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Prevention, treatment and detection of pig progressive atrophic rhinitis

A technology for atrophic rhinitis and vaccines, which can be applied to antibody medical components, medical preparations containing active ingredients, bacterial antigen components, etc., and can solve the problems of tediousness, high operation time and cost, and time-consuming steps of culturing cells.

Inactive Publication Date: 2006-02-22
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 pig progressive atrophic rhinitis
  • Prevention, treatment and detection of pig progressive atrophic rhinitis
  • Prevention, treatment and detection of pig progressive atrophic rhinitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1. Molecular-level identification of Type A and Type D Pasteurella multocida and nucleotide sequence alignment of PMT-encoding genes

[0120] I. Materials and methods:

[0121] A, strain source:

[0122]The applicant took the nursery pigs culled from the monitoring pig farms in the four counties and cities of Taichung City, Taichung County, Changhua County and Nantou County in central Taiwan as the object to carry out the epidemic investigation, sampling and pathological examination of porcine atrophic rhinitis. Among the isolates biochemically identified as Pasteurella multocida, D-type toxigenic strains (PMD 14, PMD 21, PMD 48) and D-type non-toxigenic strains (PMD) were selected by the CPE effect test of Vero cells. 20, PMD 27), A-type toxigenic strains (PMA 2, PMA 10, PMA 23) and A-type non-toxigenic strains (PMA 33, PMA 59), a total of 10 isolates, and additionally used standard reference strains (D type The toxigenic strain NCTC 12178 was purchased from ...

Embodiment 2

[0176] Example 2. Establishment of DNA clones encoding PMT subunits

[0177] A. Cloning of PMT-encoding gene:

[0178] PCR reaction of PMT-encoding gene:

[0179] According to item 1 in Example 1, item F in materials and methods, extraction of chromosomal DNA and determination of concentration, the total DNA extract was obtained from the D-type Pasteurella multocida PMD-48 strain culture, and used Absorbance A 260 / 280 Determine the concentration and purity of DNA.

[0180] In the cloning of the PMT-encoding gene, PCR was performed using the above-obtained DNA total extract as a template and a set of primer pairs A1B2 shown in Table 1:

[0181] forward primer

[0182] A1: 5′-agaggttat ggatcc gaaaacaaaacatttt-3' (SEQ ID NO: 3)

[0183] BamHI

[0184] reverse primer

[0185] B2: 5′-ctcttgttaa gctagc ctttgtgaaaagaggag-3' (SEQ ID NO: 10)

[0186] NheI

[0187] Since there is no BamHI and NheI restriction enzyme cleavage sites in the P...

Embodiment 3

[0217] Example 3. Toxicity test of PMT recombinant subunit (rsPMT)

[0218] Vero Cytotoxicity Assay:

[0219] The rsPMT cloned strains induced by IPTG were centrifuged at 5,000×g for 10 minutes to collect bacteria, washed repeatedly with sterile PBS to suspend the bacteria, and sonicated (output power 20% for 30 minutes; MisonixXL2020, Heat Systems) Inc.) crushed the bacteria followed by high speed centrifugation at 10,000 xg for 30 minutes. Take the supernatant and filter it through 0.45 μm and 0.22 μm pore size filter membranes. The filtrate filtered twice is the crude soluble protein of the expression product, and the Vero cytotoxicity test is carried out. The experimental method is as in Example 1. I. Item D in Materials and Methods, Determination of cytopathic effect (CPE) of PMT protein on Vero cell line of African green ape. The results of the Vero cytotoxicity assay are shown in Figures 15-17.

[0220]In this experiment, the crude PMT toxin protein expressed by Past...

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Abstract

Disclosed is the animal vaccine for the prevention, treatment and detection of pig progressive atrophic rhinitis, which comprises three polypeptides generated by antibody against Pasteurella multocida related to progressive atrophic rhinitis, these polypeptides all have a amino acid sequence, which mainly corresponds to the 2-486, 486-986 or 986-1281 amino acid residue of the PMT protein. The invention also discloses the multivalent vaccine for the prevention and treatment of at least PAR for animals.

Description

technical field [0001] The present invention relates to the prevention, treatment and detection of progressive atrophic rhinitis (PAR), wherein three subunits of Pasteurella multocida toxin (PMT) are used as the prevention, treatment and detection of PAR. Active ingredient for treatment and detection. The present invention also relates to a multivalent vaccine for animals capable of preventing at least PAR, the vaccine comprising three PMT subunits against PAR and at least one pathogenic antigen or epitope thereof associated with other animal diseases as the vaccine. Active ingredient. Background technique [0002] Porcine atrophic rhinitis (AR), also known as porcine progressive atrophic rhinitis (PAR), is a common bacterial disease of the upper respiratory tract in pigs. Affects the development of the whole body skeleton, which in turn leads to stunted growth of pigs, and makes pigs susceptible to secondary complex pneumonia, resulting in great losses to pig farmers. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61P11/02
CPCC07K14/285A61K39/102A61P11/02
Inventor 简茂盛廖志明刘正义
Owner 简茂盛
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