Puccinia triticina f.sp.tritic monoclonal antibody and preparation method and application thereof

A monoclonal antibody, technology of wheat leaf rust, applied in biochemical equipment and methods, anti-fungal/algae/lichen immunoglobulin, instruments, etc., can solve the problem of the preparation method of monoclonal antibody of wheat leaf rust, etc. question

Active Publication Date: 2010-05-19
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As our partner, the Danish Academy of Agricultural Sciences has made some progress in the research of monoclonal antibodies against wheat stripe rust (Skottrup et al., 2007), but it has not yet developed a method for preparing monoclonal antibodies against wheat stripe rust. report

Method used

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  • Puccinia triticina f.sp.tritic monoclonal antibody and preparation method and application thereof
  • Puccinia triticina f.sp.tritic monoclonal antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the propagation of wheat leaf rust

[0033] According to the current prevalence of wheat rust in China, the present invention selects the main popular races PHT and THT of wheat leaf rust as experimental materials, and conducts identification and mass propagation in the high-temperature chamber of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0034]Specific steps are as follows:

[0035] 1. Plant Zhengmai 5389, a susceptible variety of wheat leaf rust, and inoculate with PHT and THT when the second new leaf grows.

[0036] 2. Spray the wheat leaves with a small watering can filled with clean water before inoculation. After the hands of the operator are disinfected with alcohol, dip in clean water and gently wipe off the wax paper layer on the leaves. On the leaves, wipe back and forth several times to evenly spread.

[0037] 3. After wiping the rust, the hands of the experimenter are first rinsed with 70% alcohol, and the...

Embodiment 2

[0042] Embodiment 2: wheat leaf rust fungus immune mice

[0043] 1. Collect freshly multiplied spores of two kinds of wheat leaf rust fungus, count them under a microscope, weigh 0.15 mg each of wheat leaf rust fungus PHT and THT uredospores (5×10 5 spores / ml), add 675 μl normal saline and 675 μl Freund’s complete adjuvant (US sigma company, product number F5881), put it into the syringe, connect the two syringes, push back and forth to mix well to obtain the emulsified antigen.

[0044] 2. Take female Balb / C mice aged 4-8 weeks, and take 200 μl of emulsified antigen to immunize by subcutaneous multi-point injection. After 3 weeks, the uredia spore suspension was mixed and emulsified with Freund's incomplete adjuvant, and after 3 weeks, the Freund's complete adjuvant (product number F5506) was used instead of Freund's complete adjuvant and the bacterial solution was mixed and emulsified according to Immunization was carried out with the same dose and method, and subsequent im...

Embodiment 3

[0052] Example 3: Screening of wheat leaf rust hybridoma cell line and preparation of monoclonal antibody

[0053] Take the mouse with higher serum titer in Example 2, boost immunization with 150 μl of the uredospore suspension in Example 2, take the mouse spleen and carry out cell fusion with myeloma cells after 5 days, and screen by "two-step screening method" Positive clones, at the same time, for the cells detected in the positive wells of specific antibodies, they were transplanted and cloned in time. The "limiting dilution method" is used for cloning of hybridoma cells. The cell suspension is serially diluted to statistically contain only a single cell in each well, and then inoculated into a culture plate, so that a single cell can be propagated to form a homogeneous cell clone. , when there are multiple positive cells, try to take single clone wells for re-cloning, transfer them to 24-well plates, and then transfer them to culture flasks for expanded culture until the ...

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Abstract

The invention relates to a puccinia triticina f.sp.tritic monoclonal antibody and a preparation method and application thereof, which belong to the field of biotechnology. The preparation method for the puccinia triticina f.sp.tritic monoclonal antibody comprises three steps of immunizing animals with an antigen, preparing hybridoma through cell fusion, and excreting the monoclonal antibody from the hybridoma. The preparation method is characterized in that the antigen is a urediospore of puccinia triticina f.sp.tritic. The puccinia triticina f.sp.tritic monoclonal antibody is used for detecting the puccinia triticina f.sp.tritic and has the advantages of good specificity, high sensitivity, and simple, convenient and quick operation.

Description

technical field [0001] The invention relates to biotechnology, in particular to a monoclonal antibody against wheat leaf rust and its preparation method and application. Background technique [0002] Wheat rust includes pathogenic bacteria including wheat stripe rust (Puccinia striiformis f.sp.tritici), wheat leaf rust (Puccinia triticina f.sp.tritic) and wheat stem rust (Puccicinia graminis f.sp.tritici), is An important disease of a class of worldwide cereal crops. Wheat rust has the characteristics of wide occurrence area, strong outbreak, high epidemic frequency, and heavy damage and loss, which seriously threatens the production safety of wheat in China. In China, wheat stripe rust occurs most widely and is the primary factor affecting the production safety of wheat in China. The disease mainly occurs in relevant provinces (municipalities, autonomous regions) such as the northwest, southwest, middle and lower reaches of the Yangtze River, and North China. The annual oc...

Claims

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Application Information

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IPC IPC(8): C07K16/14G01N33/577C12N5/20
Inventor 高利陈万权刘太国
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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