Test paper for detecting African swine fever virus antibody

An African swine fever virus and antibody detection technology, applied in the field of immune protein preparation and its application, can solve the problems of low specificity of colloidal gold immunochromatographic test strips, the accuracy rate needs to be improved, the sensitivity needs to be improved, etc. The effect of strong specificity, high sensitivity and easy portability

Active Publication Date: 2019-11-08
ZHENGZHOU UNIV +2
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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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  • Test paper for detecting African swine fever virus antibody
  • Test paper for detecting African swine fever virus antibody
  • Test paper for detecting African swine fever virus antibody

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Gene Design and Vector Construction

[0044] 1) Gene 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 for serum antibodies; p54 protein is a transmembrane protein, of which amino acids 1 to 29 are extracellular The 30th to 52nd amino acid is the transmembrane region, and the 53rd to 184th amino acid is the intracellular region;

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

Embodiment 2

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

[0075] The bacmid Bacmid that embodiment 1 gains and through identifying uses Cellfectin ® II transfection kit into sf21 insect cells, the specific steps are as follows:

[0076] Prepare cells at a density of 1 x 10 6 Cells / mL sf21 cell suspension was added to a six-well plate for culture, 2 mL was added to each well, incubated for 1 hour, and transfection samples were prepared at the same time; l-2ng of recombinant bacmid Bacmid (about 5-10 μL) was diluted in 100 μL of Sf- In 900II SFM, take the transfection reagent Cellfectin ® Dilute II in 100 μL of Sf-900 II SFM, mix the above two dilutions, incubate at room temperature for 15-45 minutes, add 800 μL of Sf-900 II SFM to make a transfection mixture; Add the above transfection mixture and incubate at 28°C for 5 hours; discard the transfection mixture, add 2 mL of Sf-900II SFM, and incubate in a constant temperature incubator at 28°C for 3 to 4 days...

Embodiment 3

[0089] Example 3 Antigen Purification and Detection

[0090] The P3 generation cells identified in Example 2 were used to infect sf21 suspension cells at 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; cultured for 72 hours after inoculation, the cell supernatant was harvested and the protein was purified by nickel-affinity chromatography: the cell-disrupted bacterial solution was loaded through a nickel column, and then 20mM PB (150mMNaCL, 25mM imidazole) to wash away the non-specific binding protein, and then use 20mM PB (150mM NaCL, 250mM imidazole) to elute the protein with his tag bound to the nickel filler, to obtain that;

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

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Abstract

The invention belongs to the technical field of immune protein preparation and applications thereof, and particularly relates to a test paper for detecting African swine fever virus antibody. The invention provides a gene sequence E183L-1 for encoding an extracellular region of an African swine fever virus p54 protein. Based on a general inventive concept, the invention also proposes a primer pairfor amplifying the gene sequence and a synthetic protein encoded by the gene sequence. In order to solve the problems existing in the detection of the African swine fever virus, the invention prepares the test paper for detecting the antibody of the African swine fever virus by utilizing the synthetic protein encoded by the gene. The test paper can rapidly and accurately detect the antibody of the African swine fever virus, and is very suitable for basic-level and on-site rapid detection and diagnosis.

Description

technical field [0001] The invention belongs to the technical field of immune protein preparation and application thereof, and in particular relates to a test paper for detecting antibodies against African swine fever virus. Background technique [0002] African swine fever (ASF) is a highly fatal infectious disease of pigs. The disease has been prevalent in African countries. In recent years, African swine fever has also seen continuous outbreaks in other parts of the world, causing huge losses to the global pig 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 (Open reading frames, ORF), encoding 54 structural proteins and More than 100 nonstructural proteins. Under the electron microscope, ASFV is a regular hexagon with a diameter of about 200 nm. Partial composition. The p54 protein of African swine fever virus exists in the inner layer of the vi...

Claims

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Application Information

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Patent Type & Authority Applications(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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