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Schmallenberg virus nucleocapsid protein monoclonal antibody, and preparation method thereof

A technology of monoclonal antibody and nucleocapsid protein, which is applied in the field of genetic engineering and immunity to achieve the effect of abundant expression, high purity and good repeatability

Active Publication Date: 2013-09-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Since Schmallenberg disease is a newly discovered animal disease, there is currently no domestic detection technology reserve for this disease

Method used

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  • Schmallenberg virus nucleocapsid protein monoclonal antibody, and preparation method thereof
  • Schmallenberg virus nucleocapsid protein monoclonal antibody, and preparation method thereof
  • Schmallenberg virus nucleocapsid protein monoclonal antibody, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0052] 2 Preparation of immune splenocytes

[0053] After 3 days of booster immunization, aseptically collect splenocytes to prepare splenocyte suspension, the specific operation is as follows:

[0054] 1) Mice were killed by dislocation;

[0055]2) Soak the mouse in 75% alcohol for disinfection, cut its abdomen with sterile scissors, take out the spleen, put it in a sterile plate containing a small amount of culture solution (1640 culture solution) and put it on a stainless steel drying net. The needle core was ground into a cell suspension and counted;

[0056] 3) Collect the spleen cell suspension on ice, centrifuge to remove the supernatant;

[0057] 4) Wash the pellet again by centrifugation with fresh RPMI-1640 culture medium;

[0058] 5) Add fresh RPMI-1640 culture medium; count spleen cells.

[0059] 3 Recovery of myeloma cells

[0060] The myeloma cell SP2 / 0 was stored in a liquid nitrogen tank at -196°C, and it was sufficient to recover, proliferate, and passage...

Embodiment 3

[0105] Example 3 Anti-SBV N Protein Monoclonal Antibody Titer Determination——ELISA Detection

[0106] 1. Coating: The purified recombinant N protein was used as the detection antigen, the coating concentration was 2 μg / ml, 100 μL / well, overnight at 4°C, and washed 3 times with the washing solution.

[0107] 2. Blocking: add 150 μL / well blocking solution, wash 3 times after 2 hours at 37°C, and pat dry. Store in a 4°C refrigerator for later use.

[0108] 3. Add the sample to be tested:

[0109] 1) For serum / antibody / ascitic fluid titer detection, dilute the first well at 1:1000, then dilute downward at a gradient ratio of 1:2, incubate at 37°C for 30 minutes, wash the plate 4 times, and pat dry.

[0110]2) For cell supernatant detection, pipette 100 μL of cell supernatant, add it to the corresponding microplate, incubate at 37°C for 30 min, wash the plate 4 times, and pat dry.

[0111] 3) Add secondary antibody: take horseradish-enzyme-labeled goat anti-mouse IgG (IgG-specif...

Embodiment 4

[0117] Example 4 Western blot identification of anti-Schmallenberg virus nucleocapsid protein N monoclonal antibody (2C8)

[0118] 1 Purpose of the test

[0119] To verify whether the monoclonal antibody 2C8 can react with the recombinant N protein and the natural nucleocapsid protein (N) of SBV.

[0120] 2 test material

[0121] Cell line: BHK-21.

[0122] SBV strains: BH80 / 11-4, BH652 / 12-1, BH619 / 12, D512 / 12, D495 / 12-1, and D495 / 12-2; viral stock was 10 6 TCID 50 / mL.

[0123] HRP-goat anti-mouse IgG (product number P0447): purchased from Dako Company in Denmark.

[0124] 3 operation steps

[0125] 3.1 Preparation of protein samples

[0126] Cells infected by different strains of SBV and control cells (approximately 2.0×10 6 cells / bottle) were digested from the wall of the cell flask, transferred to a 15mL centrifuge tube with a pipette, centrifuged at 300×g at 4°C for 6min, poured off the cell supernatant, and resuspended the cell pellet with 2mL PBS. Store in a 4°...

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Abstract

The invention provides a schmallenberg virus nucleocapsid protein monoclonal antibody which is produced and secreted by hybridoma SBV-N McAb2C8 with a preservation number of CGMCC No. 7617. The preparation method of the hybridoma comprises the following steps of extracting genomic RNA of SBV, obtaining N gene sequences by RT-PCR amplification; inserting the N gene sequences into prokaryotic expression vectors; transforming into Escherichia coli; carrying out inducible expression and protein purification; immunizing an animal by using purified protein as an antigen; fusing splenocytes of the immunized animal with mouse myeloma cells to prepare the hybridoma; and screening to obtain the hybridoma capable of stably secreting the schmallenberg virus nucleocapsid protein monoclonal antibody. The monoclonal antibody provides a material basis for establishment of an ELISA detection method for schmallenberg diseases.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and immunization, in particular to a monoclonal antibody to Schmallenberg virus nucleocapsid protein and a preparation method thereof. Background technique [0002] In the summer and autumn of 2011, an unknown disease began to appear in some cattle farms in Germany and the Netherlands. The sick cows showed clinical symptoms such as fever (>40°C), physical decline, loss of appetite, anorexia, diarrhea, and decreased milk production. miscarriage symptoms. On November 18, 2011, the Friedrich Loeffler Institute (FLI) in Germany used Metagenomics technology (Metagenomic technique) and virus isolation technology to finally confirm that the pathogen that caused the outbreak was a new type of Bunia virus. Because the virus was first isolated and identified from the blood of a sick cow in Schmallenberg, a town in western Germany, it was tentatively named "Schmallenberg virus (SBV)" accord...

Claims

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Application Information

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IPC IPC(8): C07K16/10G01N33/577C12N5/20C12R1/91
Inventor 张永宁吴绍强林祥梅吕继洲王彩霞邓俊花袁向芬景宏丽
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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