not limited to Gly-Gly, Gly-Ser, or sequences as shown in SEQ ID NOs:11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. In one embodiment, the linker has the amino acid sequence shown in SEQ ID NO:11. However, the amino acid sequences of each of the stated antigenic determinants are not necessarily linked by linkers.

[0022] The recombinant Pasteurella multocida toxin protein (re-PmT) provided by this invention can be represented by the following formula:

[0023] (Antigen determinant 1)-(Connector 1) m -(antigen determinant 2)-(connector 2) n -(Antigenic determinant 3) In formula (I), one of antigenic determinant 1, antigenic determinant 2, and antigenic determinant 3 has an amino acid sequence as shown in SEQ ID NO:2, and the other two antigenic determinants have amino acid sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; 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, and 19;

[0024] Where m represents an integer from 0 to approximately 10;

[0025] Where n represents an integer from 0 to 10.

[0026] In some embodiments, the recombinant Pasteurella multocida toxin protein (re-PmT) provided by the present invention has at least one amino acid sequence as shown in SEQ ID NOs:5, 22, 23, 24, 25 and 26.

[0027] In some embodiments, the recombinant Pasteurella multocida toxin protein (re-PmT) provided by the present invention has at least about 80% sequence homology with the amino acid sequence represented by the above formula (I), preferably about 85% sequence homology, more preferably about 90% sequence homology, or even about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence homology.

[0028] This invention also provides a virus-like particle (re-PmT VLP) containing recombinant Pasteurella multocida toxin protein. This is achieved by inserting or replacing three epitopes of Pasteurella multocida toxin protein (PmT) into or replacing one of the major immunodominant regions (MIR) of the hepatitis B virus core protein (HBc), thus forming a virus-like particle (re-PmT VLP) containing recombinant Pasteurella multocida toxin protein. The epitopes of the three Pasteurella multocida toxin proteins (PmT) are as follows:

[0029] Antigenic determinant A: SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO:2);

[0030] Antigenic determinant B: SISPDDPPREITD (SEQ ID NO:3); and

[0031] Antigenic determinant C: LNSTPGTGRPMP (SEQ ID NO:4).

[0032] In some preferred embodiments, the hepatitis B virus core protein (HBc) has the amino acid sequence shown in SEQ ID NO:6.

[0033] In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 73-94 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 73-82 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 75-81 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 78-79 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 78-81 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 78-82 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 78-86 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 78-89 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 78-94 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 81-82 of the protein. In some embodiments, the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc) is located at amino acid positions 82-83 of the protein.

[0034] In some preferred embodiments, the amino acid sequences of each antigenic determinant and the amino acid sequences of the antigenic determinants may be further linked by linkers containing at least one glycine (Gly) residue, including but not limited to: Gly-Gly, Gly-Ser, SEQ ID NOs:11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21. In one embodiment, the linker has an amino acid sequence as shown in SEQ ID NOs:11 and / or 12. However, the amino acid sequences of each antigenic determinant are not necessarily linked by linkers.

[0035] The virus-like particle (re-PmT VLP) containing recombinant Pasteurella multocida toxin provided by this invention can be represented by the following formula:

[0036] (HBc-N end segment) - (connector 3) p -(antigen determinant 1)-(connector 1) m -(antigen determinant 2)-(connector 2) n -(antigen determinant 3)-(connector 4) q -(HBc-C end segment) formula (II)

[0037] One of the antigenic determinants 1, 2, and 3 has an amino acid sequence as shown in SEQ ID NO:2, and the other two antigenic determinants have amino acid sequences as shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; the linkers 1, 2, 3, and 4 are each independently selected from Gly-Gly, Gly-Ser, and SEQ ID NOs:11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21.

[0038] Where m represents an integer from 0 to approximately 10;

[0039] Where n represents an integer from 0 to 10;

[0040] Where p represents an integer from 0 to 10;

[0041] Where q represents an integer from 0 to approximately 10.

[0042] In some embodiments, the N-terminal segment of the hepatitis B virus core protein (HBc-N-terminal segment) has the sequence of amino acids 1 to 73 of the HBc protein, while the C-terminal segment of the hepatitis B virus core protein (HBc-C-terminal segment) has the sequence of amino acids 83 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 75 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 82 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 78 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 79 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 78 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 82 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 78 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 83 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 78 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 87 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 78 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 90 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 78 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 95 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 81 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 82 to 144 of the HBc protein. In some embodiments, the HBc-N-terminal segment has the sequence of amino acids 1 to 82 of the HBc protein, while the HBc-C-terminal segment has the sequence of amino acids 83 to 144 of the HBc protein.

[0043] In some embodiments, the virus-like particles (re-PmT VLP) containing recombinant Pasteurella multocida toxin provided by the present invention have at least one of the amino acid sequences shown in SEQ ID NOs:9, 10, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36.

[0044] In some embodiments, the virus-like particles (re-PmT VLP) containing recombinant Pasteurella multocida toxin provided by the present invention have at least about 80% sequence homology with the amino acid sequence represented by formula (II) above, preferably about 85% sequence homology, more preferably about 90% sequence homology, or even about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence homology.

[0045] This invention also provides a nucleic acid sequence encoding the recombinant Pasteurella multocida toxin protein (re-PmT) of this invention and a nucleic acid sequence encoding a virus-like particle (re-PmTVLP) containing the recombinant Pasteurella multocida toxin protein of this invention. The recombinant Pasteurella multocida toxin protein (re-PmT) includes antigenic determinants as shown in SEQ ID NOs:2, 3, and 4. The virus-like particle (re-PmTVLP) containing the recombinant Pasteurella multocida toxin protein includes antigenic determinants as shown in SEQ ID NOs:2, 3, and 4 and a hepatitis B virus core protein (HBc) as shown in SEQ ID NO:6, wherein the antigenic localization inserts into or replaces the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc).

