Monoclonal antibody of specificity detection of bluetongue viruses and hybridoma cell strain and application of monoclonal antibody

A technology of hybridoma cell line and bluetongue virus, applied in the field of basic research and prevention of bluetongue disease, can solve the problems of insufficient specificity, delayed bluetongue disease, high homology, etc., and achieve simple and reliable operation High, reliable and accurate results

Active Publication Date: 2019-07-16
YUNNAN ANIMAL SCI & VETERINARY INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing detection technology for researching BTV NS2 antibody has the defect of insufficient specificity. For example, Chinese patent 201310478429.7 discloses a bluetongue virus NS2 protein monoclonal antibody BTV-4D4 and the B cell epitope it recognizes and its application , providing a way to detect whether animals in bluetongue endemic areas are infected by wild strains, but in practice, it is found that the specificity of this method is not strong enough, especially with deer epidemic hemorrhagic fever virus infection, it is easy to misjudge and delay The timing of the prevention and treatment of bluetongue disease in the epidemic area, and the root of the misjudgment is that the B cell epitope recognized by the monoclonal antibody BTV-4D4 disclosed in the patent has a high homology with the corresponding segment of the NS2 protein of EHDV

Method used

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  • Monoclonal antibody of specificity detection of bluetongue viruses and hybridoma cell strain and application of monoclonal antibody
  • Monoclonal antibody of specificity detection of bluetongue viruses and hybridoma cell strain and application of monoclonal antibody
  • Monoclonal antibody of specificity detection of bluetongue viruses and hybridoma cell strain and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 culturing and screening hybridoma cell lines

[0068] 1. Main experimental materials

[0069] 1.1 Main biological living materials

[0070] BHK cells, SP2 / 0 cells, and BTV-1 strains are preserved by our institute.

[0071] Eight-week-old female Balb / c mice were provided by the Experimental Animal Department of Kunming Medical University.

[0072] 1.2 Main reagents and consumables

[0073] MEM medium and RPMI-1640 medium (supplemented with 10% FBS and 100U / ml penicillin-streptomycin, both purchased from Giboc); hybridoma screening reagents HAT and HT, polyethylene glycol fusion agent (PEG1500) were purchased from Sigma. RNA extraction kit (Takara, #9767), SuperScript III reverse transcription kit (Invitrogen, #18080051), Pfu enzyme (Tiangen, #EP101-01), 2×PCR Mix (Tiangen, #KT201-02), DNA marker DL 5 000 (Tiangen), agarose, DNA purification kit (Tiangen, #DP203-02), endonuclease BamH I and Xho I (Thermo), T4 DNA ligase (Shenggong, #B600009) , pET-32a p...

Embodiment 2

[0114] Example 2 Monoclonal Antibody Subclass Identification

[0115] Using the antibody subtype identification kit (Beijing Boaolong Immunotechnology Co., Ltd., BF16002X), the monoclonal antibody secreted by the cross-tumor cell line BTV-2A4 obtained in the examples was identified according to the instructions.

[0116] The result shows that the monoclonal antibody BTV-2A4 subclass of the present invention is IgG1, and the light chain subtype is κ chain.

Embodiment 3

[0117] Antigen epitope identification of embodiment 3 monoclonal antibody

[0118] The antigen peptide gene was divided into three segments x, y, and z and cloned into the pET-28a vector (inserted through BamHI and XhoI restriction sites), and then constructed to express x (theoretical molecular weight 15kDa, NS2 protein amino acid range 1-90AA ), y (theoretical molecular weight 20kDa, NS2 protein amino acid range 46-183AA), z (theoretical molecular weight 15kDa, NS2 protein amino acid range 139-228AA) peptide fragments of Rosetta bacteria. Select clones x1, x2, y3, y4, z5, z6 with correct recombinant plasmid DNA sequences. 0.2mM IPTG was used to induce protein expression in 1ml of bacterial liquid for 4h, and the bacterial pellet was collected and lysed with 200μl cell lysate. After 1h in ice bath, it was centrifuged (4°C, 12 000rpm, 10min), and the supernatant was collected as a protein sample. Add equal volumes of x, y, z protein samples to 1 / 4 volume of 5×loading buffer, ...

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Abstract

The invention relates to a hybridoma cell strain BTV-2A4, a monoclonal antibody secreted by the same, and BTV-NS2 protein B cell epitope polypeptide discriminated by the monoclonal antibody. The monoclonal antibody provided by the invention and the BTV-NS2 protein B cell epitope polypeptide are in specific binding. Through experimental verification, sibling viruses of bluetongue viruses, such as Zhongshan viruses CHUV, deer epidemic hemorrhagic fever viruses EHDV-1 and EHDV-6, Guangxi orbiviruses GXOV and the like do not generate a reaction. Therefore, during detection and identification of bluetongue virus infection, the monoclonal antibody is high in degree of reliability, and has good directive significance for prevention and treatment of epidemic diseases. Besides, the invention provides a C-ELISA reagent kit for specificity detection of bluetongue virus NS2 protein, prepared by the monoclonal antibody. Because the monoclonal antibody is adopted, antigenic protein containing specific B cell epitope can be subjected to specificity distinguishing highly. Therefore, the testing results are reliable and accurate, and the method is simple to operate. Epidemic disease prevention andtreatment are facilitated.

Description

technical field [0001] The present invention relates to the field of basic research and prevention of bluetongue disease, in particular to a hybridoma cell line BTV-2A4 and its secreted monoclonal antibody, and the BTV-NS2 protein B cell epitope polypeptide recognized by the monoclonal antibody The present invention also relates to a C-ELISA kit for diagnosing 1-24 types of bluetongue virus NS2 antibody detection, and the hybridoma cell line, monoclonal antibody and B cell epitope polypeptide in the detection and prevention of bluetongue virus on the application. Background technique [0002] Bluetongue is an insect-borne animal disease caused by bluetongue virus (BTV). The virus mainly infects ruminants such as cattle, sheep, and deer. The onset features are: fever, oral congestion and ulcers, tongue congestion and cyanosis, hoof inflammation, which can cause lameness, deformity of newborn animals, abortion, and death. Bluetongue is classified as a category A infectious ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10C07K14/14G01N33/577G01N33/569
CPCC07K16/10C07K14/005G01N33/577G01N33/56983C12N2720/12122G01N2333/14G01N2469/10
Inventor 段莹亮李占鸿李乐李华春
Owner YUNNAN ANIMAL SCI & VETERINARY INST
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