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Pasteurella multocida toxin recombinant protein, virus-like particle thereof and use thereof

A virus-like particle and Pasteurella technology, applied in the direction of anti-bacterial immunoglobulin, virus, application, etc., can solve problems such as death, high pollution rate, and liver damage

Pending Publication Date: 2019-04-30
SCHWEITZER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Inoculation of PMT toxin into four-week-old piglets by means of muscle, intraperitoneal and nasal inoculation, etc., can cause turbinate and interosseous atrophy of lesions, in addition, it will also affect the general bone development and cause growth retardation, even at high doses of inoculation, Can cause liver damage leading to jaundice and death in pigs
[0004] Porcine atrophic rhinitis (AR) spreads all over the pig raising areas in the world, and the affected pigs grow slowly and the feed utilization efficiency decreases
Although the mortality rate is not high when infected alone, the pollution rate is very high, and it is easy to induce other complications or infection of pathogens, resulting in high mortality and increased production costs

Method used

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  • Pasteurella multocida toxin recombinant protein, virus-like particle thereof and use thereof
  • Pasteurella multocida toxin recombinant protein, virus-like particle thereof and use thereof
  • Pasteurella multocida toxin recombinant protein, virus-like particle thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

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

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

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

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

[0073] Each epitope A (SEQ ID NO: 2), epitope B (SEQ ID NO: 3), and epitope C (SEQ ID NO: 4) are respectively connected with a linker, and the connection The sub has the amino acid sequence shown in SEQ ID NO:11. According to the difference in sequence combination of antigenic determinants A, B, and C from the N-terminus of the recombinant protein from the C-terminus, the amino acid sequences of the obtained Pasteurella septica toxin recombinant protein (re...

Embodiment 2

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

[0080] 1. Culture of Bordetella bronchiseptica (B.bronchiseptica)

[0081] First inoculate B.bronchiseptica on TSB solid medium [containing 5% (v / v) yeast extract, 10% (v / v) serum, soybean decomposed protein Casein medium (tryptic soy broth, TSB, BD Company, USA)], after culturing overnight at 37°C, a single colony was selected and inoculated in brain heart infusion (BHI) liquid medium (BD Company, USA) ), shake culture overnight at 37°C; then inoculate the bacterial liquid in BHI liquid medium, shake culture overnight at 37°C, and calculate the colony forming unit (CFU) value; finally add formaldehyde (formaldehyde ), shake at room temperature for 24 to 36 hours, and inactivate the bacterial solution.

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

[0083] Pasteurella septica type A (PmA) and Pasteurella septica type D (PmD) with the ability to produce toxins were first inoculated o...

Embodiment 3

[0084] Example 3 Preparation of porcine atrophic rhinitis immune composition

[0085] Pasteurella septica toxin recombinant protein (re-PmT) (SEQ ID NO: 5) (final concentration: 250 μg / ml) obtained in Example 1 or virus-like particles containing Pasteurella septica toxin recombinant protein ( re-PmT VLP) (SEQID NO:10) (final concentration is 250 μ g / ml) and the inactivated bronchiseptic Bordetella (B. bronchiseptica) obtained in Example 2 (final concentration is 1×10 9 CFU / ml), inactivated Pasteurella septica type A bacteria (PmA) (final concentration is 1x10 9 CFU / ml) and inactivated Pasteurella septicemia type D bacteria (PmD) (final concentration is 1×10 9 CFU / ml) was mixed evenly with phosphate buffered solution (PBS), and aluminum gel [final concentration was 30% (v / v)] was added as an adjuvant to prepare a porcine atrophic rhinitis immune composition.

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Abstract

The invention provides a Pasteurella multocida toxin recombinant protein, comprising three epitopes of the Pasteurella multocida toxin protein. The three epitopes have the sequences as shown in SEQ IDNO:2, 3 and 4 respectively. Also provided are a virus-like particle containing the Pasteurella multocida toxin recombinant protein, a nucleic acid sequence encoding the Pasteurella multocida toxin recombinant protein or encoding the virus-like particle containing the Pasteurella multocida toxin recombinant protein, and an immune composition of swine atrophic rhinitis comprising the Pasteurella multocida toxin recombinant protein and / or the virus-like particle containing the Pasteurella multocida toxin recombinant protein.

Description

technical field [0001] The present invention relates to the recombinant protein of Pasteurella septica toxin, in particular to the recombinant protein containing the epitope of Pasteurella septica toxin and the virus-like particles containing the recombinant protein. Background technique [0002] Atrophic Rhinitis (AR) is one of the three major infectious diseases of swine respiratory system. Porcine atrophic rhinitis causes facial bone deformities and chronic suppurative rhinitis in infected pigs, resulting in atrophy of the turbinates. In severe infection, lesions will also occur in the nasal cavity, jaw, and maxilla. The downstream fossa of the inferior turbinate is most commonly infected. The superior turbinate, inferior turbinate, nasal septum, or ethmoid bone can all be infected. [0003] Porcine atrophic rhinitis (AR) is caused by Bordetella bronchiseptica (B.b) and Pasteurella multocida type A (PmA) and type D (PmD), especially Pasteurella multocida toxin (PMT) p...

Claims

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Application Information

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IPC IPC(8): C07K14/285C07K14/02C07K16/12C12N15/31C12N15/36A61K39/102A61P31/04A61P11/02G01N33/569
CPCA61K39/102C07K14/005C07K14/285C07K16/1242C12N2730/10123C12N2730/10134G01N33/56911G01N2469/10G01N2800/14A61K39/116A61K39/295A61P11/02A61P31/04C07K14/02C07K16/12G01N33/569G01N33/68
Inventor 杨滢臻陈灿坚
Owner SCHWEITZER
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