[0046] The nucleotide sequence encoding the recombinant Pasteurella multocida toxin protein (re-PmT) of the present invention and the nucleic acid sequence encoding the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein of the present invention are derived from the amino acid sequences of the recombinant Pasteurella multocida toxin protein (re-PmT) and the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein of the present invention, respectively. The nucleotide sequence provided by the present invention can be obtained by replacing each amino acid in the amino acid sequences of the recombinant Pasteurella multocida toxin protein (re-PmT) and the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein of the present invention with the nucleotide sequences encoding those amino acids listed in the genetic code table (including various degenerate codons, or synonymous codons). For example, the serine residues in the amino acid sequences of the recombinant Pasteurella multocida toxin protein (re-PmT) of the present invention and the virus-like particles (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein of the present invention can be encoded by nucleotide sequences such as TCT, TCC, TCA, TCG, AGT, and AGC. Each amino acid in the amino acid sequence of the recombinant Pasteurella multocida toxin protein (rPMT) of the present invention and the virus-like particles (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein of the present invention can be encoded by the following nucleotide sequences:

[0047]

[0048]

[0049] Furthermore, the present invention also provides an immune composition for porcine atrophic rhinitis. The porcine atrophic rhinitis immune composition contains a recombinant Pasteurella multocida toxin protein (re-PmT) and / or a virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein. The recombinant Pasteurella multocida toxin protein (re-PmT) includes antigenic determinants as shown in SEQ ID NOs:2, 3, and 4. The virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein includes antigenic determinants as shown in SEQ ID NOs:2, 3, and 4 and a hepatitis B virus core protein (HBc) as shown in SEQ ID NO:6, wherein the antigenic localization inserts into or replaces the major immunodominant region (MIR) of the hepatitis B virus core protein (HBc).

[0050] In some embodiments, the recombinant Pasteurella multocida toxin protein (re-PmT) has an amino acid sequence as shown in SEQ ID NOs:5, 22, 23, 24, 25, or 26. In one specific embodiment, the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein has an amino acid sequence as shown in SEQ ID NO:9. In another specific embodiment, the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein has an amino acid sequence as shown in SEQ ID NO:10. In yet another embodiment, the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein has one of the amino acid sequences shown in SEQ ID NOs:27, 28, 29, 30, 31, 32, 33, 34, 35, or 36.

[0051] The recombinant Pasteurella multocida toxin protein (re-PmT) and the virus-like particles (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein provided by the present invention include, but are not limited to, obtaining them by gene selection or by using a peptide synthesizer. The recombinant protein obtained by gene selection may include, but is not limited to: selecting the nucleic acid sequence encoding the recombinant Pasteurella multocida toxin protein (re-PmT) or the nucleic acid sequence encoding the virus-like particles (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein into an expression vector, thereby forming a plasmid containing the nucleic acid sequence encoding the recombinant Pasteurella multocida toxin protein (re-PmT) or a plasmid containing the nucleic acid sequence encoding the virus-like particles (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein; then transfecting the plasmid into a biological expression host, and obtaining the antigen protein after protein expression.

[0052] The expression vector system includes, but is not limited to, the pET vector system and the pGEX vector system; the biological expression system (host) includes, but is not limited to: prokaryotic expression systems (e.g., Escherichia coli), eukaryotic expression systems (e.g., animal cells (insect cells or mammalian cells), plant cells).

[0053] In one embodiment, the porcine atrophic rhinitis immune composition provided by the present invention further contains *B. bronchiseptica*, *Pasteurella multocida* type A (PmA), and *Pasteurella multocida* type D (PmD). The *B. bronchiseptica* may be sourced from, for example, but not limited to, strains from the American Type Culture Collection (ATCC) ATCC31437; the *Pasteurella multocida* type A (PmA) may be sourced from, for example, but not limited to, strains from the National Collection of Type Cultures (NCTC) NCTC 12177; and the *Pasteurella multocida* type D (PmD) may be sourced from, for example, but not limited to, strains from the National Collection of Type Cultures (NCTC) NCTC 12178, or strains isolated from the wild.

[0054] The porcine atrophic rhinitis immune composition provided by the present invention may further contain other pathogen antigens, including, but not limited to: porcine circovirus type 2 (PCV2) antigen, swine influenza virus (SIV) antigen, porcine reproductive and respiratory syndrome virus (PRRSV) antigen, mycoplasma, porcine parvovirus (PPV), swine erysipelas, Aujeszky's disease, and / or Actinobacillus pleuropneumonia (APP).

[0055] In addition, the porcine atrophic rhinitis immune composition provided by the present invention may further comprise one or more carriers selected from the following pharmaceutically acceptable carriers, including: solvents, emulsifiers, suspending agents, disintegrants, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, adjuvants, biotype carriers, etc.

[0056] The pharmaceutically acceptable carrier comprises one or more agents selected from the following: solvent, emulsifier, suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, surfactant, adjuvant, and other similar or applicable carriers of the present invention.

[0057] The pharmaceutically acceptable excipient may be a pharmaceutically acceptable organic or inorganic carrier substance suitable for parenteral, enteral, or nasal administration, and the excipient shall not produce an adverse reaction with the active composition. Suitable excipients include, but are not limited to, water, saline solutions, vegetable oils, polyethylene glycol, gelatin, amylose, lactose, magnesium stearate, talc, silica, viscous paraffin, fatty acid monoglycerides and glycerol, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0058] The pharmaceutically acceptable adjuvant includes, but is not limited to, aqueous aluminum hydroxide gel, alum, Freund's incomplete adjuvant, oil adjuvant, water-soluble adjuvant, or water-in-oil-in-water (W / O / W) biphasic adjuvant; in one embodiment, the adjuvant is aqueous aluminum hydroxide gel.

[0059] Furthermore, the present invention provides a method for animals to combat swine atrophic rhinitis, comprising administering an effective amount of the above-mentioned immune composition to the animal to enhance the animal's immunity against swine atrophic rhinitis, thereby improving its clinical symptoms, survival rate, and weight gain trend.

