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Detection reagent strip utilizing recombinant fluorescent phycobiliprotein subunit, and preparation method thereof

A technology for phycobiliprotein and detection reagents, which can be used in measurement devices, instruments, scientific instruments, etc., and can solve the problems of secondary environmental pollution, inability to recycle and use colloidal gold particles, and environmental pollution.

Inactive Publication Date: 2017-08-18
YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation process of colloidal gold itself is relatively cumbersome, requires a variety of chemical reagents, and has certain pollution to the environment.
At the same time, colloidal gold reagent strips are mainly discarded after use, and the colloidal gold particles in them cannot be recycled at present, causing secondary pollution to the environment

Method used

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  • Detection reagent strip utilizing recombinant fluorescent phycobiliprotein subunit, and preparation method thereof
  • Detection reagent strip utilizing recombinant fluorescent phycobiliprotein subunit, and preparation method thereof
  • Detection reagent strip utilizing recombinant fluorescent phycobiliprotein subunit, and preparation method thereof

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Experimental program
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Effect test

preparation example Construction

[0035] Please refer to figure 2 , a method for preparing a detection reagent strip utilizing recombinant fluorescent phycobiliprotein subunits, comprising the steps of:

[0036] S10. Incubate the prepared mixture of the phycobiliprotein subunit fused with streptavidin and the biotinylated detection antibody to obtain the fluorescent probe, and place it at 4-8° C. for later use.

[0037] The recombinant fluorescent phycobiliprotein subunit is one or more of recombinant phycocyanin, recombinant phycoerythrin and recombinant allophycocyanin subunit. The recombinant fluorescent phycobiliprotein subunit is obtained through the method of recombinant fermentation preparation.

[0038] Specifically, the recombinant fluorescent phycobiliprotein subunit is prepared by the following method:

[0039](1) Using genetic engineering methods, the phycocyanin-like apoprotein subunit gene (cpcA) or its homologous or similar gene and the core streptavidin gene (sa) or its homologous or other t...

Embodiment 1

[0058] Example 1 Preparation of Fluorescent Reagent Strips for Rapid Detection of Vibrio splendidus in Aquaculture

[0059] 1.1 Preparation of fluorescent probe: The prepared phycobiliprotein subunit fused with 2 mg / mL streptavidin and 3 mg / mL biotinylated rabbit anti-Vibrio brilliant antibody mixture were incubated at 20°C for 15 min. Store at 4°C and use within 24h-48h.

[0060] 1.2 Preparation of fluorescent sample pad: Spray the fluorescent probe prepared above evenly on the glass fiber membrane at a rate of 1mL / min, fix at 10°C for 30min, and dry at 20°C for later use.

[0061] 1.3 Preparation of detection protein-binding membrane: After diluting the rabbit-derived Vibrio splendidus antibody with carbonate buffer solution, draw a line at the T line of the detection line at 1 mg / mL with an automatic scribing machine at 1 m / min; The goat anti-rabbit secondary antibody was drawn at line C of the control line according to the above method.

[0062] 1.4 Preparation of the de...

Embodiment 2

[0068] 2.1 Preparation of fluorescent probe: The prepared phycobiliprotein subunit fused with 1 mg / mL streptavidin and 2 mg / mL biotinylated rabbit anti-Vibrio brilliant antibody mixture were incubated at 25°C for 10 min. Store at 8°C and use within 24h-48h.

[0069] 2.2 Preparation of fluorescent sample pad: Spray the fluorescent probe prepared above evenly on the glass fiber membrane at a rate of 1.5mL / min, fix at 15°C for 45min, and dry at 25°C for later use.

[0070] 2.3 Preparation of detection protein-binding membrane: After diluting the prepared rabbit-derived Vibrio resplendent antibody with carbonate buffer, draw a line at the T line of the detection line at 2 mg / mL with an automatic scribing machine at 1.5 m / min; The goat anti-rabbit secondary antibody was drawn on line C of the control line according to the above method.

[0071] 2.4 Preparation of the detection plate: Paste the non-fluorescent filter paper, fluorescent sample pad, detection protein binding membrane...

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Abstract

A detection reagent strip utilizing a recombinant fluorescent phycobiliprotein subunit comprises a non-fluorescence bottom plate, first non-fluorescence filter paper, a detection protein binding membrane, a fluorescent sample pad and second non-fluorescence filter paper, wherein a detection line and a control line are arranged on the detection protein binding membrane; a detection antibody is marked on the detection line; a control antibody is marked on the control line; the fluorescent sample pad comprises a glass fiber membrane and a mixture of a streptavidin blended phycobiliprotein subunit and a biotinylated antibody; the mixture covers the glass fiber membrane. In addition, the invention further provides a preparation method of the detection reagent strip utilizing the recombinant fluorescent phycobiliprotein subunit. The detection reagent strip utilizing the recombinant fluorescent phycobiliprotein subunit can perform qualitative and quantitative detection on antigens or antibodies of diseases such as tumor and microbial infection, and the recombinant fluorescent phycobiliprotein subunit used by the reagent strip is environmentally friendly. The preparation method is low in purification preparation cost and environmentally friendly.

Description

technical field [0001] The invention relates to the technical field of preparation of reagent strips, in particular to a detection reagent strip utilizing recombinant fluorescent phycobiliprotein subunits and a preparation method thereof. Background technique [0002] Phycobiliprotein is a protein derived from algae with specific fluorescent properties. Mainly include phycocyanin, phycoerythrin, allophycocyanin. Natural phycobiliproteins exist in the form of phycobilisomes in algal cells. Many phycobiliprotein subunits with similar molecular weights constitute the supermolecules—phycobilisomes with a weight of up to one million Daltons. It mainly exists on the surface of thylakoids and plays the role of receiving light energy. [0003] Due to the advantages of good solubility, high fluorescence quantum yield, large Stokes shift, and obvious difference from the fluorescence background of biological materials, phycobiliprotein has a good application prospect in fluorescence...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/543G01N33/558
CPCG01N33/533G01N33/543G01N33/558
Inventor 焦绪栋李文军秦松
Owner YANTAI INST OF COASTAL ZONE RES CHINESE ACAD OF SCI
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