Fusion protein 3AN for capturing PRRSV nucleocapsid protein antibody and application thereof

A technology of nucleocapsid protein and fusion protein, which is applied in the direction of immunoglobulin, antiviral immunoglobulin, positive-sense single-stranded RNA virus, etc., and can solve the problems of detection of N protein antibody obstruction and lack of N protein antibody, etc.

Active Publication Date: 2021-08-17
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Competitive ELISA is an effective method to detect N protein antibody of PRRSV, but currently there is a lack of antigens that c

Method used

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  • Fusion protein 3AN for capturing PRRSV nucleocapsid protein antibody and application thereof
  • Fusion protein 3AN for capturing PRRSV nucleocapsid protein antibody and application thereof
  • Fusion protein 3AN for capturing PRRSV nucleocapsid protein antibody and application thereof

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preparation example Construction

[0038]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.

[0039] In the present...

Embodiment 1

[0063] Preparation and Identification of Single B Cell Antibody to PRRSVN Protein from Porcine

[0064] 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, c...

Embodiment 2

[0068] Expression and purification of PRRSV nucleocapsid protein fused with foot-and-mouth disease virus 3A protein epitope

[0069]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 (EDTA10mmoXXl / L, Tris-HCl 20mmol / L, pH 7.5, TritonX-1001%) to resuspend the precipitate, sonicate until the bacteria are completely lysed, and collect the supernatant by centrifugation. Add 50% High Affinity Ni-Charged ResinFF 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 0.5 time...

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Abstract

The invention provides a fusion protein 3AN for capturing a PRRSV nucleocapsid protein antibody and application thereof, and belongs to the technical field of virus detection. The invention provides a fusion protein 3AN for capturing a porcine reproductive and respiratory syndrome virus nucleocapsid protein antibody. The amino acid sequence of the fusion protein 3AN is shown as SEQ ID NO: 3. Tests show that a 3A epitope monoclonal antibody 3A24 of FMDV serves as a coating antibody, a coating ELISA plate is formed after 3AN fusion protein is captured, a C8 monoclonal antibody serves as a detection antibody, a porcine serum PRRSV antibody is detected in a competitive ELISA reaction mode, 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 fusion protein 3AN for capturing PRRSV nucleocapsid protein antibody and its application. 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 epidem...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/10G01N33/577G01N33/569G01N33/58G01N33/543
CPCC07K14/005C07K16/10C07K16/1009G01N33/577G01N33/56983G01N33/58G01N33/54306C12N2770/10022C12N2770/32122C07K2319/21C07K2317/56G01N2469/20G01N2333/08G01N2333/09
Inventor 张婧卢曾军李坤孙普王健张海霞白兴文付元芳曹轶梅刘在新
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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