[0060] This invention also provides an antibody against Pasteurella multocida type D toxin (PmT), said antibody being prepared or derived from the recombinant Pasteurella multocida toxin protein (re-PmT) and / or virus-like particles (re-PmT VLPs) containing the recombinant Pasteurella multocida toxin protein provided by this invention; said antibody includes, but is not limited to: monoclonal antibodies, polyclonal antibodies, and genetically recombinant antibodies. In one embodiment, said antibody is a polyclonal antibody obtained by injecting the recombinant Pasteurella multocida toxin protein (rPMT) provided by this invention into an animal.

[0061] The present invention also provides a detection kit for porcine atrophic rhinitis, the detection kit being used to detect whether a test sample contains Pasteurella multocida type D toxin (PmT) or whether a test sample contains an antibody against Pasteurella multocida type D toxin (PmT). The detection kit includes, but is not limited to: (1) an antigen, the antigen being the recombinant Pasteurella multocida toxin protein (re-PmT) and / or a virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein provided by the present invention, wherein in one embodiment, the antigen is placed on an antigen tray; and / or (2) an antibody, the antibody being a monoclonal antibody or polyclonal antibody derived and prepared from the recombinant Pasteurella multocida toxin protein (re-PmT) and / or the virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein provided by the present invention.

[0062] The detection kits include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) kits, microchip kits, immunofluorescence assay (IFA) kits, or other detection kits prepared using the recombinant Pasteurella multocida toxin protein (re-PmT) and / or virus-like particles (re-PmT VLPs) containing the recombinant Pasteurella multocida toxin protein. In one embodiment, the detection kit includes at least an antigen disc containing the recombinant Pasteurella multocida toxin protein (re-PmT) and / or virus-like particles (re-PmT VLPs) containing the recombinant Pasteurella multocida toxin protein provided by the present invention, which can be used to test whether the sample contains antibodies against Pasteurella multocida toxin (PmT).

[0063] All technical and scientific terms used in this specification, unless otherwise defined, shall have the meaning commonly understood by one of ordinary skill in the art.

[0064] As used herein and in the claims of the appended patent applications, the terms “about,” “approximately,” or “nearly” substantially mean that the stated value or range is within 20%, preferably within 10%, and more preferably within 5%. The numerical values ​​provided herein are approximate and are intended to be inferred even if the terms “about,” “approximately,” or “nearly” are not used.

[0065] Unless the context clearly indicates otherwise, the terms "a," "an," and "described" as used herein and in the appended claims have the meaning of the plural. Furthermore, for ease of reading, headings or subheadings may be used in this specification, which do not affect the scope of the invention.

[0066] The following embodiments will describe the invention in more detail. These embodiments are merely illustrative, as many modifications and variations will become apparent to those skilled in the art. Various specific embodiments of the invention will be described in detail below.

[0067] Example 1: Construction of recombinant Pasteurella multocida toxin protein (re-PmT)

[0068] 1. Design of the amino acid sequence of recombinant Pasteurella multocida toxin (re-PmT)

[0069] Three epitopes were selected from the full-length amino acid sequence of Pasteurella multocida toxin protein (PmT) (as shown in SEQ ID NO:1), namely:

[0070] Antigenic determinant A: SVGKEGAYYPDHDYGPEYNPVWGPNEQI (SEQ ID NO:2);

[0071] Antigen determinant B: SISPDDPPREITD (SEQ ID NO:3);

[0072] Antigenic determinant C: LNSTPGTGRPMP (SEQ ID NO:4).

[0073] Each of the antigenic determinants A (SEQ ID NO:2), B (SEQ ID NO:3), and C (SEQ ID NO:4) is connected by a linker having an amino acid sequence as shown in SEQ ID NO:11. Depending on the different combinations of antigenic determinants A, B, and C from the N-terminus to the C-terminus of the recombinant protein, the amino acid sequences of the recombinant Pasteurella multocida toxin protein (re-PmT) are shown in SEQ ID NOs:5, 22, 23, 24, 25, and 26, respectively. These amino acid sequences are synthesized using a synthesizer or obtained through gene selection and expression.

[0074] 2. Design of the amino acid sequence of a virus-like particle (re-PmT VLP) containing recombinant Pasteurella multocida toxin protein.

[0075] The recombinant Pasteurella multocida toxin protein (re-PmT) (SEQ ID NO:5) constructed above was inserted between amino acids 78 and 79 of the hepatitis B virus core protein (HBc) (SEQ ID NO:6). Furthermore, a linker was used between the N-terminal segment of the hepatitis B virus core protein (HBc) (amino acids 1-78; SEQ ID NO:7) and the recombinant Pasteurella multocida toxin protein (re-PmT) (SEQ ID NO:5), and between the recombinant Pasteurella multocida toxin protein (re-PmT) (SEQ ID NO:5) and the C-terminal segment of the hepatitis B virus core protein (HBc) (amino acids 79-144; SEQ ID NO:8), respectively. The linkers have the amino acid sequence shown in SEQ ID NO:12. The amino acid sequence of the resulting virus-like particle (re-PmT VLP) containing the recombinant Pasteurella multocida toxin protein is shown in SEQ ID NO:9. The amino acid sequence can be synthesized using a synthesizer, or a nucleic acid sequence encoding the amino acid sequence can be synthesized first, and the nucleic acid sequence can be colonized into a performance vector, expressed in a biological performance host, and purified.

