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A kind of monoclonal antibody for detecting cabbage catalase and its application

A catalase and monoclonal antibody technology, applied in the field of plant immunology, can solve the problems of inability to cultivate, lack of antibody tools, and low accuracy, and achieve the effect of early prevention and control

Active Publication Date: 2021-03-19
云南省农业科学院园艺作物研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Plasmodium brassicae cannot be cultured on artificially synthesized media at present, so it is difficult to identify and make an early diagnosis of Plasmodium brassicae with traditional research methods
With the rapid development of molecular biology technology, the application of polymerase chain reaction (PCR) amplification technology has become an important means for molecular identification of plant diseases and ecological monitoring, but the accuracy of this method is not high. The primers are targeted, and one primer cannot complete the detection for all the physiological races contained in the clubroot pathogen, and there are certain limitations
[0005] At present, there is no catalase-targeting antibody specifically for Chinese cabbage research on the market. Therefore, at the proteomic level, there is a lack of effective antibody tools for this research field. application in

Method used

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  • A kind of monoclonal antibody for detecting cabbage catalase and its application
  • A kind of monoclonal antibody for detecting cabbage catalase and its application
  • A kind of monoclonal antibody for detecting cabbage catalase and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The preparation of the monoclonal antibody hybridoma cell of embodiment 1 catalase and the preparation and the purification of anti-catalase monoclonal antibody

[0027]The present invention synthesizes three groups of catalase polypeptides, that is, three groups of 'VRPSSAHDSP', 'DSDRQERFVK' and 'GERYRSWDSD', respectively use them as immunogens, immunize mice to obtain lymphocytes, and use hybridoma cell fusion technology to obtain fusion cells , by Western blotting, limiting dilution and ELISA, a cell line capable of secreting high-affinity and high-specificity monoclonal antibodies, and the monoclonal antibodies secreted by the cell line were obtained, and immunoenzyme-linked, protein Imprinting, co-immunoprecipitation plus mass spectrometry and antibody chip detection and other verifications to determine the most effective antibody.

[0028] 1.1 Antigen preparation

[0029] Synthesized three groups of catalase polypeptides: 'VRPSSAHDSP', 'DSDRQERFVK' and 'GERYRSWDS...

Embodiment 2

[0040] Embodiment 2 Verification of anti-CAT1 monoclonal antibody

[0041] The 8 obtained monoclonal antibody cell lines were verified by ELISA, Western blotting, antibody chip, etc. to determine the most effective antibody.

[0042] 2.1 Elisa (immunoenzyme-linked) pairing verification of antibody and antigen polypeptide

[0043] Coat the 96-well ELISA plate with ascitic fluid antibody to be paired, incubate, wash and block overnight with skim milk, wash with PBS, and store at 4°C until use. Antigen peptides were incubated, washed with PBS, and controls were set at the same time. HRP-labeled detection antibody was added to the previously incubated ELISA plate. TMB color reaction, microplate reader reading. The titers of the 8 cell lines obtained by screening are shown in Table 1:

[0044] Table 1 Antibody ELISA detection data

[0045] Antibody name clone number 3.125K 6.25K 12.5K 25K 50K 100K 200K 400K 800K 1600K PC NC Whether to rework poten...

Embodiment 3

[0051] Example 3 Application of a monoclonal antibody for detecting catalase of the present invention in different stages of cabbage infection with clubroot

[0052] Because the antibody of the present invention has unique specificity to catalase, it can be used as a detection antibody for the different infection stages of cabbage infection clubroot, or it can be further developed into a detection kit according to conventional technical means in the art .

[0053] First, the antibody of the present invention was tested with a fixed concentration (1mg / ml) and two varieties of Chinese cabbage (Minihuang No. 1 susceptible to clubroot and Kangda No. 3 resistant to clubroot) at different stages of infection. Dot blot experiment, obtain the corresponding expression value, and draw the standard curve, the abscissa is the time coordinate of cabbage infection clubroot, and the ordinate is the dot blot detection value, as shown in Table 2, image 3 , Figure 4 shown.

[0054] Table 2...

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Abstract

The invention discloses a monoclonal antibody for detecting catalase and an application of the monoclonal antibody. A heavy chain variable region of the monoclonal antibody comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO.2, 4 and 6 respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO.9, 11 and 13 respectively. The monoclonal antibody provided bythe invention can be used for specifically detecting the content of catalase in the early stage of clubroot infection of Chinese cabbages, and can be used for preparing a preparation or a kit for early diagnosis of clubroot infection of cruciferous crops.

Description

technical field [0001] The invention belongs to the technical field of plant immunology, and specifically relates to a monoclonal antibody of catalase and an application thereof. Background technique [0002] Catalase (CAT1) is an enzyme that catalyzes the decomposition of hydrogen peroxide into oxygen and water, and exists in the peroxide body of cells. Catalase is the marker enzyme of peroxisome, accounting for about 40% of the total amount of peroxisome enzymes. Hydrogen peroxide is a metabolic waste product that can cause damage to the body. To avoid this damage, hydrogen peroxide must be quickly converted to other harmless or less toxic substances. Catalase is a tool often used by cells to catalyze the decomposition of hydrogen peroxide. Catalase is normally localized in an organelle called the peroxisome. Peroxisomes in plant cells are involved in photorespiration (utilization of oxygen and production of carbon dioxide) and symbiotic nitrogen fixation (conversion o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40G01N33/573G01N33/577
CPCC07K16/40C07K2317/56C07K2317/565G01N33/573G01N33/577
Inventor 兰梅和江明胡靖锋杨红丽张丽琴徐学忠
Owner 云南省农业科学院园艺作物研究所
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