Prevention, treatment and detection of progressive atrophic rhinitis of pig

A technique for atrophic rhinitis and musculocidal, applied in the direction of antibody medical ingredients, medical preparations containing active ingredients, bacterial antigen ingredients, etc., can solve the problems of high operating time and cost, cumbersome, time-consuming steps for culturing cells, etc.

Active Publication Date: 2009-08-05
简茂盛
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since EBL cells are not cell lines, this poses a major limitation in detection
Although Vero cells are cell lines, the steps of cultivating cells are time-consuming and cumbersome, and the operation time and cost required are high, and the judgment is easily affected by human subjectivity and cell state
When using the mouse lethal test (MLT), it is necessary to inject the mixture of the serum to be tested and PMT to the mice, so the operation time is long and the animals must be sacrificed, so there are also disadvantages

Method used

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  • Prevention, treatment and detection of progressive atrophic rhinitis of pig
  • Prevention, treatment and detection of progressive atrophic rhinitis of pig
  • Prevention, treatment and detection of progressive atrophic rhinitis of pig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Embodiment 1.Molecular-level identification of type A and D type Pasteurella multocida (Pasteurella multocida) and the nucleotide sequence alignment of the PMT encoding gene

[0121] I. Materials and methods:

[0122] A. Source of the strain:

[0123] The applicant conducted investigation, sampling and pathological examination of swine atrophic rhinitis on pigs eliminated from pig farms in Taichung City, Taichung County, Changhua County, and Nantou County in central Taiwan. Among the isolates identified as Pasteurella multocida biochemically, D-type toxin-producing strains (PMD 14, PMD 21, PMD 48), D-type non-toxin-producing strains (PMD 20, PMD 27), A-type toxin-producing strains (PMA 2, PMA 10, PMA 23) and A-type non-toxin-producing strains (PMA 33, PMA 59), a total of 10 isolates, and additional standard reference strains (D-type The toxin-producing strain NCTC 12178 was purchased from the Culture Center of the Food Industry Development Institute, and the type A no...

Embodiment 2

[0179] Example 2. Establishment of DNA clones encoding PMT subunits

[0180] A, the cloning of PMT coding gene:

[0181] PCR reaction for PMT-encoding gene:

[0182] According to the extraction and concentration determination of I in embodiment 1, the F item in the material and method, chromosomal DNA, and obtain DNA total extract from D type Pasteurella multocida PMD-48 bacterial strain culture, and with Absorbance A 260 / 280 Determine the concentration and purity of DNA.

[0183] When carrying out the cloning of PMT coding gene, take the DNA total extract obtained above as template, and use a group of primer pair A1B2 shown in Table 1 to carry out PCR:

[0184] forward primer

[0185] A1: 5′-agaggttat ggatcc gaaaacaaaacatttt-3' (SEQ ID NO: 3)

[0186] BamHI

[0187] reverse primer

[0188] B2: 5′-ctcttgttaa gctagc ctttgtgaaaagaggag-3' (SEQ ID NO: 10)

[0189] NheI

[0190] Since there are no restriction enzyme cleavage sites of BamHI and NheI in the PMT coding ge...

Embodiment 3

[0221] The toxicity test of embodiment 3.PMT recombinant subunit (rsPMT)

[0222] Vero cytotoxicity test:

[0223] The rsPMT clone strain induced by IPTG was centrifuged at 5,000xg for 10 minutes to collect the bacteria, then washed repeatedly with sterile PBS to suspend the bacteria, and ultrasonic (20% output power, 30 minutes; MisonixXL2020, Heat Systems Inc.) to crush the bacteria followed by high speed centrifugation at 10,000 xg for 30 minutes. Take the supernatant and filter it through filter membranes with a pore size of 0.45 μm and 0.22 μm. The filtrate filtered twice is the crude soluble protein of the expression product, and the Vero cell toxicity test is carried out in this way. The experimental method is according to Example 1. Item I, item D in materials and methods, determination of cytopathic effect (cytopathic effect, CPE) of PMT protein on African green ape kidney cell line (Vero cell). Vero cytotoxicity assay results are shown in Figures 15-17.

[0224] I...

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Abstract

The invention discloses a method for manufacturing a vaccine for animals with progressive atrophic rhinitis (PAR), which comprises the following steps that: nucleic acid fragments of polypeptides with three respective amino acid sequences encoded by which are 2-486, 486-986 or 986-1281 amino acid residues corresponding to Pasteurella multocida toxin (PMT) protein are expressed in Escherichia coli in a large amount respectively, and the polypeptides are separated and used for preparing a vaccine which can excite the generation of an antibody against Pasteurella multocida (related to the progressive atrophic rhinitis). The invention also discloses a method for manufacturing a multivalent vaccine capable of at least preventing the PAR for the animals, which comprises the following steps that: the multivalent vaccine is obtained by mixing the three polypeptides for the PAR prepared by the method and at least one pathogenic antigen related with other animal diseases or an epitope thereof.

Description

[0001] This application is a divisional application of an invention patent application with an application date of August 20, 2004, an application number of 200410057590.8, and an invention title of "Prevention, Treatment and Detection of Pig Progressive Atrophic Rhinitis". technical field [0002] The present invention relates to the manufacture method of the animal vaccine of progressive atrophicrhinitis (PAR), wherein three subunits of Pasteurella multocida toxin (PMT) are used as the prevention of PAR , Active ingredients for treatment and testing. The present invention also relates to a method for the manufacture of a multivalent vaccine for animals capable of preventing and treating at least PAR, the vaccine comprising three PMT subunits directed against PAR and at least one pathogenic antigen or antigenic epitope associated with other animal diseases active ingredient in vaccines. Background technique [0003] Porcine atrophic rhinitis (AR), also known as porcine pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/102A61K39/116A61K39/245A61P11/02
Inventor 简茂盛廖志明刘正义
Owner 简茂盛
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