[0076] The aforementioned virus-like particles (re-PmT VLPs) containing recombinant Pasteurella multocida toxin proteins can also be obtained using gene selection expression. When constructing the nucleic acid sequence encoding virus-like particles (re-PmT VLPs) containing recombinant Pasteurella multocida toxin proteins using gene selection, restriction enzyme sites can be used to ligate the individual fragments. For example, but not limited to, the nucleic acid sequences encoding the N-terminal segment (amino acids 1-78; SEQ ID NO:7) of the hepatitis B virus core protein (HBc), the nucleic acid sequence encoding the linker (SEQ ID NO:12), the nucleic acid sequence encoding the recombinant pasteurellosis toxin protein (re-PmT) (SEQ ID NO:5), the nucleic acid sequence encoding the linker (SEQ ID NO:12), and the nucleic acid sequence encoding the C-terminal segment (amino acids 79-144; SEQ ID NO:8) of the hepatitis B virus core protein (HBc) are sequentially inserted into multiple restriction enzyme cleavage sites (MCS) of the vector pET24, and the above nucleic acid sequences are linked by restriction enzyme cleavage sites such as SacI, NcoI, HindIII, and KpnI, respectively, to construct a virus-like particle (re-PmT) encoding the recombinant pasteurellosis toxin protein. The nucleic acid sequence of the VLP is obtained, and then the vector containing this nucleic acid sequence is transferred into the biological expression host. After protein expression, the recombinant protein of Pasteurella multocida toxin is obtained as a virus-like particle (re-PmT VLP), and its amino acid sequence is shown in SEQ ID NO:10.

[0077] 3. Observation of virus-like particles (re-PmT VLP) containing recombinant Pasteurella multocida toxin protein.

[0078] After purifying the recombinant Pasteurella multocida particles (re-PmT VLP) containing the septicemic pasteurellosis toxin, the protein concentration was adjusted to 1 mg / ml. The sample was then filtered through a 0.22 mm filter membrane and observed using a JEM-1400 electron microscope (JEOL Corporation, Japan) with negative staining. First, a 300-mesh carbon-coated copper grid was prepared. Then, 8 ml of sample was dropped onto each grid, allowed to stand for 3-5 minutes, and excess liquid was absorbed with filter paper. Next, the sample was stained with 1% uranium acetate solution (pH 4.5) for 30 seconds, and after absorbing excess liquid with filter paper, it was observed under a microscope. The voltage was set to 80 kV during observation. The results of the microscopic observation are as follows: Figure 1 As shown, the virus-like particles (re-PmT VLP) containing recombinant Pasteurella multocida toxin of the present invention exhibit a virus-like particle shape (e.g., Figure 1 (As indicated by the arrow)

[0079] Example 2: Culture of *Bordetella bronchiseptica* and *Pasteurella multocida*

[0080] 1. Culture of *B. bronchiseptica*

[0081] First, *B. bronchiseptica* was inoculated onto TSB solid medium [containing 5% (v / v) yeast extract, 10% (v / v) serum, and tryptic soy broth (TSB, BD Biosciences, USA)] and cultured overnight at 37°C. Then, a single colony was selected and inoculated onto brain heart infusion (BHI) liquid medium (BD Biosciences, USA) and cultured overnight at 37°C with shaking. Next, the bacterial culture was inoculated again onto BHI liquid medium and cultured overnight at 37°C with shaking, and the colony forming unit (CFU) value was calculated. Finally, formaldehyde was added and the culture was shaken at room temperature for 24 to 36 hours to inactivate the bacteria.

[0082] 2. Culture of Pasteurella multocida (P. multocida)

[0083] First, *Pasteurella multocida* type A (PmA) and *Pasteurella multocida* type D (PmD), which are capable of producing toxins, were inoculated separately onto TSB solid medium [containing 5% (v / v) yeast extract, 10% (v / v) serum, and soybean casein medium (TSB, BD Biosciences, USA)]. After incubation overnight at 37°C, a single colony was selected and inoculated into brain heart extract (BHI) liquid medium (BD Biosciences, USA) and incubated overnight with shaking at 37°C. Then, 0.1% (v / v) of the bacterial solution was inoculated into BHI liquid medium and incubated overnight with shaking at 37°C, and the colony forming unit (CFU) value was calculated. Finally, formaldehyde was added to inactivate the bacterial solution.

[0084] Example 3: Formulation of an immunomodulatory composition for porcine atrophic rhinitis

[0085] The recombinant Pasteurella multocida toxin protein (re-PmT) (SEQ ID NO:5) (final concentration 250 μg / ml) obtained in Example 1 or virus-like particles containing recombinant Pasteurella multocida toxin protein (re-PmT VLP) (SEQ ID NO:10) (final concentration 250 μg / ml) were compared with the inactivated Bordetella bronchiseptica (B. bronchiseptica) obtained in Example 2 (final concentration 1 x 10⁻⁶). 9 CFU / ml), inactivated Pasteurella multocida type A (PmA) (final concentration 1x10⁻⁶ CFU / ml). 9 CFU / ml) and inactivated Pasteurella multocida type D (PmD) (final concentration 1x10⁻⁶ CFU / ml) 9 The mixture of CFU / ml was homogenized with phosphate buffered solution (PBS), and aluminum gel [final concentration 30% (v / v)] was added as an adjuvant to prepare an immune composition for porcine atrophic rhinitis.

[0086] Example 4: Immunogenicity and Neutralizing Antibody Analysis of an Immunotherapy Composition for Porcine Atrophic Rhinitis

[0087] 1. Mouse immunization test

[0088] Three-week-old healthy BALB / c mice (Experimental Animal Center, Taiwan) that were negative for Pasteurella multocida antibodies were randomly divided into 5 groups; Group 1 was the control group, and Groups 2-5 were the immunization test groups; each mouse in each group was injected intraperitoneally (ip.) with 0.2 ml of the following substance:

[0089] Group 1: PBS buffer solution containing 30% (v / v) aluminum gel (negative control group);

[0090] Group 2: *B. bronchiseptica* obtained in Example 2 (1x10⁻¹¹) 9 CFU / ml), Pasteurella multocida type A (PmA) (1x10) 9 CFU / ml) and Pasteurella multocida type D (PmD) (1x10) 9 An immunotherapy composition containing 30% (v / v) aluminum gel adjuvant (B.b+PmA+PmD group);

[0091] Group 3: Virus-like particles of recombinant Pasteurella multocida toxin obtained in Example 1 (SEQ ID NO: 10) (concentration of 250 μg / ml) (re-PmT VLP group);

[0092] Group 4: The porcine atrophic rhinitis immune composition (B.b+PmA+PmD+re-PmT VLP group) containing recombinant Pasteurella multocida toxin protein (SEQ ID NO: 10) obtained in Example 3; and

[0093] Group 5: Commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group).

