ELISA kit for detecting porcine toxigenic pasteurella multocida toxin antibody and application

A Pasteurella multocida technology, which is applied in the field of ELISA kits for detecting swine toxin-producing Pasteurella multocida toxin antibodies, can solve the problem of not finding antibodies for detecting swine toxin-producing Pasteurella multocida toxins. ELISA kits, biological materials that cannot obtain core reagents, etc., achieve the effects of low production cost, high biological safety, and wide application prospects

Inactive Publication Date: 2009-08-05
武汉科前动物生物制品有限贵任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, no ELISA kits for detecting antibodies to Pasteurella multocida toxin in pigs have been retrieved, especially the public literature on the preparation method of the core reagent, and the biological materials for preparing the core reagent have not been obtained.

Method used

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  • ELISA kit for detecting porcine toxigenic pasteurella multocida toxin antibody and application
  • ELISA kit for detecting porcine toxigenic pasteurella multocida toxin antibody and application
  • ELISA kit for detecting porcine toxigenic pasteurella multocida toxin antibody and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Cloning of the C' end of the toxA gene of Pasteurella multocida

[0050] 1. Design of primers A pair of C’-terminal subcloning primers were designed with reference to the toxA gene sequence published on GenBank (accession number: Z28388). The primer sequences are as follows:

[0051] P 1 : 5’-TA G GAT CC T TTC CGT ATT GGA TTA G-3’

[0052] P 2 : 5’-AA G TCG AC T TAA GAA AGT TGT ATT GG-3'

[0053] Among them, the underlined parts of P1 and P2 represent the restriction sites of BamH I and Sal I, which were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. The sequence amplified by the primers is 2257bp, of which the C' terminal coding region of the toxA gene is 2115bp, encoding the toxin r-PMT protein from the 580th amino acid to the terminator, a total of 705 amino acids.

[0054] 2. Cloning of the C' end of the toxA gene of the porcine toxin-producing Pasteurella multocida toxA

[0055] Toxigenic Pasteurella multocida was isol...

Embodiment 2

[0056] Example 2 Construction of the C'-terminal expression vector of the toxA gene of porcine toxin-producing Pasteurella multocida

[0057] The PCR product obtained in Example 1 was recovered, connected to the cloning vector pMD18-T (purchased from Treasure Bioengineering (Dalian) Co., Ltd.), transformed into Escherichia coli DH5α (purchased from Novagen), and screened for positive clones. Extract the plasmid, use restriction endonucleases BamH I and Sal I to double-digest and identify the recombinant plasmid, and use 0.8% agarose gel electrophoresis to confirm that its size is consistent with the expected target fragment size; it is confirmed by sequencing that the fragment has no bases Mismatched, containing the 2115bp sequence at the C' end of the toxA gene. The recombinant plasmid was named pT toxA-C2115.

[0058] Digest pT toxA-C2115 and vector pET28b with BamH I and Sal1 respectively, recover the C' end of toxA gene and vector pET28b; then connect with T4ligase, and t...

Embodiment 3

[0059] Example 3 Mass preparation of plasmid pET28b-C2115

[0060] (1) Pick the DH5α colony containing the plasmid pET28b-C2115 and inoculate it in 75mL LB culture medium, culture at 37°C 300r / min overnight, 6000rpm for 5min, collect the cell pellet, and use 3mL solution I (50mmol / L glucose, 10mmol / L ethylene glycol) Tetrasodium amine tetraacetate (EDTA), 25mmol / L Tris-Cl (pH8.0)) suspension;

[0061] (2) Add 6mL of solution II (0.2mol / L NaOH, 1% sodium dodecyl sulfate (SDS), ready to use), and ice-bath for 7-10min;

[0062] (3) Add 4.5mL solution III (3mol / L potassium acetate, adjust the pH value to 4.8 with glacial acetic acid), and ice-bath for 7-10min. 4℃10000r / min 10-15min;

[0063] (4) Take the supernatant, add 0.6 times the volume of isopropanol, mix well, and then 10000r / min for 6-15min at room temperature for 5min;

[0064] (5) Discard the supernatant, rinse once with 75% ethanol, vacuum dry or dry naturally, add 1.5mL TE (10.0mmol / LTris-HCl, 1.0mmol / L EDTA) to sus...

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Abstract

The invention belongs to the technical field of animal bacteriology and zoonotic disease detection, and in particular relates to an ELISA kit for detecting antibodies to toxin-producing Pasteurella multocida in pigs and its use in preventing and treating progressive atrophic rhinitis (progressive atrophic rhinitis, PAR). The kit includes a box body, a microtiter plate coated with the purified rPMT-C protein at the C' end of the toxA gene expressed as an antigen, negative and positive standard serum, rabbit anti-pig IgG enzyme-labeled secondary antibody, sample dilution Solution, substrate chromogenic solution A, substrate chromogenic solution B, stop solution and washing solution. Also disclosed is the preparation of a recombinant Escherichia coli BL21 / pET28b-C2115 (preservation number: CCTCC NO: M207012) expressing the rPMT-C protein at the C' end of the toxA gene of Pasteurella multocida toxA and the preparation of the rPMT-C protein expression and purification methods. The kit of the invention has the advantages of simple operation, fast and accurate diagnosis, low cost, etc., is suitable for large-scale clinical serological investigation of T+Pm, and can provide technical support for the purification of PAR in pigs.

Description

technical field [0001] The invention relates to the technical field of animal bacteriology and animal infectious disease detection. Specifically, the present invention relates to an ELISA kit and application for detecting antibodies to the toxin-producing Pasteurella multocida toxin in pigs. Background technique [0002] Toxigenic Pasteurella multocida (T. + Pm) is the main pathogen causing progressive atrophic rhinitis (PAR) in pigs. The bacterium secretes and produces a skin necrosis toxin (Pasteurella multocida toxin, PMT) of about 146kD, encoded by the toxA gene, which can directly cause swine rhinitis, nose bridge deformation, turbinate atrophy or even disappearance, systemic metabolic disorders, and production performance decline. Induce infection of other pathogenic microorganisms, even lead to death (Chanter, N et al., Interactions between Bordetella bronchiseptica and toxic Pasteurella multocida in atrophic rhinitis of pigs [J]. Res. Vet. Sci, 1989, 47 (1): 48-53;...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/70G01N33/543C12R1/19
Inventor 吴斌汤细彪王大鹏罗勇卢顺陈焕春
Owner 武汉科前动物生物制品有限贵任公司
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