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ELISA kit for detecting hog cholera virus Erns IgM antibody

A swine fever virus and kit technology are applied in the fields of biotechnology and animal infectious disease diagnosis and research to achieve the effects of improving expression, sensitivity and specificity

Inactive Publication Date: 2014-08-13
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the Elisa kits in the market are kits for detecting anti-E2 antibodies, and there is no research on Erns IgM antibodies

Method used

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  • ELISA kit for detecting hog cholera virus Erns IgM antibody
  • ELISA kit for detecting hog cholera virus Erns IgM antibody
  • ELISA kit for detecting hog cholera virus Erns IgM antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] The preparation of embodiment 1 recombinant swine fever virus Erns protein

[0040] (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 E2 major antigen region B / C and D / A regions The gene sequence was used as a template to design synthetic primers,

[0041] The upstream primer of CSFV-Erns gene is: 5'-CG GGATCC GAAAATATAACTCAGTGGAACCTGA-3',

[0042] The downstream primer of CSFV-Erns gene is: 5'-CG CTCGAG GGCATAGGCACCAAACCAG-3';

[0043] Wherein, the underlined part is the restriction site introduced, the restriction site introduced by the upstream primer is BamHI, and the restriction site introduced by the downstream primer is XhoI. The RNA extracted from CSFV Shimen strain was used as template to carry out reverse transcription PCR to obtain the target gene of Erns protein.

[0044] (2) Construction of a recombinant expression ve...

Embodiment 2

[0052] The preparation of embodiment 2 anti-swine fever virus Erns protein (CSFV-Erns) IgG monoclonal antibody

[0053] (1) The recombinant CSFV-Erns protein purified in Example 1 was immunized with Balb / c mice. After three immunizations, mouse splenocytes whose Elisa titer reached 1:10000 were fused with mouse myeloma cells SP2 / 0, HAT medium at 37°C, saturated humidity, 5% CO 2 Culture confluent cells.

[0054] (2) Colonies were observed on the fourth day after fusion. When the colonies grew to about 1 / 6 of the bottom of the well, half of the HT medium was replaced. The next day, the recombinant CSFV-Erns protein-coated plate was used for indirect Elisa detection, and positive wells were selected for testing. subcloning.

[0055] (3) Subcloning: observe the cells in the positive wells under a microscope, and select colonies with good cell morphology for subcloning by the limiting dilution method. On the 5th day of the subcloning plate, the plate was coated with CSFV positi...

Embodiment 3

[0063] Embodiment 3 detects the preparation of the ELISA kit of swine fever virus (CSFV) Erns IgM antibody

[0064] (1) ELISA plate coated with anti-porcine IgM monoclonal antibody: Dilute anti-porcine IgM monoclonal antibody to 1 μg / mL with coating buffer, add 100 μL to each well, and coat overnight at 4°C; wash with washing solution for 2 ~3 times, pat dry; add 150 μL of blocking solution to each well, incubate at 37°C for 1 h, wash with washing solution 2-3 times, and pat dry. Among them, the coating buffer is 0.05mol / L pH9.6 carbonate buffer, which is prepared as follows: accurately weigh 1.59g sodium carbonate, 2.93g sodium bicarbonate, add distilled water to 1000mL; L, 5% BSA dissolved in phosphate buffered saline (PBS) with a pH of 7.2-7.4; the washing solution is a working solution prepared by diluting the concentrated washing solution 10 times.

[0065] (2) Sample diluent: add 1% (m / v) BSA, 0.1% (m / v) deep-sea fish gelatin, 0.02% (m / v) sodium azide (NaN3) prepared. ...

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Abstract

The invention discloses an ELISA kit for detecting a hog cholera virus Erns IgM antibody, and belongs to the fields of a biotechnology and diagnosis research of an animal-borne disease. The ELISA kit comprises an elisa plate of enveloping an anti-swine IgM monoclonal antibody, a sample diluent, a positive control and a negative control, a detection antigen (hog cholera virus Erns protein), washing concentrate, an enzyme conjugate, an enzyme chromogenic substrate and a stopping solution. The IgM antibody is detected by adopting a capture method, the specific hog cholera virus Erns IgM antibody is detected by adopting a sandwich method, the specificity of the prepared kit can be up to 100%, the sensitivity is 1:800, and the ELISA kit can be applied to diagnosis of swine fever virus infection and assessment of immune efficacy of a hog cholera vaccine.

Description

technical field [0001] The invention belongs to the field of biotechnology and diagnosis and research of animal infectious diseases, in particular to an ELISA kit for detecting swine fever virus Erns IgM antibody. 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 un...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/535G01N33/56983
Inventor 李建漆世华韩兴舒银辉廖园园刘洁谢红玲秦红刚徐松牟林琳朱薇温文生
Owner WUHAN CHOPPER BIOLOGY
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