[0094] Blood was collected from each mouse 24 hours before the initial immunization (day 0). Blood was collected again on day 13 after the initial immunization (day 1), followed by a second immunization on day 14 with the same dose, and then blood was collected again on day 24. Serum was separated from the blood samples for enzyme-linked immunosorbent assay (ELISA) of toxin antibodies and neutralizing antibody assays.

[0095] 2. Enzyme-linked immunosorbent assay (ELISA) for toxin antibodies

[0096] Commercially available Pasteurella multocida toxin (PMT) (Abcam, USA) was used as the antigen. The antigen was coated onto a 96-well ELISA plate (Thermo Scientific, USA) and incubated at 4°C for 16 hours. After removing excess antigen, wash buffer (0.9% NaCl; 0.1% Tween 20) was added, and the plate was washed three times before being drained. Next, blocking buffer (wash buffer containing 1% BSA) was added, and the mixture was allowed to stand at room temperature for 1 hour. Then, the samples were washed with washing buffer. Serum samples collected from each group of mice were then diluted with PBS buffer, and diluted mouse serum was added to each well. After standing at room temperature for 1 hour, the serum samples were removed, and the mixture was washed with washing buffer. Then, horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (goat anti-mouse conjugated HRP, GeneTex, USA) was added. This secondary antibody was first diluted 5,000 times with blocking buffer before being added to a 96-well plate (100 μl / well). After standing at room temperature for 1 hour, the secondary antibody was removed, and the mixture was washed with washing buffer. Finally, 100 μl of the diluted antibody was added to each well. A solution of 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB, KPL, USA) was used for color development in the dark for 10 minutes, followed by immunoassay using an enzyme-linked immunosorbent assay (ELISA) reader. The M2 / M2ELISAReader (Molecular Devices, USA) reads the absorbance at a wavelength of 650 nm.

[0097] The results of enzyme-linked immunoassay are as follows: Figure 2As shown. After secondary immunization, the serum of mice immunized with the porcine atrophic rhinitis immunization composition containing recombinant Pasteurella multocida toxin protein (Group 4, i.e., B.b+PmA+PmD+re-PmT VLP group) had the highest antibody titer against Pasteurella multocida toxin (PMT), followed by the serum of mice immunized with the recombinant Pasteurella multocida toxin protein recombinant protein obtained in Example 1 (Group 3, i.e., re-PmT VLP group). However, the antibody titer against Pasteurella multocida toxin (PMT) in the serum of mice immunized with commercially available porcine atrophic rhinitis vaccine (Group 5, i.e., commercially available vaccine group) was not significantly different from the antibody titers in the serum of mice immunized with Bordetella bronchiseptica, Pasteurella multocida type A, and Pasteurella multocida type D obtained in Example 2 (Group 2, i.e., B.b+PmA+PmD group). The anti-PMT antibody titers in the serum of mice in each immunization test group (groups 2-5) were significantly higher than those in the serum of mice in group 1 (negative control group) (p<0.05 or p<0.01). Therefore, the recombinant PMT protein recombinant particle (re-PmTVLP) provided by this invention can effectively induce anti-PMT antibodies in animals, exhibiting immunogenicity, and its immunization effect is better than that of commercially available porcine atrophic rhinitis vaccine.

[0098] 3. Neutralizing antibody titer test

[0099] Using Vero cells as experimental material, this study tested whether the anti-Pasteurella multocida toxin (PMT) antibodies in the serum of mice immunized with a porcine atrophic rhinitis immunization composition (Group 4, i.e., B.b+PmA+PmD+re-PmT VLP group) containing recombinant Pasteurella multocida toxin protein were neutralizing antibodies capable of neutralizing PMT toxicity. Before testing the neutralizing antibody titer, the minimum toxindose (MTD) of Vero cells was determined.

[0100] (a) Minimum toxic dose (MTD) determination of Vero cells

[0101] Vero cells were seeded in 96-well culture dishes. After the cells grew into a monolayer, the culture medium was removed, and septicemia pasteurelloma toxin (PMT, Abcam) diluted in serum-free DMEM medium (GIBCO, USA) was added for treatment (0, 20, 50, 100 ng). DMEM medium containing fetal bovine serum (FBS) was used as a negative control. Cell morphology changes were observed after culture. The lowest concentration of PMT that induced cytopathic effect (CPE) in Vero cells was defined as the minimum toxic dose (MTD). The results showed that the minimum toxic dose of PMT for Vero cells was 50 ng.

[0102] (b) Neutralizing antibody titer assay

[0103] First, mouse serum immunized with a porcine atrophic rhinitis immunization composition containing recombinant Pasteurella multocida toxin protein (Group 4, i.e., B.b+PmA+PmD+re-PmT VLP group) and mouse serum immunized with a commercially available porcine atrophic rhinitis vaccine (Group 5, i.e., commercially available vaccine group) were diluted 10-fold and then serially diluted (40, 80, 120, 160, 200, 240-fold). The diluted mouse serum was added to each well of a 96-well culture dish, along with Pasteurella multocida toxin (PMT) containing 4 times the minimum toxic dose (MTD), and the mixture was incubated at 37°C for 1 hour. Next, the reaction solution was added to Vero cells cultured in the 96-well dish and incubated at 37°C in a 5% CO2 incubator. The presence of the serum in these cells was then observed to determine if it could inhibit cytopathic effects (CPE) in Vero cells.

