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Envelope protein VP28 idiotype monoclonal antibody against shrimp white spot syndrome virus (WSSV) and preparation method thereof

A monoclonal antibody, envelope protein technology, applied in the field of molecular immunology and virology, can solve the problems of anti-idiotype antibody research that has not been reported and rarely applied.

Inactive Publication Date: 2010-04-07
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Anti-idiotypic antibodies have the characteristics of mimicking antigens, and are used in medicine for receptor research, autoimmune disease pathogenesis research, and new vaccine research, but are rarely used in aquatic animal disease research. Regarding WSSV or its related proteins Studies of anti-idiotypic antibodies have not been reported

Method used

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  • Envelope protein VP28 idiotype monoclonal antibody against shrimp white spot syndrome virus (WSSV) and preparation method thereof
  • Envelope protein VP28 idiotype monoclonal antibody against shrimp white spot syndrome virus (WSSV) and preparation method thereof
  • Envelope protein VP28 idiotype monoclonal antibody against shrimp white spot syndrome virus (WSSV) and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A hybridoma cell that secretes the anti-WSSV-VP28 monoclonal antibody (Ab1), its preparation method is as follows:

[0026] 1. Antigen preparation

[0027] (1) Purification of the virus: take the head and chest of the dying Penaeus sinensis infected with WSSV (remove the hepatopancreas), cut it into pieces and add it to prepare with TNE buffer (0.05M Tris-Hcl, 0.1M NaCl, 1mM EDTA, pH7.4) Grinding in 25% (W / W) sucrose solution, the sample after grinding was centrifuged at 600g for 20min, and the supernatant was taken; 800g was centrifuged for 20min, and the supernatant was taken; 20000g was centrifuged for 2h, and the supernatant was discarded; After suspension, spread on a discontinuous sucrose gradient and centrifuge at 20000g for 2h; take the virus zone after centrifugation, dilute it 6 times with TNE and centrifuge at 20000g for 90min; 2 HP0 4 , 1.47mM KH 2 PO 4 , pH 7.4), observed under a negative stain electron microscope, aliquoted, and frozen at -80°C for lat...

Embodiment 2

[0077] Preparation of anti-WSSV-VP28 idiotype monoclonal antibody:

[0078] 1. Antigen preparation

[0079] Each Balb / c mouse was intraperitoneally injected with 0.5ml of liquid paraffin, and 10 days later, it was intraperitoneally injected with hybridoma cells secreting anti-WSSV-VP28 monoclonal antibody (Ab1). The mice were reared and closely observed. After about 14 days, the abdomen of the mice was enlarged and extracted. ascites. The ascites was initially purified by caprylic acid-ammonium sulfate method and then purified by Protein G Agarose (Amersham) to adjust the concentration to 2mg / ml, and then divided into equipment for use.

[0080] 2. Immunization of mice

[0081] Using Ab1 purified in Example 1 as the antigen, immunize Balb / c mice. The immunizations are divided into 5 times. The interval between the first two immunizations is 2 weeks, and the interval between the next three immunizations is 1 week. The first 2 immunizations are intraperitoneal injections. , t...

Embodiment 3

[0113] Identification of the anti-WSSV-VP28 idiotype monoclonal antibody of the present invention by competitive enzyme-linked immunosorbent assay:

[0114] ① Step 1 of Example 1 (1) Dilute the purified WSSV with carbonate coating solution 1:40, add 100 μl per well into a 96-well microtiter plate, and coat overnight at 4°C;

[0115] ② Aspirate the coating solution, wash with PBST 3 times, 5min each time;

[0116] ③Add 200 μl 3% bovine serum albumin to each well, and block at 37°C for 1 hour;

[0117] ④ Wash with step ②;

[0118] 5. The monoclonal antibody of the present invention purified by octanoic acid-ammonium sulfate method is used as an antigen, and the antiserum (Ab3) is obtained by immunizing Balb / c mice. 320, 640, 1 280, 2 560, 5120 times, the gradient dilutions of the monoclonal antibody of the present invention and Ab3 were mixed with the rabbit anti-WSSV-VP28 antibody with a concentration of 40 μg / ml in equal volumes to form the experimental group, after overnigh...

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Abstract

The invention discloses an envelope protein VP28 idiotype monoclonal antibody against shrimp white spot syndrome virus (WSSV) and a preparation method thereof. The antibody is secreted by a hybridoma cells with the collection number of CCTCC-CT200938, is prepared by taking anti-WSSV-VP28 monoclonal antibody (Ab1) as antigen, can bind with anti-WSSV-VP28 antibody of hare, and has the capability of competing with WSSV to bind with the anti-WSSV-VP28 antibody of the hare. The anti-WSSV-VP28 idiotype monoclonal antibody (Ab3) prepared by taking the antibody as antigen can bind with the WSSV, the binding site of the anti-WSSV-VP28 idiotype monoclonal antibody (Ab3) and the WSSV is located on an envelope, and the Ab3 can neutralize WSSV infection and has Ab1 properties. In the invention, the idiotype antibody is applied in the research of WSSV for the first time; a screening system is established, which uses an indirect enzyme-linked immunosorbent assay (ELISA) method and a competitive enzyme-linked immunosorbent assay (ELISA) method for detection; the fact that Ab3 has properties of Ab1 is proved by adopting an indirect immnnofluotesent method (IIF), a gold labeling immunoelectron microscopic method and crayfish in vivo neutralization tests, thus proving that the monoclonal antibody in the invention has the property to simulate original antigen WSSV-VP28.

Description

technical field [0001] The present invention relates to a monoclonal antibody (monoclonal antibody) and a preparation method thereof, in particular to an idiotype monoclonal antibody against shrimp white spot disease virus (Whitespot syndrome virus, WSSV) envelope protein VP28 (virus protein 28) and a preparation method thereof, It belongs to the cross technical field of molecular immunology and virology. Background technique [0002] WSSV is an enveloped double-stranded DNA virus with high infection and replication capabilities and a wide range of hosts. The existing research at home and abroad involves the morphological structure, chemical composition and physical and chemical characteristics of WSSV, genome and its encoded polypeptide, host and transmission route, and epidemiology, etc., to find out the molecular mechanism of WSSV infection, and find ways to prevent and control WSSV infection. Effective methods and measures are the hotspots of current research. One of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/08C12N15/06C12P21/08
CPCC07K16/081C12N2710/18022C07K2317/76A61P31/12
Inventor 战文斌韦秀梅绳秀珍
Owner OCEAN UNIV OF CHINA
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