Application of truncated fragment of porcine nlrp3 as antigenic structural protein

A protein and monoclonal antibody technology, applied in anti-animal/human immunoglobulin, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems that do not exist, and achieve stable performance and high antigen titer. Effect

Active Publication Date: 2021-03-30
海南省农业科学院畜牧兽医研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Rapid and accurate diagnosis of pig inflammation can provide a time reference for the correct treatment and prevention of inflammatory diseases caused by viruses and bacteria, colloidal gold test strips for accurate diagnosis of inflammation in pigs, and antigen ELISA kits for checking NLRP3 expression , are inseparable from the NLRP3 monoclonal antibody with stable performance and high antigenic titer. There is no good monoclonal antibody against pigs at home and abroad, so there is no diagnostic reagent for NLRP3.

Method used

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  • Application of truncated fragment of porcine nlrp3 as antigenic structural protein
  • Application of truncated fragment of porcine nlrp3 as antigenic structural protein
  • Application of truncated fragment of porcine nlrp3 as antigenic structural protein

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1, antigen preparation

[0027] In order to study new therapeutic targets for porcine inflammatory diseases, correctly treat, prevent and control inflammatory diseases caused by viruses and bacteria, etc., it is planned to prepare porcine NLRP3 monoclonal antibody. First, a truncated porcine NLRP3 fragment consisting of 288 amino acid residues is selected. The molecular weight is about 50KDa. As the NLRP3 antigen structural protein, the amino acid sequence of its NLRP3 truncated fragment is shown in SEQ ID NO.1, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO.2.

[0028] The nucleotide sequence shown in SEQ ID NO.2 was connected into the expression vector pET-32a through BamH I and Xho I to obtain the recombinant expression vector pET32a-NLRP3. After sequencing and verification, the expression vector with the correct target gene sequence was selected for protein expression. Express.

[0029] The specific expression method is as foll...

Embodiment 2

[0031] Embodiment 2, animal immunization

[0032] Experimental animals and immunization methods: select 5 Balb / C mice aged 5-8 weeks, use NLRP3 recombinant protein prepared in Example 1 as antigen, inject 100 μg antigen / experimental mouse at multiple points on the back, and inject 50 μg antigen for booster immunization For experimental mice, Freund's complete adjuvant was mixed with an equal volume of antigen for the first injection, and Freund's incomplete adjuvant was mixed with an equal volume of antigen for booster injection. The specific immunization time and cycle are shown in Table 1.

[0033] Table 1. Immunization cycle

[0034] Experiment process date primary exemption 2018.07.15 first booster 2018.07.23 second booster 2018.07.31 third booster 2018.08.08 fusion 2018.08.16 filter 2018.08.24 sub-screening 2018.09.01 Secondary screening 2018.09.09 ascites 2018.09.24

[0035] Antiserum testing:

[003...

Embodiment 3

[0043] Embodiment 3, cell fusion and subcloning

[0044] (1) Preparation of myeloma cells: One week before fusion, SP2 / 0 cells were revived and cultured to logarithmic phase normally.

[0045] (2) Spleen cell preparation: the mice to be fused were selected, sacrificed by cervical dislocation on the day of fusion, the spleen was taken, and the splenocytes were collected and counted according to the standard procedure.

[0046] (3) Cell fusion: mix myeloma cells and spleen cells at a ratio of 1:3-1:10, perform cell fusion operation according to the standard procedure, and then culture with HAT DMEM complete medium. Hybridomas can be seen 3 days after fusion Cells were replaced with 1 / 2HAT complete medium on the 7th day, and 1 / 2HT medium on the 8th day, and the screening test was started about 10 days after fusion.

[0047] Results of cell fusion: after fusion, cultured with HAT selective medium, observed under a microscope, many growing hybridoma cells were seen, proving that t...

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Abstract

The invention discloses a porcine NLRP3 antigenic structural protein, a porcine NLRP3 monoclonal antibody and its preparation method and application. The porcine NLRP3 antigenic structural protein has good immunogenicity, and is used as an antigen to immunize mice, and is screened by IFA after cell fusion Hybridoma cells were screened to obtain 7 cell lines secreting NLRP3 monoclonal antibodies, and the monoclonal antibodies were purified to obtain porcine NLRP3 monoclonal antibodies; among them, the 9H5 hybridoma cell line NLRP3 had the best stability, and the monoclonal antibody secreted by it was more effective. It has the highest price and the strongest specificity, and its biological deposit number is CCTCC NO: C201973. The present invention further provides the hybridoma cell line porcine NLRP3 and the application of the secreted monoclonal antibody in detection or purification of NLRP3 protein.

Description

technical field [0001] The invention relates to the antigen-antibody field of immunology, in particular to the application of porcine NLRP3 truncated fragments as antigenic structural proteins, and also to porcine NLRP3 monoclonal antibodies, and the preparation method and application of the monoclonal antibodies. Background technique [0002] NLRP3, also known as inflammasome, is a cytokine produced by white blood cells that mediates intercellular interactions and plays a regulatory role in cell activation, proliferation, and differentiation. As one of the main constituent proteins of NLRP3 inflammasome, NLRP3 protein plays an important role in the body's defense against pathogenic microorganisms and inflammatory responses. It provides basic data for in-depth exploration of the mechanism of inflammasome in porcine inflammatory diseases. Inflammasome is a multi-protein complex assembled by innate immune recognition receptors in the cytoplasm, which is crucial for the occurre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C07K16/18C12N5/20C12N15/70G01N33/577G01N33/68
CPCC07K14/47C07K16/18C12N15/70G01N33/577G01N33/68
Inventor 张艳刘海隆王文秀曹宗喜谭树义黄丽丽
Owner 海南省农业科学院畜牧兽医研究所
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