[0104] The results are shown in Figure 3. Vero cells treated with 4 times the minimum toxic dose (MTD) of Pasteurella multocida toxin (PMT) served as a positive control group, and their cell morphology is as follows: Figure 3B As shown, the cells exhibit a typical nodular appearance (e.g. Figure 3B (As indicated by the arrow). In contrast, Vero cells cultured in DMEM medium containing fetal bovine serum (FBS) served as a negative control group, and their cell morphology was as follows. Figure 3A As shown; mouse serum from mice immunized with a porcine atrophic rhinitis immunization composition (Group 4, i.e., B.b+PmA+PmD+re-PmT VLP group) containing recombinant Pasteurella multocida toxin protein was diluted 160-fold and neutralized with 4 times the minimum toxic dose (MTD) of Pasteurella multocida toxin (PMT). The resulting cells were then co-cultured with Vero cells, and the cell morphology was as shown. Figure 3CAs shown. Mouse serum immunized with a commercially available porcine atrophic rhinitis vaccine (Group 5, i.e., the commercially available vaccine group) diluted 160-fold and neutralized with 4 times the minimum toxic dose (MTD) of Pasteurella multocida toxin (PMT), was then added to Vero cells for co-culture. The cell morphology is shown in the figure. Figure 3D As shown. Figure 3A and Figure 3C None of the cells shown exhibited the same characteristics as described above. Figure 3B The image shown is a typical nodular pattern. Figure 3D A few nodular cell morphologies were still observed. Serum from mice immunized with a porcine atrophic rhinitis immunization composition containing recombinant Pasteurella multocida toxin protein (Group 4, i.e., B.b+PmA+PmD+re-PmT VLP group) and serum from mice immunized with a commercially available porcine atrophic rhinitis vaccine (Group 5, i.e., commercially available vaccine group) both contained neutralizing antibodies against Pasteurella multocida toxin (PMT), with the former showing a higher level of neutralizing antibodies than the latter. These results indicate that, compared to commercially available porcine atrophic rhinitis vaccines, the porcine atrophic rhinitis immunization composition containing recombinant Pasteurella multocida toxin protein provided by this invention can induce the production of more neutralizing antibodies in test animals.

[0105] 4. Statistical Methods

[0106] All experimental data were statistically analyzed using SigmaState software and one-way ANOVA. Results are expressed as mean ± standard deviation (mean ± SEM). The Student-Newman-Keuls test was used for comparisons between groups; * and ** indicate significant differences compared to Group 1 (negative control group) (p < 0.05 and p < 0.01, respectively). # and ## indicate significant differences compared to Group 2 (B.b+PmA+PmD group) (p < 0.05 and p < 0.01, respectively). ++ indicates significant differences compared to Group 5 (commercially available vaccine group) (p < 0.01).

[0107] Example 5: Immunogenicity and protective efficacy analysis of a porcine atrophic rhinitis immunization composition containing recombinant Pasteurella multocida toxin protein (re-PmT VLP) 1 – Taiwan Porcine Atrophic Rhinitis Vaccine Testing Standards (Pasteurella multocida potency test)

[0108] According to the testing standards for Pasteurella multocida vaccine in Taiwan, 3-week-old BALB / c mice (Experimental Animal Center, Taiwan) that were negative for Pasteurella antibodies were randomly divided into 3 groups: Group 1 was the control group, Group 2 was the B.b+PmA+PmD+re-PmTVLP immunization test group, and Group 3 was the commercially available vaccine immunization test group. Each mouse was injected intraperitoneally (ip.) with 0.5 ml of a 10-fold diluted test sample. The results for each group were as follows:

[0109] Group 1: PBS buffer solution containing 30% (v / v) aluminum gel (control group);

[0110] Group 2: The porcine atrophic rhinitis immune composition (B.b+PmA+PmD+re-PmT VLP group) containing recombinant Pasteurella multocida toxin protein (SEQ ID NO: 10) obtained in Example 3; and

[0111] Group 3: Commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group).

[0112] The immunization test groups (groups 2 and 3) were each divided into 3 subgroups on day 14 post-immunization. The subgroups were then treated with 1 x 10^6 highly virulent strains of Pasteurella multocida type D (PmD) capable of producing toxins (same as in Example 2). 6 CFU / ml, 1x10 7 CFU / ml, 1x10 8 Mice were intraperitoneally injected with 0.1 ml of live bacterial solutions at three concentrations of CFU / ml. Simultaneously, the control group (Group 1) mice were also divided into three subgroups, and each subgroup was injected with 1 x 10^6 CFU / ml of virulent Pasteurella multocida strain, according to the group order. 5 CFU / ml, 1x10 6 CFU / ml, 1x10 7 Three concentrations of live bacterial solutions (CFU / ml) were administered via intraperitoneal injection at 0.1 ml. After a 10-day observation period, the LD50 of each immunization experimental group and the control group was calculated using the Beherens-Karber method. 50 Furthermore, the defense index of each immunization test group must be 1x10 higher than that of the control group. 0.5 The Becquerel II defense index calculation method is as follows:

[0113] LD 50 =Minimum dilution factor of the challenge dose - [(Total mortality rate of all groups / 100) - 0.5] x 1

[0114] Defense index = [Minimum dilution factor of challenge dose in control group - [(Total mortality rate of each group / 100) - 0.5] x 1] - [Minimum dilution factor of challenge dose in immunized group - [(Total mortality rate of each group / 100) - 0.5] x 1].

[0115] The results are shown in Table 1. The porcine atrophic rhinitis immune composition containing recombinant Pasteurella multocida toxin particles (Group 2, i.e., B.b+PmA+PmD+re-PmT VLP group) did indeed induce protective efficacy in mice and resistant them to challenge with virulent Pasteurella multocida type D (PmD) strains, with a defense index greater than 1x10. 2.8 It exceeds Taiwan's testing standards (1x10). 0.5 ) and the defense index (1x10) of commercially available porcine atrophic rhinitis vaccine (commercially available vaccine group). 2.5 ).

