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Specific IgY antibody for resisting African swine fever virus as well as preparation method and application thereof

An African swine fever virus and specific technology, applied in the field of anti-African swine fever virus p54 protein-specific IgY antibody and preparation, to achieve the effects of stable physical properties, easy storage and transportation, and reduced false positive rate

Inactive Publication Date: 2015-01-07
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on ASFV p54 protein mainly focuses on the prokaryotic expression and the preparation of mouse monoclonal antibody and the ELISA method established based on this. There is no report on the preparation of specific anti-ASFV p54 protein chicken IgY antibody

Method used

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  • Specific IgY antibody for resisting African swine fever virus as well as preparation method and application thereof
  • Specific IgY antibody for resisting African swine fever virus as well as preparation method and application thereof
  • Specific IgY antibody for resisting African swine fever virus as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: Expression and purification of African swine fever virus p54 protein

[0040] 1.1 Expression of African swine fever virus p54 protein

[0041] According to the instructions of the pET-52b(+)3C / LIC plasmid kit, design the upstream and downstream primers of the p54 gene that introduce the specific sequence, PCR amplify the gene from the pMD18-T-p54 plasmid template containing the ASFV-p54 gene, and pass T4DNA The role of polymerase, the end sticky p54 gene (GenBank accession number: EU874328) cloned into the pET-52b prokaryotic expression vector, transformed into competent cells E.coli BL21 (DE3) strain, in the presence of 100mg / L ampicillin (Amp ) on LB plates, pick a single colony, inoculate in 2 mL of LB culture medium containing 100 mg / L ampicillin, shake at 37°C and 200 rpm on a constant temperature shaker until the OD600 value is about 0.6, and add isotropic acid at a final concentration of 1 mM Propylthio-β-D-galactoside (IPTG), induced for 2 hours. ...

Embodiment 2

[0063] Example 2: Preparation of p54 protein-specific IgY antibody

[0064] 2.1 Animal immunization

[0065] Grouping of experimental animals: A total of 12 healthy 23-week-old Hy-Line laying hens were randomly divided into 3 groups, 4 in each group. The experimental groups were as follows: group A was the p54 antigen immunization group; group B was the pET-52b(+)3C / LIC negative plasmid antigen control group; group C was the PBS control group.

[0066] Immunization schedule:

[0067] The immunogen for the first immunization is the emulsified solution of p54 antigen and negative plasmid antigen respectively with equal volume of Freund’s complete adjuvant, and the immunogen for the second and third immunization is p54 antigen and negative plasmid antigen respectively with equal volume of Freund’s adjuvant For the emulsified liquid after incomplete adjuvant emulsification, the fourth immunity is p54 antigen and negative plasmid antigen without adjuvant.

[0068] (1) Group A (p...

Embodiment 3

[0096] Example 3: Application of p54 protein-specific IgY antibody in detection of African swine fever antibody competition ELISA

[0097] A competitive ELISA method for detecting African swine fever antibody was established by using prokaryotically expressed p54 protein and specific anti-ASFV-p54 protein IgY polyclonal antibody. The specific steps are as follows:

[0098] 1) Dilute the ASFV-p54 protein antigen to the working concentration with 0.05mol / L carbonate buffer solution with a pH value of 9.6, coat the microtiter plate with 100 μL / well (2.5 μg / well protein crude extract), and overnight at 4°C.

[0099] 2) Take out the 96-well plate, discard the coating solution, add blocking solution, incubate at 37°C for 2 hours, and wash 5 times with PBST.

[0100] 3) Add 1:10 dilution of ASFV positive pig serum, negative serum and serum to be tested, 100 μL / well, act at 37°C for 1 hour, and wash 5 times with PBST.

[0101] 4) Add 1:400 diluted purified ASFV-specific chicken IgY a...

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Abstract

The invention discloses a specific IgY antibody for resisting African swine fever virus p54 protein and a preparation method thereof, and application of the IgY antibody prepared by the method to specific detection of an African swine fever virus antibody. The preparation method comprises the following steps: preparing p54 protein antigen by utilizing a genetic recombination expression method, immunizing Hy-Line laying hens aged 23 weeks for 4 times, collecting eggs, and extracting and purifying immunoglobulin (IgY antibody) of egg yolk by utilizing a saturated ammonium sulfate precipitation method. A competitive ELISA method for detecting African swine fever serum antibodies established based on the specific IgY antibody is good in effect, only the positive serum detection result of African swine fever is positive, and the detection result is consistent with the parallel detection result of a commercialized ASFV antibody detection ELISA kit. The competitive ELISA method can be used for generally surveying and monitoring the serum epidemiology of African swine fever so as to prevent and control the disease from being spread to China from entry and exit ports.

Description

technical field [0001] The invention relates to the field of animal virus antibody detection, in particular to an anti-African swine fever virus p54 protein-specific IgY antibody, its preparation method and application. Background technique [0002] African swine fever (African swine fever, ASF) is a severe viral disease of pigs. Since it was first discovered in Africa in 1909, it has spread to countries and regions throughout Africa, Europe, South America and Asia. Suffered huge economic losses, and the disease was listed as an important animal disease by the International Organization for Animal Health (OIE). Although my country has not found the African swine fever epidemic, according to the incidence of neighboring countries and the outbreak of swine epidemic disease in some provinces and cities in my country since 2006, it has sounded the alarm for us. my country is already facing the serious threat of the introduction and prevalence of ASF . [0003] African swine fev...

Claims

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Application Information

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IPC IPC(8): C07K16/08C07K16/02G01N33/569
Inventor 张彩虹花群义刘建利曹琛福陶虹宗卉杨俊兴吕建强高又文孙洁曾少灵廖立珊唐金明黄超华
Owner SHENZHEN AUDAQUE DATA TECH
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