Plant light system I polyclonal antibody and preparation method thereof

A polyclonal antibody and photosystem technology, applied in plant peptides, chemical instruments and methods, anti-plant immunoglobulin, etc., can solve problems such as time-consuming and laborious, hindering in-depth research of photosystem I, and many cross-reactions

Active Publication Date: 2021-02-26
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 photosystem I protein research, 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 I protein detection. 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 I proteins, which hinders the in-depth study of photosystem I Research

Method used

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  • Plant light system I polyclonal antibody and preparation method thereof
  • Plant light system I polyclonal antibody and preparation method thereof
  • Plant light system I polyclonal antibody and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Antigen epitope design

[0045]Using antheprot software, according to the Arabidopsis photosystem I protein sequence (ATCG00350 / PsaA, ATCG00340 / PsaB, ATCG01060 / PsaC, AT1G03130 / PsaD, AT4G28750 / PsaD, AT1G31330 / PsaF, AT1G55670 / PsaG, AT3G1614 published by www.arabidopsis.org / PsaH, ATCG00630 / PsaJ, AT1G30380 / PsaK, AT4G12800 / PsaL, AT5G64040 / PsaN, AT1G08380 / PsaO) for photosystem I PsaA protein, PsaB protein, PsaC protein, PsaD protein, PsaE protein, PsaF protein, PsaG protein, PsaH protein, PsaJ protein, PsaK protein, PsaL protein, PsaN protein, and PsaO protein for sequence analysis, including transmembrane distinction, signal peptide analysis, modification site analysis, and full sequence analysis to find out possible epitopes, and Cysteine ​​is added to the end of the sequence that does not contain cysteine ​​in the above antigen epitopes, such as PsaA, PsaD, PsaE, PsaG, PsaJ, and PsaO.

[0046] Finally select the epitope as described in Table 1:

[0047] Table...

Embodiment 2

[0050] Example 2: Preparation of polyclonal antibody

[0051] 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.

[0052] 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

[0055] Embodiment 3: ELISA serum titer determination

[0056] Experimental reagents: 0.01M PBS (pH 7.4); coating solution (0.1mol / L Na 2 CO 3 -NaHCO 3 buffer, pH9.6), adjust the pH value with concentrated HCl; Tween-20; enzyme-labeled secondary antibody (HRP-GAR IgG); absolute ethanol; 0.03%H 2 o 2 ;2mol / LH 2 SO 4 solution; acetate buffer (pH 5.8-6.0); antigen (coated polypeptide).

[0057] Steps:

[0058] Coating antigen: Dilute the antigen to 5 µg / ml with ELISA coating solution (i.e. 1ml ELISA coating solution + 5ul 1mg / ml polypeptide mix well), add 100µl to each well, incubate overnight at 4°C or in a 37°C incubator for 2hr ;

[0059] Washing solution: wash wells with 0.01 mol / L PBS-0.05% Tween-20 solution 3 times on a plate washer, and pat dry;

[0060] Sealed microtiter plate: Add 200µl of 3% skimmed milk / 0.01M PBS to each well, block at 37°C for 1hr; (3% skimmed milk / 0.01MPBS: 1.5g skimmed milk powder + 50ml 0.01M PBS fully dissolved - two plates);

[0061] Was...

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Abstract

The invention discloses a plant light system I polyclonal antibody and a preparation method thereof. The antigenic epitope of the light system I related protein and the synthesized corresponding KLH coupled polypeptide are designed, the series of coupled polypeptides serve as immunogens to immunize animals to obtain corresponding antibodies, the antibodies are used as detection reagents for detecting the plant light system I related protein through an ELISA method, full detection and accurate quantitative detection of the light system I related protein are achieved, and deep research on the optical system I 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 I 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] PSI is composed of a series of proteins required for light energy absorption, transmissio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C07K16/16G01N33/68G01N33/58
CPCC07K14/415C07K16/16G01N33/581G01N33/6854G01N2333/415
Inventor 赵运明刘桂山
Owner 北京启维益成科技有限公司
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