[0116] Table 1. Immunogenicity and protective efficacy analysis of swine atrophic rhinitis immunization compositions containing recombinant Pasteurella multocida toxin protein (re-PmT VLP). Results

[0117]

[0118]

[0119] Example 6: Immunogenicity and protective efficacy analysis of an immunogenic composition for porcine atrophic rhinitis containing recombinant Pasteurella multocida toxin protein (re-PmT VLP) 2

[0120] BALB / c mice (Experimental Animal Center, Taiwan) weighing 15–20 g and negative for Pasteurella multocida antibodies were randomly divided into three groups: Group 1 was the control group, and Groups 2 and 3 were the immunization test groups. Each immunization test group was further subdivided into three subgroups; mice in each group were injected intraperitoneally with the following substances:

[0121] Group 1: Each mouse was injected with 0.2 ml of PBS buffer solution (control group);

[0122] Group 2-1: Each mouse was injected with 0.2 ml of the porcine atrophic rhinitis immune composition stock solution (B.b+PmA+PmD+re-PmT VLP group) containing recombinant protein of Pasteurella multocida toxin obtained in Example 3;

[0123] Group 2-2: Each mouse was injected with 0.2 ml of the porcine atrophic rhinitis immune composition (1 / 5B.b+PmA+PmD+re-PmT VLP group) containing recombinant Pasteurella multocida toxin protein (SEQ ID NO:10) obtained in Example 3, diluted 5 times.

[0124] Groups 2-3: Each mouse was injected with 0.2 ml of the porcine atrophic rhinitis immune composition (1 / 25B.b+PmA+PmD+re-PmT VLP group) containing recombinant Pasteurella multocida toxin protein (SEQ ID NO:10) obtained in Example 3, diluted 25 times.

[0125] Group 3-1: Each mouse was injected with 0.2 ml of commercially available porcine atrophic rhinitis vaccine stock solution (commercially available vaccine group);

[0126] Groups 3-2: Each mouse was injected with 0.2 ml of commercially available porcine atrophic rhinitis vaccine diluted 5 times (1 / 5 of the commercially available vaccine group);

[0127] Group 3-3: Each mouse was injected with 0.2 ml of commercially available porcine atrophic rhinitis vaccine diluted 25 times (1 / 25 commercially available vaccine group).

[0128] The immunization test groups (groups 2 and 3) received a second immunization on day 14 after the initial immunization, at the same dose as the initial immunization; the control group (group 1) mice were injected again with 0.2 ml of PBS buffer solution; on day 10 after the second immunization, a challenge test was conducted, with each mouse injected intraperitoneally with 0.2 ml of 100 LD of a highly virulent strain of Pasteurella multocida type D (PmD) capable of producing toxins [same as in Example 2]. 50 Live bacterial suspensions were used, and the survival rate was recorded after 10 days. The survival rate of the undiluted immunization group (groups 2-1 and 3-1) must be higher than 80%, the survival rate of the 5-fold dilution immunization group (groups 2-2 and 3-2) must be higher than 50%, and the survival rate of the 25-fold dilution immunization group (groups 2-3 and 3-3) must be higher than 20%. All bacteria in the control group must die.

[0129] The results are shown in Table 2. The porcine atrophic rhinitis immunization composition containing recombinant Pasteurella multocida toxin protein (re-PmT VLP) (groups 2-1, 2-2, and 2-3) could indeed induce sufficient protective efficacy in mice. The survival rate of mice with the original immunized solution, mice with the vaccine diluted 5 times, and mice with the vaccine diluted 25 times was 100%, all meeting the above survival rate criteria.

[0130] Table 2. Immunogenicity and protective efficacy analysis of swine atrophic rhinitis immunization compositions containing recombinant Pasteurella multocida toxin protein (re-PmT VLP). Results

[0131]

[0132] Example 7: Preparation of Antibody against Pasteurella multocida toxin (PmT)

[0133] 1. Polyclonal antibodies against Pasteurella multocida toxin (PmT)

[0134] The virus-like particles (re-PmTVLP) containing recombinant Pasteurella multocida toxin obtained in Example 1 were mixed with a suitable adjuvant (e.g., aluminum gel) and administered to animals (e.g., mice, rats, pigs, goats, rabbits) for primary immunization. A secondary immunization could be performed after an appropriate time interval (e.g., 2-3 weeks), if necessary. After an appropriate time interval (e.g., 2-3 weeks), serum was collected from the immunized animals (e.g., mice, rats, pigs, goats, rabbits) to obtain polyclonal antibodies against Pasteurella multocida toxin (PmT).

[0135] The polyclonal antibody against Pasteurella multocida toxin (PmT) can be bound to a chromogenic agent or fluorescence as needed.

[0136] The animals described herein may be immunized more frequently as needed after primary and secondary immunizations to increase antibody titers.

[0137] The animals to which the treatment is administered include, but are not limited to: mice, rats, rabbits, poultry (eggs), pigs, goats, cattle, and aquatic animals.

[0138] 2. Monoclonal antibodies against Pasteurella multocida (PmT) virus

[0139] The virus-like particles (re-PmTVLP) containing recombinant Pasteurella multocida toxin obtained in Example 1 were mixed with a suitable adjuvant (e.g., aluminum gel) and administered to animals (e.g., mice, rats, pigs, goats, rabbits) for primary immunization. A secondary immunization could be performed as needed after an appropriate time interval (e.g., 2-3 weeks). After an appropriate time interval (e.g., 2-3 weeks), serum was collected from the immunized animals (e.g., mice) to evaluate mice suitable for spleen cell collection. Spleen cells were collected from the suitable mice and fused with myeloma cells (e.g., FO cell lines, NS cell lines) using PEG (Polyethylene Glycol, such as PEG1500). After screening for secretory fusion tumors from the fused cells and monoclonalizing them, a fusion cell line suitable for producing monoclonal antibodies against Pasteurella multocida toxin (PmT) was obtained.

