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Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method

A monoclonal antibody and virus technology, which is applied in the intersection of molecular immunology and virology, and can solve the problems of screening vitiligo virus restriction, no report on vitiligo virus receptors, etc.

Active Publication Date: 2005-08-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, at home and abroad, not only have not successfully established a cell line sensitive to VSV, which limits the use of the above method to screen VSV receptors, but also there is no report on the use of monoclonal antibody technology to screen and identify VSV receptors.

Method used

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  • Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method
  • Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method
  • Monoclonal antibody of virus receptor of anti leukoplakia disease and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1. Development of monoclonal antibody I:

[0034] 1) Antigen preparation:

[0035] Take healthy sea-caught prawns with a body length of 18-20 cm, disinfect the rear edge of the prawn head and breastplate with alcohol cotton balls, and absorb pre-cooled anticoagulants (sodium chloride 8.2g / L, citric acid 5.5g / L, glucose 19.8 g / L, sodium citrate 8.8g / L, pH5.6) 5ml disposable medical syringe inserted into the heart of the prawns to extract blood in equal proportions, centrifuged at 400g, 10 minutes, 4 ° C.

[0036] Remove the supernatant, resuspend the blood cell pellet with anticoagulant, and centrifuge again. This was repeated 3 times to obtain pure hemocytes of prawns. Use 0.01M phosphate buffer (137mM sodium chloride, 2.7mM potassium chloride, 8.09mM disodium hydrogen phosphate, 1.47mM potassium dihydrogen phosphate, pH7.4) to adjust the concentration of blood cells to 2×10 8 individual / ml.

[0037] 2) Immunity:

[0038] Use the purified Chinese shrimp ...

Embodiment 2

[0082] Example 2. Screening of monoclonal antibody II from monoclonal antibody I:

[0083] 1) Purification of leukoplakia virus:

[0084] (1) Get the shrimp head of the Chinese prawn infected by white spot disease virus, remove the hepatopancreas, weigh 10 grams of the disease material, add 2ml of 25% sucrose, and grind it with quartz sand for 3-5 minutes (4°C) to break the cells and free the virus into the solution, then add 100ml of 25% sucrose, and mix well.

[0085] (2) Centrifuge the ground disease material at 3000 rpm for 15 minutes.

[0086] (3) Take the supernatant and centrifuge at 5000 rpm for 15 minutes at 4°C.

[0087] (4) Take the supernatant and centrifuge at 8000 rpm for 15 minutes at 4°C.

[0088] (5) Take the supernatant and centrifuge at 25,000 rpm (P7OAT rotor) for 2 hours at 4°C.

[0089] (6) Take the precipitate, resuspend it with 8ml of 25% sucrose solution, and stir slowly with a magnetic stirrer for 1 hour at 4°C.

[0090] (7) Lay the uniformly sti...

Embodiment 3

[0137] Embodiment 3. One of the application of monoclonal antibody II of the present invention, the molecular weight of the white spot virus receptor that monoclonal antibody II recognizes on the Chinese prawn blood cell membrane is determined by transfer immunoblotting method:

[0138] Separating gel 10% concentration 16ml

Stacking gel 4.75% concentration 7.5ml

Distilled water 6.45ml

Distilled water 4.33ml

30% Acrylamide 5.3ml

30% Acrylamide 1.19ml

15M Tris-HCl 4ml

0.5M Tris-HCl 1.88ml

10% Sodium Dodecyl Sulfate 160ul

10% Sodium Dodecyl Sulfate 75ul

100% ammonium persulfate 60ul

100% ammonium persulfate 60ul

Tetramethylethylenediamine (TEMED) 15ul

Tetramethylethylenediamine (TEMED) 15ul

10 minutes after pouring the glue to solidify

2 minutes after pouring the glue to solidify

[0139] 1) The purified hemocytes of Penaeus sinensis were separated by sodium dodecyl sulfate-polyacrylami...

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Abstract

A monoclonal antibody B for the white spot virus receptor of prawn is prepared from the hybrid tumor cell (CCTCC-C200418) through conventional culture for 2-3 days to secrete a lot of said monoclonal antibody B. It can be used for preventing and treating whit spot of prawn.

Description

technical field [0001] The present invention relates to the improvement of the application technology of antibody, specifically a kind of anti-white spot syndrome virus (White Spot Syndrome Virus, WSSV) receptor monoclonal antibody and preparation method thereof secreted by hybridoma cells, which belong to molecular immunology and Cross-cutting technical field of virology. Background technique [0002] At present, white spot virus disease is one of the serious diseases in prawn farming, which causes high mortality of prawns and causes huge losses to the prawn farming industry. The white spot disease virus that causes the disease can widely infect the subcutaneous tissue, hematopoietic tissue, connective tissue, lymphoid organs, etc. of prawns. The screening of virus receptors mostly adopts virus overlay protein blot assay (virus overlay protein blot assay) technology. Prerequisites for using this technology to screen viral receptors: receptor activity must be expressed by ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P31/12C07K16/08C07K16/28C12N15/02
Inventor 战文斌张志栋
Owner OCEAN UNIV OF CHINA
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