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A kind of African swine fever virus antibody detection test strip

A technology of African swine fever virus and carrier, which is applied in the field of immune protein preparation and application, can solve the problems of colloidal gold immunochromatographic test strips such as low specificity, accuracy and sensitivity to be improved, and achieve detection specificity Strong performance, high sensitivity, easy to carry

Active Publication Date: 2022-07-05
ZHENGZHOU UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patent CN103293306A discloses a preparation method of colloidal gold immunochromatographic test strip for detection of African swine fever virus antibody, which can obtain clear diagnostic results within 5 minutes and can directly detect the virus in suspicious pig serum (or anticoagulant blood) Antibody, suitable for on-site African swine fever virus serological diagnosis, epidemiological investigation and pig trade inspection and quarantine; For serum, the accuracy rate needs to be improved, which restricts the popularization and application of this method; Zhang Xinyu et al. (Establishment of colloidal gold test paper detection method for African swine fever virus p54 antibody [J]. Chinese Journal of Preventive Veterinary Medicine, 2014) established an African pig Pestivirus p54 antibody colloidal gold test paper detection method, the prepared colloidal gold test paper is highly specific to African swine fever virus antibody-positive pig serum, and has no cross-reaction with other pig virus antibody-positive sera; The sensitivity of pestivirus antibody is only 200ng / mL, and the sensitivity needs to be improved

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  • A kind of African swine fever virus antibody detection test strip
  • A kind of African swine fever virus antibody detection test strip
  • A kind of African swine fever virus antibody detection test strip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Gene design and vector construction

[0044] 1) Genetic optimization and design

[0045] As one of the structural proteins of African swine fever virus (hereinafter referred to as ASFV), the p54 protein is encoded by the ASFV E183L gene and is one of the main binding sites of serum antibodies; p54 protein is a transmembrane protein, of which amino acids 1 to 29 are extracellular region, amino acids 30 to 52 are transmembrane regions, and amino acids 53 to 184 are intracellular regions;

[0046] The inventors improved the design on the basis of the original p54 (GenBank sequence number CBW46791.1) protein extracellular region gene: ASFV belongs to the genus African swine fever virus of the family Iridoviridae. Compared with insect cells, the two are different species. Genus, the frequency of use of codons is different, so the present invention selects insect cells to form synthetic genes with the codons with the highest frequency, and the selected codons are s...

Embodiment 2

[0074] Example 2 Eukaryotic expression and identification of E183L-1 gene

[0075] The baculovirus plasmid Bacmid obtained in Example 1 and identified with Cellfectin ® Ⅱ Transfection kit was used to transfer sf21 insect cells, and the specific steps were as follows:

[0076] Prepare cells at a density of 1 x 10 6 cells / mL of sf21 cell suspension was added to a six-well plate for culture, 2 mL was added to each well, incubated for 1 h, and samples were prepared for transfection; 1-2 ng of recombinant bacmid (about 5-10 μL) was diluted in 100 μL of Sf- 900 II SFM, take the transfection reagent Cellfectin ® II was diluted in 100 μL of Sf-900 II SFM, mixed with the above two dilutions, incubated at room temperature for 15-45 min, and then added 800 μL of Sf-900 II SFM to make a transfection mixture; after cells were incubated for 1 hour, the culture medium was removed. Add the above transfection mixture, incubate at 28 °C for 5 h; discard the transfection mixture, add 2 mL of ...

Embodiment 3

[0089] Example 3 Antigen purification and detection

[0090] The P3 passage cells identified in Example 2 were used to infect sf21 suspension cells with a multiplicity of infection of 1, and the supernatant cell density when inoculated with the virus was 0.8-1.0×10 6 cells / ml; culture for 72h after inoculation, harvest the cell supernatant and purify the protein by nickel affinity chromatography: load the cell-disrupted bacterial solution through a nickel column, and then use 20mM PB (150mM NaCl, 25mM imidazole) to wash off non-specifically bound proteins, and then use 20mM PB (150mM NaCl, 250mM imidazole) to elute the his-tagged proteins bound to the nickel filler, that is;

[0091] The obtained protein was identified by western blot, and the identification results were shown in Figure 4 , it can be seen from the figure that the reaction band is located at the 4 kDa position, indicating that the obtained protein is the target antigen protein;

[0092] The ASFV positive ser...

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Abstract

The invention belongs to the technical field of immune protein preparation and application, in particular to an African swine fever virus antibody detection test paper. The present invention proposes a gene sequence E183L-1 encoding the extracellular region of the African swine fever virus p54 protein. Based on a general inventive concept, the present invention also proposes a primer pair for amplifying the gene sequence and a synthetic protein encoded by the gene sequence; In order to solve the problems existing in the detection of African swine fever virus, the present invention uses the synthetic protein encoded by the above gene to prepare a test paper for detecting African swine fever virus antibody, which can quickly and accurately detect African swine fever virus antibody, and is very suitable for grassroots And on-site rapid detection and diagnosis.

Description

technical field [0001] The invention belongs to the technical field of immune protein preparation and application, in particular to an African swine fever virus antibody detection test paper. Background technique [0002] African Swine Fever (ASF) is a highly lethal infectious disease of pigs. The disease has been endemic in African countries. In recent years, ASF has also sustained outbreaks in other parts of the world, causing huge losses to the global swine industry. . African swine fever virus (ASFV) is a double-stranded closed linear DNA virus with a genome size of about 170kb to 190kb, containing 150–167 open reading frames (ORFs), encoding 54 structural proteins and More than 100 nonstructural proteins. ASFV is a regular hexagon with a diameter of about 200 nm under the electron microscope. part composition. African swine fever virus p54 protein exists in the inner envelope of virions and participates in the adsorption and entry of the virus. It is the main struct...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C12N15/11C12N15/866C07K14/01G01N33/558G01N33/569G01N33/68
CPCC07K14/005C12N15/86G01N33/558G01N33/56983G01N33/6854C12N2710/12022C12N2710/14043G01N2333/01
Inventor 王爱萍张改平贾蕊刘运超周景明祁元明赵建国牛艳王彦伟刘亚伟
Owner ZHENGZHOU UNIV
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