[0140] The antibodies prepared in the above manner can be used in immunoassay reagents, therapeutic agents, or added to food or feed to give consumers immunity.

[0141] The above detailed description is a specific description of one feasible embodiment of the present invention. However, this embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the spirit of the present invention should be included in the patent scope of this case. sequence list <110> Jinxie International Industrial Co., Ltd. <120> Recombinant protein of Pasteurella multocida toxin, its virus-like particles and their applications <130> P17-0222 <140> 201710985876X <141> 2017-10-20 <160> 36 <170> SIPOSequenceListing 1.0 <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 Ile 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 Tyr 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 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 Leu 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 Leu 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 Leu 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 Glu 1010 1015 1020 Ala Ile Glu Lys Leu Asp Arg Glu Gly Gln Lys Phe Val Val Phe Ala 1025 1030 1035 1040 Asp Ser Ser Leu Leu Asn Ser Thr Pro Gly Thr Gly Arg Pro Met Pro 1045 1050 1055 Gly Leu Val Gln Tyr Leu Lys Ile Pro Ala Thr Val Val Asp Ser Asp 1060 1065 1070 Gly Ala Trp Gln Phe Leu Pro Asp Val Ala Ser Ser Arg Val Pro Ile 1075 1080 1085 Glu Val Thr Glu Leu Glu Asn Trp Gln Val Leu Thr Pro Pro Gln Gly 1090 1095 1100 Lys Ile Leu Gly Leu Lys Gln Phe Lys Leu Thr Ala Gly Phe Pro Thr 1105 1110 1115 1120 Glu Gln Ser Arg Leu Pro Leu Leu Glu Asn Ser Val Ser Glu Asp Leu 1125 1130 1135 Arg Glu Glu Leu Met Gln Lys Ile Asp Ala Ile Lys Asn Asp Val Lys 1140 1145 1150 Met Asn Ser Leu Val Cys Met Glu Ala Gly Ser Cys Asp Ser Val Ser 1155 1160 1165 Pro Lys Val Ala Ala Arg Leu Lys Asp Met Gly Leu Glu Ala Gly Met 1170 1175 1180 Gly Ala Ser Ile Thr Trp Trp Arg Arg Glu Gly Gly Met Glu Phe Ser 1185 1190 1195 1200 His Gln Met His Thr Thr Ala Ser Phe Lys Phe Ala Gly Lys Glu Phe 1205 1210 1215 Ala Val Asp Ala Ser His Leu Gln Phe Val His Asp Gln Leu Asp Thr 1220 1225 1230 Thr 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 Gly 1250 1255 1260 Asn Met Leu Ala Leu Phe Met Pro Pro Leu Phe Thr Lys Pro Arg Leu 1265 1270 1275 1280 Thr <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 <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 A virus <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 A virus <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 virus <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 <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 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> 10 <211> 231 <212> PRT <213> Artificial Sequence <400> 十 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 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 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 <400> 11 Gly Ser Thr Ala 1 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <400> 12<s Gly Gly Gly Gly Ser Gly Gly Gly Gly<s 1 5 <210> 13 <211> 14 <212> PRT1] [ [[ID=z]]<213> Artificial Sequence <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 <400> 14 Gly Ser Gly Ser Gly Ser 1 5 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <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 <400> 16 Gly Gly Ser Gly 1 <210> 17 <211> 8 <212> PRT <213> Artificial Sequence <400> 17 Gly Gly Ser Gly Gly Gly Ser Gly 1 5 <210> 18 <211> 12 <212> PRT <213> Artificial 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 <400> 19 Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly 1 5 10 <210> 20 <211> 36 <212> PRT <213> Artificial Sequence <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 <400> 21 Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 1 5 10 15 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 20 25 30 Gly Gly Gly Ser 35 <210> 22 <211> 61 <212> PRT <213> Artificial Sequence <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 Asn 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 <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[[ID=3​​​​​​​​​​​​​​​ 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 Glu Tyr Asn Pro Val Trp Gly Pro Asn Glu Gln Ile 50 55 60 <210> 25 <211> 61 <212> PRT <213> Artificial 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 <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> Artificial Sequence <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 <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 <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[[ID=*]] 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 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 <400> 30 Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Thr Val Glu Leu Leu 1 5 10 15​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 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 <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 <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 <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 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 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 <400> 34 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 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 Pro 225 230 <210> 35 <211> 223 <212> PRT <213> Artificial Sequence <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 <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

1. A virus-like particle containing a recombinant protein of Pasteurella multocida toxin, characterized in that, The amino acid sequence of the virus-like particle containing the recombinant protein of Pasteurella multocida toxin is shown in SEQ ID NO:

10.

2. A nucleic acid molecule encoding a virus-like particle containing a recombinant protein of Pasteurella multocida toxin as described in claim 1.

3. An immune composition for porcine atrophic rhinitis, characterized in that, The invention comprises a virus-like particle containing a recombinant protein of Pasteurella multocida toxin as described in claim 1, and a pharmaceutically acceptable carrier.

4. The porcine atrophic rhinitis immune composition as described in claim 3, characterized in that, It further includes inactivated *Bordetella bronchiseptica*, inactivated *Pasteurella multocida* Type A, and inactivated *Pasteurella multocida* Type D.

5. The porcine atrophic rhinitis immune composition as described in claim 3, characterized in that, It further includes other inactivated pathogen antigens selected from the following groups: porcine circovirus type 2 antigen, swine influenza virus antigen, porcine reproductive and respiratory syndrome virus antigen, porcine mycoplasma, porcine parvovirus, swine erysipelas, Actinobacillus pleuropneumoniae, and pseudorabies.

6. The use of the porcine atrophic rhinitis immune composition as described in claim 3 in the preparation of a medicament for animals to combat porcine atrophic rhinitis.

7. A diagnostic kit for porcine atrophic rhinitis, characterized in that, It includes a detection unit, wherein the detection unit is a virus-like particle containing recombinant protein of Pasteurella multocida toxin as described in claim 1.

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