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ELISA kit for detecting largemouth bass ranavirus antibody and detection method thereof

A kit and antibody technology, applied in the direction of virus/bacteriophage, virus, virus peptide, etc., can solve the problems of large genome, complex pathogenicity and difficulty of frog virus

Active Publication Date: 2021-05-07
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the harm of frog virus to largemouth bass has not been taken seriously before. With the continuous improvement of the breeding level of largemouth bass, frog virus has become the biggest pain point of high-density culture of largemouth bass; the genome of frog virus is relatively large (80-120Kbp) , the pathogenicity is relatively complex, and the preparation of immunological raw materials for detecting LMBV-specific antibodies is technically difficult, and there is no accurate, simple and sensitive detection method for LMBV-specific antibodies

Method used

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  • ELISA kit for detecting largemouth bass ranavirus antibody and detection method thereof
  • ELISA kit for detecting largemouth bass ranavirus antibody and detection method thereof
  • ELISA kit for detecting largemouth bass ranavirus antibody and detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0050] Expression and purification of embodiment 1 recombinant protein

[0051] Construct the expression plasmids of the LMBV MCP full sequence (LVMCP), the N-terminal domain (LVMCPn) of the MCP protein of LMBV and the C-terminal domain (LVMCPc) of LMBV MCP, and transform them into Escherichia coli expression strain Rosetta (DE3) pLysS for Expressed to obtain recombinant proteins LVMCP, LVMCPn and LVMCPc.

[0052] Wherein, the amino acid sequences of LVMCP, LVMCPn and LVMCPc are respectively: SEQ ID NO:2, SEQ ID NO:1 and SEQ ID NO:3; LMBV MCP protein sequence structure analysis is as follows: figure 1 shown.

[0053] The expression of the three engineering bacteria was identified by Western Blot, and the results were as follows: figure 2 Shown, wherein, M is the protein Ladder, lanes 1-3 are LVMCP, LVMCPn and LVMCPc western blots respectively.

[0054] The three recombinant proteins were purified by affinity chromatography, and the protein concentration was calibrated with...

Embodiment 2

[0055] Example 2LMBV antibody positive, negative serum and quality control

[0056] 1. Preparation of LMBV antibody positive serum

[0057] The LMBV virus was cultured on carp epithelial cells (EPC), and the final concentration of 0.2% formaldehyde was added to inactivate the virus. After the quality inspection, the virus stock solution was emulsified with VSA201 adjuvant to prepare the vaccine, and the vaccine was stored at 4°C after the quality inspection. Select 50 healthy largemouth bass of 200-300 grams, and immunize them intraperitoneally with the prepared LMBV inactivated vaccine. Blood was collected from the base of the caudal fin of all fish, and the antibody titer was evaluated in combination with ELISA test. Serum samples with relatively high titers (serum samples diluted 40 times and with an OD450nm value of about 1.0) were selected as LMBV antibody-positive serum, and stored at -20°C for later use.

[0058] 2. LMBV antibody negative serum preparation

[0059] C...

Embodiment 3

[0063] The ELISA reactivity of embodiment 3 three kinds of recombinant proteins

[0064] The immunological reaction between the serum antibody of largemouth bass and three kinds of recombinant proteins (prepared in Example 1) was detected by indirect ELISA method. ELISA detection solid-phase coating were selected: LVMCP, LVMCPn and LVMCPc. specific method:

[0065] (1) Dilute the three recombinant proteins to 2μg / mL, 4μg / mL and 8μg / mL respectively with 0.05mol / L carbonate (PH=9.6) to coat the microtiter plate, 100μL / well, incubate at 37°C for 1 hour , Incubate overnight at 4°C;

[0066] (2) Wash the ELISA plate 3 times with 0.05% Tween-20 (PBST), 5 minutes each time; add 100 μL blocking solution of 5% skimmed milk powder to each well, and block at 37°C for 1 hour;

[0067] (3) Wash 3 times with PBST, 3 minutes / time; the largemouth bass serum (LMBV antibody-positive serum or LMBV antibody-negative serum) prepared in Example 2 was diluted 1:20 times, added to the well, and in...

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Abstract

The invention relates to the technical field of immunodetection, and particularly relates to an ELISA kit for detecting a largemouth bass ranavirus antibody and a detection method thereof. The kit comprises an antibody trapping agent, a solid-phase support, a first antibody, an enzyme-labeled second antibody, confining liquid, developing liquid and stop buffer, wherein the antibody trapping agent is recombinant largemouth bass ranavirus LVMCPn protein, and the amino acid sequence of the antibody trapping agent is shown as SEQ ID NO: 1. The kit has good sensitivity and specificity to an LMBV antibody and has good repeatability and stability.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to an ELISA kit for detecting antibodies against largemouth bass iridescent virus and a detection method thereof. Background technique [0002] Largemouth bass (Largemouth bass, Micropterus salmoides) is a fish of the sunfish family and the genus Black Bass, commonly known as California perch, and is an important fish species for special fish farming in South China. Largemouth bass has delicious meat and high nutritional value, and its consumption is increasing year by year. With the change of growth habits, large-mouth bass farming has gradually formed a large-scale farming model. Under the condition of no disease, large-mouth bass can produce 10,000 catties per mu, and the profit is considerable. However, under high-density breeding conditions, diseases of largemouth bass occur frequently. Among many diseases, the "rotten body disease" caused by Ranavirus is the most serious. Red...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01G01N33/569G01N33/543
CPCC07K14/005C12N2710/00022G01N33/543G01N33/56983G01N2333/01G01N2469/20
Inventor 李中圣刘文娜张钰薇王凤求黄锦炉尹兴强李涛罗律曹梦蕊
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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