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Mink canine parvovirus enteritis pathogen antigen colloidal gold detection test strip and production method thereof

A mink parvovirus and toxic enteritis technology, which is applied to measuring devices, instruments, scientific instruments, etc., can solve the problems of cumbersome HA operation, no treatment method, and high incidence and mortality of mink viral enteritis

Inactive Publication Date: 2016-05-11
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] The morbidity and mortality of mink viral enteritis caused by mink enteritis virus (MEV) are high, which has brought huge economic losses to the mink industry
However, there is no specific treatment for this disease, and it can only be prevented by vaccine immunization, so the diagnosis of mink viral enteritis is particularly important
At present, the diagnosis of MEV is mainly HA-HI and traditional PCR method. HA operation is cumbersome, time-consuming and laborious, and requires the preparation of pig red blood cells; while PCR is a molecular biological diagnosis method developed in recent years, which is mainly used for viruses. Nucleic acid detection, but the PCR method requires professional equipment, and operators need special training to operate in a specialized laboratory

Method used

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  • Mink canine parvovirus enteritis pathogen antigen colloidal gold detection test strip and production method thereof
  • Mink canine parvovirus enteritis pathogen antigen colloidal gold detection test strip and production method thereof
  • Mink canine parvovirus enteritis pathogen antigen colloidal gold detection test strip and production method thereof

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preparation example Construction

[0041]The preparation method of the mink parvovirus enteritis pathogen antigen colloidal gold detection test strip of the present invention mainly comprises the following steps:

[0042] Step 1, prokaryotic expression of mink parvovirus (MEV) VP2 protein and its purification

[0043] Step 2, prokaryotic expression of mink parvovirus (MEV) VP1 partial protein and its purification

[0044] Step 3, preparation of mink parvovirus (MEV) monoclonal antibody

[0045] Step 4, preparation of colloidal gold solution

[0046] Step 5. Preparation of gold-labeled antibody solution

[0047] Step 6. Preparation of gold standard pad

[0048] Step 7. Pretreatment of NC membrane (marking of C and T lines)

[0049] Step eight, assembly of test strips.

Embodiment 1

[0050] Example 1 Prokaryotic expression of mink parvovirus (MEV) VP2 protein and its purification

[0051] The VP2 protein of the detection line (T line) is obtained through prokaryotic expression and purification.

[0052] The specific process is as follows: (1) PCR technique is used to amplify mink parvovirus MEVB strain VP2 gene, the amplification conditions are: 95°C, 5min; 94°C, 30s, 55°C, 1min, 72°C, 2min; 72°C, 8min. The obtained gene fragments were directional cloned into the prokaryotic expression vector PET-30a, and the prokaryotic expression recombinant plasmid PET-30a-VP2 of mink parvovirus (MEV) VP2 gene was constructed.

[0053] (2) The prokaryotic expression recombinant plasmid PET-30a-VP2 of mink parvovirus (MEV) VP2 gene obtained above was transformed into the host strain BL21, and the VP2 protein was expressed in the form of inclusion bodies after IPTG induction, and analyzed by SDS-PAGE and Western blotting It shows that the VP2 protein can be expressed cor...

Embodiment 2

[0055] Example 2 Prokaryotic expression of mink parvovirus (MEV) VP1 partial protein and its purification

[0056] Mink parvovirus (MEV) VP1 protein is obtained by expressing part of the VP1 gene, and its expression and purification method refers to Example 1.

[0057] The specific process is as follows: (1) Use PCR technology to amplify part of the VP1 gene of mink parvovirus MEVB strain, the amplification conditions are: 95°C, 5min; 94°C, 30s, 55°C, 45s, 72°C, 90s; 72°C, 8min . The obtained gene fragments were directional cloned into the prokaryotic expression vector PET-30a, and the prokaryotic expression recombinant plasmid PET-30a-VP1 of mink parvovirus (MEV) VP1 gene was constructed.

[0058] (2) Transform the prokaryotic expression recombinant plasmid PET-30a-VP1 of the mink parvovirus (MEV) VP1 gene obtained above into the host strain BL21. After IPTG induction, the VP1 protein is expressed in the form of inclusion bodies, and analyzed by SDS-PAGE and Western blotting...

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Abstract

The invention discloses a mink canine parvovirus enteritis pathogen antigen colloidal gold detection test strip and a production method thereof, and relates to the technical field of detection of fur animal viral infectious disease pathogens. The colloidal gold detection test strip comprises a sample pad, a colloidal gold pad, an NC film and an absorption pad, the detection line of the NC film is sprayed with a VP2 protein, and the quality control line of the NC film is sprayed with a goat-anti-mouse second antibody. The production method of the mink canine parvovirus enteritis pathogen antigen colloidal gold detection test strip mainly comprises the following steps: carrying out prokaryotic expression to obtain mink parvovirus (MEV) VP2 and VP1 proteins, and purifying the proteins; preparing an MEV monoclonal antibody; preparing a colloidal gold solution; preparing a colloidal gold antibody solution; preparing the colloidal gold pad; preprocessing the NC film; and assembling. The test strip is helpful for purifying the mink canine parvovirus enteritis in fur animal culturing farms, and is convenient for basic veterinary personnel to operate.

Description

technical field [0001] The invention relates to the technical field of detecting pathogens of fur animal viral infectious diseases, in particular to a colloidal gold detection test strip for mink parvovirus enteritis pathogen antigen and a preparation method thereof. Background technique [0002] The morbidity and mortality of mink viral enteritis caused by mink enteritis virus (MEV) are high, which has brought huge economic losses to the mink industry. However, there is no specific treatment for this disease, and it can only be prevented by vaccine immunization, so the diagnosis of mink viral enteritis is particularly important. At present, the diagnosis of MEV is mainly HA-HI and traditional PCR method, HA operation is cumbersome, time-consuming and laborious, and requires the preparation of pig red blood cells; and PCR is a molecular biological diagnosis method developed in recent years, mainly used for virus Nucleic acid detection, but the PCR method requires profession...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569G01N33/558
CPCG01N33/558G01N33/56983G01N33/577G01N2333/015
Inventor 王建科程悦宁张淼程世鹏林鹏赵航易立仝明薇曹智刚孙亚茹闫喜军陈立志
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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