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Antibody for detecting infectious spleen and kidney necrosis viruses as well as preparation and application thereof

A spleen and kidney necrosis virus, infectious technology, applied in the field of biological immunity, can solve the problems of high false positive rate, limited promotion and use, etc., and achieve the effect of good specificity and simple operation

Active Publication Date: 2015-04-29
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diagnostic methods for this virus at home and abroad are limited to the polymerase chain reaction (PCR) technology, and there are no reports of other diagnostic methods. The PCR detection method needs to purify the DNA of diseased fish tissues, and the false positive rate is high, which limits this method. promotion and use of

Method used

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  • Antibody for detecting infectious spleen and kidney necrosis viruses as well as preparation and application thereof
  • Antibody for detecting infectious spleen and kidney necrosis viruses as well as preparation and application thereof
  • Antibody for detecting infectious spleen and kidney necrosis viruses as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] An antibody for the detection of infectious spleen-kidney necrosis virus obtained by:

[0046] 1) Obtaining polypeptide fragments: Using Antigenic and DNAstar / protean and other antigenic determinant analysis software to analyze the membrane protein VP23R encoded by infectious spleen and kidney necrosis virus (ISKNV) strain (NCBI accession number: AF371960) The amino acid sequence was analyzed, and an epitope with 14 amino acid residues was screened out: ISKNV-23P8 (SEQ ID NO: 1); bioinformatics analysis showed that the homology of this polypeptide sequence with other viruses of the genus Iridovirus was relatively high. Low, no more than 5 consecutive amino acids consistent;

[0047] 2) Preparation of anti-VP23R protein antibody

[0048] ①Synthesizing the polypeptide: using the ISKNV-23P8 polypeptide fragment obtained in step 1 as a template, using biological methods to synthesize the polypeptide, and then purifying the synthesized polypeptide by HPLC to a purity of mor...

Embodiment 2

[0065] A non-diagnostic printing immunofluorescence rapid detection method for infectious spleen and kidney necrosis virus infection, comprising the following steps:

[0066] 1) Take the spleen tissue of diseased fish suspected of ISKNV infection, cut the spleen with a razor blade, print the fresh section on a siliconized or poly-lysine-treated glass slide, and air-dry;

[0067] 2) Cover and fix with 4% paraformaldehyde solution at room temperature for 3 minutes;

[0068] 3) block with 1% bovine serum albumin (BSA) PBST solution at room temperature for 20 minutes;

[0069] 4) Dilute the rabbit anti-ISKNV-23P8 polyclonal antibody with PBST at a ratio of 1:1000, incubate at room temperature for 30 minutes, and wash thoroughly with PBST for 3 times;

[0070] 5) Add FITC fluorescently labeled goat anti-rabbit IgG secondary antibody (KLH) to incubate for 20 minutes, and wash three times with PBST;

[0071] 6) Seal the slide with a cover glass, detect it with an immunofluorescence...

Embodiment 3

[0073] A non-diagnostic printing immunofluorescence rapid detection method for infectious spleen and kidney necrosis virus infection, comprising the following steps:

[0074] 1) Take the spleen tissue of diseased fish suspected of ISKNV infection, cut the spleen with a razor blade, print the fresh section on a siliconized or poly-lysine-treated glass slide, and air-dry;

[0075] 2) Cover and fix with 4% paraformaldehyde solution at room temperature for 10 minutes;

[0076] 3) block with 1% bovine serum albumin (BSA) PBST solution at room temperature for 10 minutes;

[0077] 4) Rabbit anti-ISKNV-23P8 IgG diluted with PBST at a ratio of 1:3000, incubated at room temperature for 50 minutes, and fully washed 3 times with PBST;

[0078] 5) Add FITC fluorescently labeled goat anti-rabbit IgG secondary antibody (KLH), incubate for 40 minutes, and wash three times with PBST;

[0079] 6) Seal the slide with a cover glass, detect it with an immunofluorescence microscope, and observe t...

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Abstract

The invention discloses an antibody for detecting infectious spleen and kidney necrosis viruses as well as preparation and application thereof. The antibody for detecting infectious spleen and kidney necrosis viruses is a purified anti-rabbit ISKNV-23P8 polyclonal antibody which is capable of specifically recognizing ISKNV-23P8, obtained by adopting an amino acid residue containing a sequence having a sequence number of SEQ ID NO: 1 as an epitope. The antibody is used for detecting the infectious spleen and kidney necrosis viruses and has specificity. An immunofluorescence detection kit for the infectious spleen and kidney necrosis viruses and a non-diagnostic immunofluorescence rapid detection method for infectious spleen and kidney necrosis virus-infected imprints have the characteristics of good specificity, simple operation, quickness and sensitivity, have obvious advantages compared with a PCR method, and can be used for ISKNV infection detection during the culture of various susceptible fishes; and all operations can be completed within 1.5 hours.

Description

technical field [0001] The invention relates to the technical field of biological immunity, in particular to an antibody for detecting infectious spleen and kidney necrosis virus and its preparation and application. Background technique [0002] Infectious Spleen and Kidney Necrosis Virus (ISKNV) is a representative species of the genus Megalocytivirus in the family Iridoviridae, and is a type of cytoplasmic large double-stranded virus with an icosahedron. DNA virus, the genome consists of 111,362 bases, including 125 predicted open reading frames ORF (open reading frames). ISKNV can infect nearly 60 species of freshwater and marine fish, and is a huge threat to the aquaculture industry of mandarin fish, California perch and American redfish, and mandarin fish is its main infection reservoir. [0003] Siniperca chuatsi (Siniperca chuatsi) is a fish of the genus Siniperca in the family Perciformes, commonly known as mandarin fish, crucian carp, mandarin fish, seasonal flower...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18G01N33/569G01N33/533
Inventor 李中圣徐晓鹏乔晶鑫何永龙侯月娥
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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