Preparation method of test strip for rapidly diagnosing porcine contagious pleuropneumonia actinobacillus

A technology for actinomycetes and pleuropneumonia, applied in the field of rapid biological detection, can solve the problems of complex operation, slow detection speed, low sensitivity, etc., and achieve the effects of high sensitivity, simple operation and good stability

Inactive Publication Date: 2014-05-07
JIANGXI AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention provides a preparation method for the rapid diagnosis test strip of actinobacillus pleuropneumoniae exohemolytic toxin ApxI, which solves the problems of slow detection speed, low sensitivity and complicated operation in the prior art

Method used

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  • Preparation method of test strip for rapidly diagnosing porcine contagious pleuropneumonia actinobacillus
  • Preparation method of test strip for rapidly diagnosing porcine contagious pleuropneumonia actinobacillus

Examples

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preparation example Construction

[0024] The preparation method of rapid diagnosis test strip comprises the following steps:

[0025] (1) Preparation of polyclonal antibody for pSE380-ApxI-ApxIV fusion protein: Select healthy adult male rabbits (2-3 months old, 2-3kg) as immunized animals, and mix purified His-ApxI-ApxIV fusion protein recombinant protein with Equal volumes of Freund's complete adjuvant were mixed and emulsified, and then immunized animals several times to obtain immune serum.

[0026] (2) Preparation of monoclonal antibodies to pSE380-ApxI and pSE380-ApxIV fusion proteins: inject the fusion proteins pE380-ApxI and pE380-ApxIV into Balb / c mice, and then use cell fusion technology to screen positive clones of hybridomas and screen out Positive hybridomas were cultured in vitro and in vivo to obtain monoclonal antibodies.

[0027] (3) Preparation of analysis membrane 5: the anti-porcine infectious actinobacillus pleuropneumoniae exotoxin ApxI / ApxIV prepared in step (1) was formed on the nitroce...

Embodiment 1A

[0031] Example 1 ApxI, pSE380ApxIV, ApxI-ApxIV gene cloning, prokaryotic expression vector recombinant plasmid induced expression and protein purification

[0032] 1 Materials and methods

[0033] 1.1 Strains: Escherichia coli (DH5α / BL21).

[0034] 1.2 Main reagents and instruments: LA / LB medium, electrophoresis apparatus, electrophoresis tank, ultraviolet gel imaging system, nucleic acid and protein analyzer, ultra-high-speed refrigerated centrifuge.

[0035] 1.3 Plasmid: pSE380

[0036] 2 Experimental methods

[0037] 2.1 Cloning of Actinobacillus pleuropneumoniae exotoxin ApxI / ApxIV / ApxI-ApxIV gene

[0038] According to the sequence of ApxI / ApxIV gene searched in GenBank, two pairs of primers were designed using Premier5.0 software, and ApxI / ApxIV gene was amplified from the serum of pigs infected with porcine infectious pleuropneumonia, and then the PCR products were mixed with ApxI / ApxIV As a template, ApxI gene is used as an upstream primer, and the downstream produc...

Embodiment 2

[0043] Example 2 pSE380-ApxI-ApxIV polyclonal anti

[0044] 1 Materials and methods

[0045] 1.1 Strains: Escherichia coli (DH5α / BL21).

[0046] 1.2 Main reagents and instruments: LA / LB medium, HRP-labeled goat anti-rabbit IgG, Freund's complete adjuvant / Freund's incomplete adjuvant, dialysis bag, electrophoresis apparatus, electrophoresis tank, UV gel imaging system, nucleic acid protein Analyzer, ultra high speed refrigerated centrifuge.

[0047] 1.3 Plasmid: pSE380

[0048] 1.4 Experimental animals: New Zealand white rabbits.

[0049] 2 Experimental methods

[0050] 2.1 Immunization of experimental animals

[0051] The prepared His-ApxI-ApxIV fusion protein recombinant protein was mixed with Freund's complete adjuvant in equal volumes to immunize animals. Two New Zealand white rabbits aged 2 to 3 months and weighing 2 to 3 kg were selected as immunized animals. Exemption: 0.5 mg·mL-1His-ApxI-ApxIV fusion protein was mixed in equal proportions with Freund's complete ad...

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Abstract

The invention discloses a preparation method of a test strip for rapidly diagnosing hematoxin ApxI arranged outside porcine contagious pleuropneumonia actinobacillus, and relates to the technical field of rapid biological detection. The test strip comprises an analyzing film (5) and a gold-labelled antibody combining pad (1), wherein a detection line 1 (T1) and a detection line 2 (T2) are made from an actinobacillus-resisting exotoxin ApxI/ApxIV monoclonal antibody arranged on the analyzing film (5), a quality control line (C) is made from a goat anti rabbit IgG, and the gold-labelled antibody combining pad (1) is a fusion protein anti rabbit polyclonal antibody of p380-ApxI-ApxIV. The preparation method provided by the invention solves the problems that the inspection speed is slow, the sensitivity is low, the operation is complicated and the like in the prior art.

Description

technical field [0001] The invention relates to the technical field of rapid biological detection, in particular to a method for preparing a rapid diagnostic test strip of Actinobacillus pleuropneumoniae external hemolytic toxin ApxI. Background technique [0002] Porcine infectious pleuropneumonia is a highly contagious infectious disease of pigs caused by Actinobacillus pleuropneumoniae. one of infectious diseases. In clinical autopsy, pulmonary hemorrhage, necrosis and fibrinous exudation are the main lesions, and the fatality rate of acute cases is high. The season of onset of this disease is obvious, and it occurs frequently in harsh weather such as winter with low temperature, early spring and late autumn. Pigs of any age are susceptible to the disease, with the most susceptible being 3 months old. Pigs infected with Actinobacillus pleuropneumoniae are often prone to other bacterial diseases clinically, such as Haemophilus parasuis, Pasteurella multocida, etc., as w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/531
CPCG01N33/56911G01N33/54386G01N33/558G01N2333/195
Inventor 刘平胡国良郭小权曹华斌刘佩李麟张彩英黄爱民罗军荣
Owner JIANGXI AGRICULTURAL UNIVERSITY
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