Porcine source single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit

A nucleocapsid protein and antibody detection technology, applied in immunoglobulin, antiviral immunoglobulin, measuring devices, etc.

Active Publication Date: 2021-09-03
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Antigen-specific single B cells can usually be isolated from natural hosts and non-natural hosts to develop monoclonal antibodies, and monoclonal antibodies produced by isolating antigen-specific single B cells from natural hosts are easier to identify than monoclonal antibodies produced by non-natural hosts The natural antigenic epitopes existing in viral antigens have better application effects. However, even if antigen-specific single B-cell antibodies are isolated from natural hosts, there are differences in the specificity of recognizing PRRSV antigens. Therefore, providing an antigen-specific Sexual single B cell antibodies are essential for the preparation of ELISA detection kits

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  • Porcine source single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit
  • Porcine source single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit
  • Porcine source single B cell antibody of PRRSV nucleocapsid protein and competitive ELISA antibody detection kit

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preparation example Construction

[0033] In the present invention, the preparation method of the monoclonal antibody C8 preferably comprises the following steps: combining the coding sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody C8 with the pig IgG heavy chain and light chain respectively The constant region sequences of the chains were connected and inserted into the pcDNA3.4 eukaryotic expression vector respectively, and the heavy chain recombinant expression plasmid and the light chain recombinant expression plasmid were mixed and transfected into CHO suspension culture cells at a molar ratio of 2:3, and the complete antibody was obtained by expression. Purified by affinity chromatography. The present invention does not specifically limit the recombinant expression plasmid transfection method, expression method and affinity chromatography purification method, and the technical solutions well known in the art can be adopted.

[0034] The present i...

Embodiment 1

[0058] Preparation and Identification of Single B-cell Antibody of PRRSV N Protein from Porcine

[0059] Pigs were immunized with the commercial attenuated PRRS vaccine and the laboratory-made vaccine against the prevailing strain. At the same time, the whole virus antigen was purified, and the virus particles were labeled with biotin; after the eighth immunization, the venous blood of pig 0922# was collected, and the mononuclear cells (PBMCs) in the peripheral blood were separated, and the biotin-labeled PRRSV was used as the capture antigen, and passed Antigen-specific antibody-secreting single B cells were sorted from PBMCs by flow cytometry. Through single B cell antibody gene amplification technology, the heavy chain and light chain variable region (VH and VL) gene sequences of pig IgG antibodies were obtained, and then the variable region gene sequences were inserted into the pcDNA3.4 true region containing the constant region of pig IgG antibodies. In nuclear vectors, ...

Embodiment 2

[0063] Expression and purification of PRRSV nucleocapsid protein fused with foot-and-mouth disease virus 3A protein epitope

[0064] The coding sequence of 3AN protein was cloned into the pET-28a(+) vector by gene synthesis, transformed into BL21 competent cells, and expanded to 500ml after picking a single clone, adding IPTG to a final concentration of 1mM, and inducing expression for 5h. Collect the bacteria by centrifugation, add 50ml ice-bathed IB washing solution (EDTA 10mmoXXl / L, Tris-HCl 20mmol / L, pH 7.5, Triton X-1001%) to resuspend the precipitate, ultrasonically lyse until the bacteria are completely lysed, and collect by centrifugation clear. Add 50% High Affinity Ni-Charged Resin FF at a ratio of 4:1, and incubate at 200 r / min at 4°C overnight to allow complete specific binding of the target protein to Ni-NTA His Bind. Wash with pH 8.0 (NaH 2 PO 4 50mm / L, NaCl 300mM, Urea 8M, imidazole 10mM) wash the column 3 times, each time 2 times the volume, and then use 0....

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Abstract

The invention provides a porcine source single B cell antibody of porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein (N) and a competitive ELISA antibody detection kit, and belongs to the technical field of virus detection. The invention provides a porcine source single B cell antibody C8 of PRRSV protein. Tests show that 3A epitope monoclonal antibody 3A24 of FMDV is taken as a coating antibody, 3AN fusion protein is captured to form a coating ELISA plate, a C8 monoclonal antibody is taken as a detection antibody, a competitive ELISA reaction mode is employed for detecting a porcine serum PRRSV antibody, the prepared PRRSV antibody detection kit has good sensitivity and specificity, and a new tool is provided for PRRSV serum epidemiological investigation.

Description

technical field [0001] The invention belongs to the technical field of virus detection, in particular to a pig-derived single B cell antibody C8 of PRRSV nucleocapsid protein and a competitive ELISA kit. Background technique [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV). It is characterized by reproductive failure, respiratory symptoms in piglets and high mortality in suckling piglets. The disease has caused great economic losses to the global pig industry and is one of the most important diseases affecting the healthy development of the pig industry. PRRSV is a single-stranded positive-sense RNA virus with an envelope, which is very prone to mutation and has many epidemic strains. The use of commercial attenuated vaccines will also lead to the recombination of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/543G01N33/569G01N33/577
CPCC07K16/10G01N33/56983G01N33/543G01N33/577C07K2317/56
Inventor 张婧卢曾军李坤曹轶梅王健魏德陇张海霞孙普付元芳白兴文刘在新
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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