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Plant photosystem II polyclonal antibody and preparation method thereof

A polyclonal antibody and photosystem technology, which is applied in the fields of peptide preparation methods, plant peptides, chemical instruments and methods, etc., can solve the problems that hinder the in-depth research of photosystem II, inaccurate determination results, and lack of specificity and sensitivity of antibodies. Strong and other issues

Pending Publication Date: 2021-08-06
北京启维益成科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When conducting research on photosystem II proteins, it is often necessary to determine the type and content of the extracted photosystem protein subunits. The traditional method of determination is to use a single protein extraction and measurement method, which is time-consuming and laborious, and the measurement results are not accurate. The method of protein detection using antigen antibody is a relatively accurate method, but corresponding standard antibodies need to be prepared. At present, there are very few reports on antibodies for photosystem II protein detection, and the most common ones are subunit protein whole protein immune preparation The specificity and sensitivity of these antibodies are not strong, there are too many cross-reactions, the detection results are unreliable, and there is no antibody for the detection of all types of photosystem II proteins, which hinders the in-depth study of photosystem II Research

Method used

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  • Plant photosystem II polyclonal antibody and preparation method thereof
  • Plant photosystem II polyclonal antibody and preparation method thereof
  • Plant photosystem II polyclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Antigen epitope design

[0042] Using antheprot software, according to the Arabidopsis photosystem II protein sequence published by www.arabidopsis.org (ATCG00020 / PsbA, ATCG00680 / PsbB, ATCG00280 / PsbC, ATCG00270 / PsbD, ATCG00580 / PsbE, ATCG00570 / PsbF, ATCG00430 / PsbG, 1ATCG00430 / PsbG, / PsbH、 ATCG00080 / PsbI、 ATCG00550 / PsbJ、ATCG00070 / PsbK、ATCG00560 / PsbL、ATCG00700 / PsbN、AT5G66570 / PsbO、AT1G06680 / PsbP、AT4G21280 / PsbQ、AT1G79040 / PsbR、AT1G44575 / PsbS、ATCG00690 / PsbT、AT2G30570 / PsbW , AT2G06520 / PsbX, AT1G67740 / PsbY, ATCG00300 / PsbZ) PsbA protein, PsbB protein, PsbC protein, PsbD protein, PsbE protein, PsbF protein, PsbG protein, PsbH protein, PsbI protein, PsbJ protein, PsbK protein , PsbL protein, PsbN protein, PsbO protein, PsbP protein, PsbQ protein, PsbR protein, PsbS protein, PsbT protein, PsbW protein, PsbX protein, PsbY protein, and PsbZ protein for sequence analysis, including transmembrane differentiation and signal peptide analysis , modification site analysis a...

Embodiment 2

[0047] Example 2: Preparation of polyclonal antibody

[0048] Animals for Immunization Select healthy male New Zealand white rabbits aged 3 months, weighing 2.5±0.1 kg, and immunize them with the antigen prepared in Example 1.

[0049] Before the first immunization, blood was taken from the ear vein as a negative control. The antigen was washed with PBS (137mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 2mM KH 2 PO 4 , pH7.4) diluted to 1mg / mL, aliquoted and stored at -20°C. Take 500 μL of 1 mg / mL antigen (0.5 mg), add 300 μL PBS to dilute again, and then add an equal volume of Freund’s complete adjuvant (first immunization) or Freund’s incomplete adjuvant (2nd to 4th immunization). Multi-point injections were performed subcutaneously on the limbs, underarms and back of rabbits, and the second immunization was performed three weeks after the first immunization, and then boosted every two weeks (a total of 4 immunizations). Seven days after the third immunization, the ear Take b...

Embodiment 3

[0052] Embodiment 3: western blot detection antibody

[0053] Extraction method of total protein from Arabidopsis:

[0054] 1. Take about 50 mg of tissue and put it in a centrifuge tube, and freeze it in liquid nitrogen;

[0055] 2. Grind the sample to powder;

[0056] 3. Add protein extraction solution equivalent to 2 times the volume of the sample (for example: 100 μl extraction solution / 50 mg tissue), and mix well;

[0057] 4. Denature samples in boiling water, 5 min;

[0058] 5. Centrifuge at the maximum speed for 10 minutes to separate the supernatant and the precipitate; Note: If the protein is soluble or has obvious dissolution, add homogenate at a ratio of 1:1 (for example: 50 μl homogenate / 50 mg tissue), and then Centrifuge at maximum speed for 10 min at 4°C.

[0059] 6. Estimate the protein concentration in the supernatant using the Bradford test using BSA as a standard; Note: The Bradford test cannot detect protein solutions containing SDS.

[0060] 7. Use SDS ...

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Abstract

The invention discloses a plant photosystem II polyclonal antibody and a preparation method thereof. Epitopes of related proteins in the photosystem II and synthesized corresponding KLH coupling polypeptides are designed, the series of the coupling polypeptides are adopted as an immunogen to immunize animals to obtain a corresponding antibody, and a detection reagent for detecting the related proteins in the plant photosystem II by a western blot method realizes full detection, accurate quantitative detection of the related proteins in the photosystem II and in-depth research on the photosystem II is facilitated.

Description

technical field [0001] The invention belongs to the technical field of antibody preparation for detection, and in particular relates to a plant photosystem II polyclonal antibody and a preparation method thereof. Background technique [0002] The photosystem is the functional unit for light absorption, which is a complex composed of chlorophyll, carotenoids, lipids and proteins. Including photosystem I (PSI) and photosystem II (PSII), each photosystem contains two main components: light-harvesting complex and photoreaction center complex. These two photosystems work together in series. The photochemical reaction of PSI is a long light wave reaction, the main feature of which is NADP + restoration. The photochemical reaction of PSII is a short-wave reaction, and its main feature is the photolysis and oxygen release of water, which captures electrons in water and supplies PSI. [0003] The function of PSII is to use the energy absorbed from light to split water and transfe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K16/16C07K16/06C07K1/36C07K1/30C07K1/22G01N33/68
CPCC07K14/415C07K16/16C07K16/065G01N33/68G01N2333/415
Inventor 赵运明刘桂山
Owner 北京启维益成科技有限公司
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