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Chicken Nrf2 protein antibody and immunogen thereof, immunogenic polypeptide, detection kit and application

A technology of immunogenic polypeptide and detection kit, applied in the field of virus immunology

Inactive Publication Date: 2019-05-21
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Nrf2-related research focuses on humans and mammals, and research on poultry including chicken Nrf2 is basically blank
There is no antibody that recognizes chicken Nrf2 protein and suitable reagents can detect the expression and distribution of chicken Nrf2 protein

Method used

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  • Chicken Nrf2 protein antibody and immunogen thereof, immunogenic polypeptide, detection kit and application
  • Chicken Nrf2 protein antibody and immunogen thereof, immunogenic polypeptide, detection kit and application
  • Chicken Nrf2 protein antibody and immunogen thereof, immunogenic polypeptide, detection kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Immunogens used to obtain antibodies to chicken Nrf2 protein.

[0039] The immunogen described in this example is prepared by the following method:

[0040] 1) According to the chicken Nrf2 protein sequence information (the sequence number in GenBank is BAA08364.1), comprehensively apply bioinformatics analysis methods to predict and screen peptides with good antigenicity, high solubility and easy synthesis. Finally, the polypeptide from amino acid 569 to amino acid 582 is selected as the immunogen, and the amino acid sequence is shown in SEQ ID NO:1. The polypeptide is at the C-terminus of chicken Nrf2 protein, and its amino acid sequence is: IFLVPKSRKAETKL.

[0041] 2) chemically synthesize the above polypeptide, use 4-(N-maleimidomethyl)cyclohexane-1-carboxylate sulfosuccinimide sodium salt as a coupling agent, mix the above 10mg polypeptide with 25mg Hemocyanin is coupled at room temperature, and then the free uncoupled polypeptide is removed by dialysi...

Embodiment 2

[0042] Example 2. Preparation method of chicken Nrf2 protein antibody.

[0043] The chicken Nrf2 protein antibody described in this example is prepared by mixing and emulsifying the immunogen KLH569 prepared in Example 1 with an adjuvant, immunizing rabbits, and taking the blood of the immunized rabbits after purification. The preparation method is as follows:

[0044] 1) Preparation of the immunogen: The polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 was coupled with a carrier protein through a coupling agent to obtain the chicken Nrf2 immunogen KLH569, see the preparation method described in Example 1.

[0045] 2) Immunization: Mix and emulsify the chicken Nrf2 immunogen obtained in step 1) with an adjuvant, and inoculate rabbits for immunization; details are as follows: mix 0.4 mg KLH569 with 0.1 mg muramyl dipeptide adjuvant, add PBS to volume to 1 mL. Then mix and emulsify with an equal volume of Freund's complete adjuvant (1mL), and subcutaneously immun...

Embodiment 3

[0050] Example 3. Detection of antibody quality.

[0051] The purpose of this example is to detect the quality of the chicken Nrf2 protein antibody obtained in Example 2. The specific methods and steps are as follows.

[0052] 1) Antigen coating:

[0053] Using glutaraldehyde as a coupling agent, 10 mg of the polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 was coupled with 20 mg of bovine serum albumin by shaking at room temperature as the detection antigen. Dissolve the detection antigen with 50mM carbonate coating buffer (pH 9.6), adjust the antigen concentration to 4μg / mL, add 100μL / well to each well of a 96-well microtiter plate, and place it overnight at 4°C.

[0054] 2) closed:

[0055] The next day, the coating solution was discarded, washed 3 times with PBS, 150 μL of 1% BSA solution was added to each well, and blocked for 1 hour at 37°C.

[0056] 3) Antigen-antibody binding:

[0057] Discard the blocking solution, wash with PBS for 3 times, add 100 ...

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Abstract

The invention discloses a chicken Nrf2 protein antibody and immunogen thereof, immunogenic polypeptide, a detection kit and application, and belongs to the technical field of viral immunology. Aimingat the situation that in the prior art, an antibody for effectively detecting the expression condition of chicken Nrf2 protein and a related detection kit are in lack, the immunogenic polypeptide is provided, the amino acid sequence of the immunogenic polypeptide is shown as SEQ ID NO:1. The immunogenic polypeptide is coupled with carrier protein through a coupling agent, the immunogen is obtained, the immunogen and an adjuvant are mixed to immunize an inoculated rabbit, the blood of the immunize rabbit is taken to be purified, the chicken Nrf2 protein antibody is prepared, and the ELISA detection kit for detecting the chicken Nrf2 protein is provided. The detection kit can be used for monitoring the expression and distribution of the chicken Nrf2 protein in cells.

Description

technical field [0001] The invention belongs to the technical field of virus immunology, and in particular relates to a chicken Nrf2 protein antibody and its immunogen, immunogenic polypeptide, detection kit and application. Background technique [0002] Intracellular redox balance has important and complex effects on the health of the organism. On the one hand, energy metabolism generates a large number of active oxygen free radicals, and the accumulation of these active oxygen free radicals in the body seriously damages cells, which is the pathophysiological basis of many diseases such as aging, allergic reactions, diabetes, liver disease, and cardiovascular disease. On the other hand, when pathogenic microorganisms infect the body, the cells will trigger an appropriate level of oxidative stress response, and the reactive oxygen species produced can not only directly kill the pathogen, but also indirectly induce and regulate the body's innate immune response system. In or...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K16/18C07K16/06C07K1/22G01N33/68G01N33/543
Inventor 李海刘胜旺
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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