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Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke, and preparation method

A technology of lymphocyst virus and monoclonal antibody, which is applied in the monoclonal antibody of flounder against lymphocyst virus immunoglobulin and its preparation, and the improvement of immunology application technology, which can solve the problem that virus infection is difficult to detect and the host has not been studied Issues such as effective drugs for cells to achieve novel effects of design

Active Publication Date: 2009-11-18
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pathogen of lymphocystis disease is lymphocyst virus, which is a kind of non-cellular ultramicrobial group that parasitizes in host cells, so no effective drugs that can kill viruses without damaging host cells have been developed so far
In addition, the recessive infection of the virus and the special aquatic environment of fish make the infection of the virus difficult to detect, and often once the symptoms are found, it is irreparable

Method used

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  • Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke, and preparation method
  • Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke, and preparation method
  • Monoclonal antibody of immunoglobulin of anti lymphocyst vitos of Pacific fluke, and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Development of monoclonal antibody against lymphocyst virus immunoglobulin in flounder

[0028] 1. Antigen preparation

[0029] (1) Preparation of flounder lymphocyst virus

[0030] ①Take the diseased flounder, first use 70% alcohol cotton ball to disinfect the affected part, then cut off the cyst with a sterilized scalpel blade, add an appropriate amount of quartz sand and TNE (50mM Tris, 00mM NaCl, 1mM EDTA, pH 7.4) buffer, and mix well. pulp;

[0031] ② Centrifuge the homogenate (4°C, 500g, 20min), and take the supernatant;

[0032] ③ Centrifuge the supernatant (4°C, 1800g, 20min), and take the supernatant;

[0033] ④ Make 30% (W / W) sucrose solution from the supernatant and sucrose, centrifuge at 78500g for 120min at 4°C, discard the supernatant;

[0034] ⑤ Add TNE to the precipitate to 1ml, mix well, place gently on top of the sucrose gradient solution (37%, 40%, 47%, 52%, 57%, 62%), centrifuge at 78500g for 120min at 4°C;

[0035] ⑥ Aspirate the virus band in th...

Embodiment 2

[0085] The indirect ELISA identification of monoclonal antibody of the present invention:

[0086] (1) Coating antigen: Lymphocyst virus was diluted 1:10 with carbonate coating solution (pH 9.6), added to a 96-well microtiter plate (50 μl / well), and coated overnight at 4°C;

[0087] (2) Aspirate the coating solution, wash with PBST, each time for 5min, and wash three times;

[0088] (3) 200 μl of 3% bovine serum albumin (with PBS) was added to each well to block for 1 hour at 37°C;

[0089] (4) Wash three times with method ②;

[0090] (5) Add flounder antiserum (diluted 1:10) 50 μl to each well, and incubate at 37°C for 2 hours;

[0091] (6) Wash three times with method ②;

[0092] (7) Add the culture supernatant of the hybridoma cells screened and cloned above as the primary antibody to the microtiter plate at 50 μl per well, and incubate in a 37°C incubator for 1 hour;

[0093] (8) Wash three times with method ②;

[0094] (9) Alkaline phosphatase-labeled goat anti-mouse I...

Embodiment 3

[0099] Identification of the transfer immunoblotting method of the monoclonal antibody of the present invention:

[0100] (1) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis:

[0101] ① Add the extracted flounder immunoglobulin to the sample buffer solution containing sodium dodecylsulfonate in equal proportions, and boil in boiling water for 3-5 minutes;

[0102] ②Put the sample treated in ① into the sample well, add 10 μl of sample into each well, under constant current condition, use low current (30-40mA) at the beginning, after the sample is concentrated into a line in the stacking gel, Increase the current (50-70mA), electrophoresis until the bromophenol blue indicator reaches the bottom edge, then stop the electrophoresis and take out the gel;

[0103] ③Cut a piece of nitrocellulose membrane (pore size 0.22 μm) with the same size as the electrophoresis gel to use the electrotransfer buffer (electrotransfer buffer: 25mmol / L Tris-Base, 192mmol / L glycine, 20% met...

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Abstract

The invention is the monoclonal antibody of flounder against lymphocyst virus (LCDV) immunoglobulin. It is secreted by the hybridoma cell named as hybridoma cell JF-IgM-H, and the preservation number is CCTCC-C200631. The preparation method of the monoclonal antibody is as follows: prepare antiserum by immunizing flounder with formalin-inactivated LCDV, then purify the flounder immunoglobulin as an antigen and immunize Balb / c mice, and use cell engineering to generate fusion hybrid Tumor cells, the monoclonal antibody was screened out by immunological detection method. Indirect enzyme-linked immunosorbent method and indirect immunofluorescence antibody method comprehensively determine: the specific binding reaction between the monoclonal antibody of the present invention and the flounder anti-LCDV immunoglobulin really exists; transfer immunoblotting method determines: the epitope of the monoclonal antibody of the present invention is located at the molecular weight On the heavy chain of the flounder anti-LCDV immunoglobulin in the range 70-80 kDa. The invention will be used in the preparation of reagents for early detection and judgment of LCDV infection in flounder and evaluation of the immune effect of LCDV formalin inactivated vaccine on flounder.

Description

technical field [0001] The invention relates to the improvement of immunology application technology, specifically a monoclonal antibody against lymphocyst virus (LCDV) immunoglobulin of flounder (Paralichthys olivaceus) and a preparation method thereof, which belong to the technical field of fish molecular immunology. Background technique [0002] In recent years, flounder has become an important marine aquaculture economic fish in my country and even in Asia, and its position in the entire aquaculture industry has become increasingly prominent. However, with the expansion of breeding scale, disease problems have become increasingly prominent, seriously affecting the sustainable and healthy development of the industry, among which lymphocystosis is one of the most serious diseases. The pathogen of lymphocystis disease is lymphocyst virus, which is a kind of non-cellular ultramicrobial group that parasitizes in host cells, so no effective drugs that can kill viruses without ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18G01N33/53
Inventor 战文斌林颖博李强
Owner OCEAN UNIV OF CHINA
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