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BAS-ELISA kit for detecting hog cholera virus Erns and E2 antigen

A swine fever virus and kit technology, applied in the field of biotechnology and animal infectious disease diagnosis research, can solve problems such as false negative results, achieve stable results, convenient use, and improve sensitivity

Active Publication Date: 2014-04-30
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Elisa kits in the market only detect a single antigen, and false negative results will occur when the sample is not well preserved

Method used

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  • BAS-ELISA kit for detecting hog cholera virus Erns and E2 antigen
  • BAS-ELISA kit for detecting hog cholera virus Erns and E2 antigen
  • BAS-ELISA kit for detecting hog cholera virus Erns and E2 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The preparation of embodiment 1 swine fever virus Erns and E2 recombinant protein

[0046] (1) According to the complete genome sequence of CSFV Shimen strain in NCBI GeneBank (Accession No. AF333000.1), the complete sequence of envelope protein Erns of CSFV Shimen strain and the B / C region and D / A region of E2 main antigen region The gene sequence is used as a template to design synthetic primers, and the two primers are:

[0047] Erns (F): 5'-CGGGATCCGAAAATATAACTCAGTGGAACCTGA-3',

[0048] Erns(R): 5'-CGCTCGAGGGCATAGGCACCAAACCAG-3',

[0049] E2(F): 5'-CGGGATCCCGTCTAGCCTGCAAGGAAG-3',

[0050] E2(R): 5'-CGCTCGAGTAGATCTTCATTTTCCACTGTGG-3'.

[0051] Wherein, the underlined part is the restriction restriction site that introduces, and Erns (F) is CSFV-Erns gene upstream amplification primer, and the restriction restriction restriction site that introduces is BamHI, and Erns (R) is CSFV-Erns gene downstream amplification primer, introduces The enzyme cutting site is XhoI...

Embodiment 2

[0061] Example 2 Preparation and biotin labeling of polyclonal antibody against CSFV-Erns protein (CSFV-Erns) and polyclonal antibody against CSFV-E2 protein (CSFV-E2)

[0062] (1) The recombinant CSFV-Erns and CSFV-E2 proteins purified in Example 1 were used to immunize the experimental rabbits respectively, and blood was collected 7 days after the third immunization to detect the Elisa titer. After the rabbit serum titer reached 1:100,000, shock immunization was carried out , collect rabbit serum after 7 days;

[0063] (2) Recombinant CSFV-Erns and CSFV-E2 proteins were coupled to CNBr-activated Sepharose4B beads (purchased from GE) to prepare antigen affinity purification columns;

[0064] (3) Purify the rabbit serum prepared in step (1) with the affinity purification column prepared in step (2) to obtain rabbit anti-CSFV-Erns polyclonal antibody and rabbit anti-CSFV-Erns polyclonal antibody;

[0065] (4) The rabbit anti-CSFV-Erns polyclonal antibody and rabbit anti-CSFV-E...

Embodiment 3

[0069] Example 3 Preparation of Anti-CSFV Erns Protein (CSFV-Erns) Monoclonal Antibody and Anti-CSFV E2 Protein (CSFV-E2) Monoclonal Antibody

[0070] (1) Immunize Balb / c mice with recombinant CSFV-Erns and CSFV-E2 proteins purified in Example 1. After three immunizations, take mouse splenocytes and mouse myeloma cells SP2 with an Elisa titer of 1:10000 / 0 fusion, HAT medium 37 ℃, saturated humidity, 5% CO 2 Culture confluent cells.

[0071] (2) The colonies were observed on the fourth day after fusion, and when the colonies grew to about 1 / 6 of the bottom of the well, half of the HT medium was replaced. The next day, recombinant CSFV-Erns and CSFV-E2 protein-coated plates were used for indirect Elisa detection, and selection Positive wells were used for subcloning.

[0072] (3) Subcloning: Subcloning was carried out by conventional limiting dilution method. On the 5th day, the subcloning plate was coated with CSFV positive serum 1:1000 for indirect Elisa detection, and the...

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Abstract

The invention discloses a BAS-ELISA kit for detecting hog cholera virus Erns and E2 antigen, and belongs to the field of biological technologies and diagnosis and research of animal-borne diseases. The kit comprises an ELISA plate coated with a CSFV-Erns monoclonal antibody and a resistant-CSFV-E2 monoclonal antibody, a sample diluent, CSFV positive control and CSFV negative control, a scrubbing solution, a biotinylation rabbit-resistant CSFV-Erns polyclonal antibody and a biotinylation rabbit-resistant CSFV-E2 polyclonal antibody mixed solution, ELISA streptavidin, an enzyme developing substrate and a stop solution, wherein the two monoclonal antibodies and corresponding polyclonal antibodies have different epitopes. The kit adopts a biotin-avidin detecting system, and implements the combined detection for the hog cholera virus Erns and E2 protein, so that the sensitivity, specificity and repeatability in the detection of the hog cholera virus are greatly improved; the kit can be applied to the diagnosis and research of the hog cholera.

Description

technical field [0001] The invention relates to the fields of biotechnology and diagnosis and research of animal infectious diseases, in particular to a BAS-ELISA kit for detecting CSFV Erns and E2 antigens. Background technique [0002] Classical swine fever virus (CSFV) is a highly contagious infectious disease caused by classical swine fever virus (CSFV), which is seriously harmful to the pig industry. It is one of the main infectious diseases closely monitored and quarantined by the World Food and Agriculture Organization and governments of various countries. [0003] CSFV is a member of the Pestivirus genus of the Flaviviridae family. It is an enveloped single-stranded positive-sense RNA virus with a genome size of about 12.3 kb and only one large open reading frame (ORF). This ORF is translated into a polyprotein containing 3898 amino acid residues and a molecular weight of about 438kDa, and further processed into structural proteins and nonstructural proteins under th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/56983G01N33/6803G01N2333/183
Inventor 李建曹娟廖园园秦伟朱薇刘洁漆世华谢红玲冯钊
Owner WUHAN CHOPPER BIOLOGY
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