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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However, Nrf2-related research focuses on humans and mammals, and research on poultry including chicken Nrf2 is basically blank

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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.

Application Domain

Serum immunoglobulinsImmunoglobulins against animals/humans +3

Technology Topic

Viral immunologyImmunogenicity +7

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Examples

Experimental program(6)

Example Embodiment

[0038] Example 1. Immunogens for obtaining chicken Nrf2 protein antibodies.

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

[0040] 1) According to the sequence information of chicken Nrf2 protein (the sequence number in GenBank is BAA08364.1), a comprehensive application of bioinformatics analysis method is used to predict and screen the polypeptides with good antigenicity, high solubility and easy synthesis. Finally, the polypeptide from amino acid 569 to amino acid 582 was selected as the immunogen, and the amino acid sequence is shown in SEQ ID NO: 1. This segment of polypeptide is at the C-terminus of chicken Nrf2 protein, amino acid sequence: IFLVPKSRKAETKL.

[0041] 2) Chemically synthesize the above-mentioned polypeptide, use 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt as a coupling agent, combine the above-mentioned 10mg polypeptide with 25mg Yuekong Qi limpet hemocyanin was coupled at room temperature, and then dialyzed with a 10kD dialysis bag in PBS (pH 7.2) to remove free uncoupled polypeptides to obtain the immunogen used to obtain chicken Nrf2 protein antibody, denoted as : KLH569.

Example Embodiment

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

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

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

[0045] 2) Immunization: after the chicken Nrf2 immunogen obtained in step 1) was mixed and emulsified with the adjuvant, the rabbit was inoculated for immunization; the details were as follows: 0.4 mg KLH569 was mixed with 0.1 mg muramyl dipeptide adjuvant, and PBS was added to the volume to 1 mL. Then it was mixed with an equal volume of Freund's complete adjuvant (1mL) to emulsify, and 2-month-old female New Zealand white rabbits were vaccinated subcutaneously at four points on both sides of the groin, under the armpit and four points on the back of the neck. The inoculation dose was 0.1mg immunogen/ Only.

[0046] 3) One month after inoculation, the second immunization is performed with reference to the dose used for the first immunization halved.

[0047] 4) Preparation of crude antibody: One month after the second immunization, PBS was injected intravenously to make up to 0.1 mL of 0.1 mg KLH569 without adjuvant, and thereafter, booster immunization was performed every two weeks, with a total of 5 booster immunizations. 1 week after each booster immunization, 20mL of rabbit blood was taken for centrifugation, and the supernatant was collected to obtain the crude antibody of chicken Nrf2 protein; the booster immunization refers to re-inoculation according to the previous dose, and the dose of the booster immunization in this example is: 0.1 mL KLH569 (containing 0.1 mg KLH569).

[0048] 5) Purified antibody: the polypeptide whose amino acid sequence is shown in SEQ ID NO: 1 is coupled to the pre-activated thiol protein agarose coupling resin in the column to prepare an antigen affinity column, and then the column is equilibrated with PBS buffer; The filtered crude chicken Nrf2 protein antibody was added to the equilibrated antigen affinity column, and then the column was equilibrated with PBS buffer again, and the chicken Nrf2 protein antibody was prepared after elution. Specifically:

[0049] First, the polypeptide with the amino acid sequence shown in SEQ ID NO: 1 was coupled to the pre-activated thiol protein agarose coupling resin in the column to prepare the antigen affinity column; then the filtered crude antibody of chicken Nrf2 protein was added with PBS The buffer-equilibrated antigen affinity column was then equilibrated again with PBS buffer; finally, 100mM glycine solution with pH 2.5 was used as the antibody eluent to elute the antigen affinity column, and the absorbance at 280nm wavelength was more than The eluate of 1.0 was dialyzed with PBS buffer as dialysate to obtain the chicken Nrf2 protein antibody, which is also referred to as chicken Nrf2-569 protein antibody in the present invention.

Example Embodiment

[0050] Example 3. Antibody quality detection.

[0051] The purpose of this example is to detect the quality of the chicken Nrf2 protein antibody obtained in Example 2, and 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 a detection antigen. Dissolve the detection antigen with 50mM carbonate coating buffer (pH 9.6), adjust the antigen concentration to 4 μg/mL, add 100 μL/well to each well of a 96-well microtiter plate, and place at 4°C overnight.

[0054] 2) Closure:

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

[0056] 3) Antigen-antibody binding:

[0057] Discard the blocking solution, wash 3 times with PBS, add 100 μL of purified anti-chicken Nrf2 protein antibody or commercial human and mouse Nrf2 antibody at different dilutions to each well, unimmunized rabbit serum as a negative control, incubate at 37°C 2 hours.

[0058] 4) Incubation with enzyme-labeled secondary antibody:

[0059] After washing 5 times with PBS, tap the ELISA plate several times, add 100 μL of horseradish peroxidase-labeled goat anti-rabbit antibody Fc segment secondary antibody diluted 1:200 with blocking solution, and incubate at 37°C for 1 hour.

[0060] 5) Color rendering:

[0061] After washing 5 times with PBS, tap the ELISA plate several times, add 100 μL of TMB color developing solution, and incubate at 37°C for 20 minutes.

[0062] 6) Reading:

[0063] Add 50 μL of 2M sulfuric acid stop solution to each well, read the absorbance value at 450 nm on the microplate reader after the termination of the reaction, and the detection results are shown in the following table.

[0064] Table 1. ELISA test results of chicken Nrf2-569 antibody

[0065]

[0066] From the test data in Table 1, it can be seen that the antibodies isolated by the method of the present invention have good specificity and sensitivity, and the antibody titers obtained by immunizing this section of polypeptides are all more than 1:512,000 times.

PUM

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