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Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycoerythrobilin and application thereof

A technology of phycoerythrocyanin and phycoerythrobilin, which is applied in the fields of application, algae/bryopeptides, hybrid peptides, etc., can solve the problems of complex technical procedures, high background, and difficult quantitative determination of fluorescence immunoassays, and achieve fluorescence High efficiency, good sensitivity, and easy purification effect

Inactive Publication Date: 2010-06-30
GUANGZHOU TEBSUN BIO TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages are: the problem of non-specific staining has not been completely solved, and the technical procedures are still relatively complicated
At the same time, due to the high background in general fluorescence measurement, it is difficult to use fluorescence immunoassay for quantitative determination.

Method used

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  • Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycoerythrobilin and application thereof
  • Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycoerythrobilin and application thereof
  • Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycoerythrobilin and application thereof

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Experimental program
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Embodiment 1

[0056] The amino acid sequence of the protein is shown in Sequence 1. The gene encoding the beta subunit of phycoerythrin was cloned into the expression plasmid, and the beta subunit of phycoerythrin was obtained by expression. Purification also helps to improve its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 201 (equivalent to position 153 of the beta subunit of original phycoerythrin) through a thioether bond. Its spectrum is figure 2 As shown, the absorption peak is at 547 nm and the fluorescence emission peak is at 566 nm.

Embodiment 2

[0058] The amino acid sequence of the protein is shown in Sequence 2. The phycoerythrin beta subunit encoding gene is cloned into the expression plasmid, and it is mutated by genetic engineering methods to express the phycoerythrin beta subunit mutant, whose N-terminal band is Has a His-tag, which not only facilitates its purification, but also helps to improve its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 201 (equivalent to position 153 of the beta subunit of original phycoerythrin) through a thioether bond. Its spectrum is image 3 As shown, the absorption peak is at 547 nm and the fluorescence emission peak is at 566 nm.

Embodiment 3

[0060] The amino acid sequence of the protein is shown in sequence 3. The gene encoding streptavidin and the beta subunit of phycoerythrin are spliced ​​and cloned into the expression plasmid, and the obtained streptavidin and beta subunit of phycoerythrin are expressed. The fusion protein of phycoerythrin directly realizes the labeling of the beta subunit of phycoerythrin by streptavidin; and the N-terminus is tagged with His-tag, which is not only conducive to its purification, but also helps to improve its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 329 (equivalent to position 153 of the beta subunit of primitive phycoerythrin) through a thioether bond. Its spectrum is Figure 4 As shown, the absorption peak is at 547 nm and the fluorescence emission peak is at 566 nm.

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Abstract

The invention relates to a molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycoerythrobilin, fusion protein formed by the phycoerythrocyanin beta subunit fluorescent protein and streptavidin, and mutant thereof, having the sequences 1, 2, 3 and 4. The invention also discloses a method for directly using the fluorescent protein fused with the streptavidin for fluorescence immunoassay; and due to thioether bond, 153th cysteine residue conserved by phycoerythrocyanin beta subunit not only can be combined with phycocyanobilin (PCB), but also can be combined with the phycoerythrobilin (PEB) through genetic engineering, so that a novel fluorescent phycoerythrocyanin can be obtained. The spectrum property of the phycoerythrocyanin is completely different from that of the phycoerythrocyanin beta subunit fluorescent protein combined with the PCB, has higher fluorescence efficiency, and is convenient for purification due to being provided with His-tag label. Furthermore, the dissolubility can be improved, the fusion protein can be formed by the phycoerythrocyanin beta subunit fluorescent protein and the streptavidin, the fluorescent protein can be directly used for fluorescence immunoassay, and the invention is beneficial to the application in various fields.

Description

technical field [0001] The invention relates to a molecularly designed phycoerythrin beta subunit-like fluorescent protein combined with phycoerythrin and application thereof, belonging to the field of pigment protein materials in biotechnology, and in particular to phycoerythrin combined with phycoerythrin PEB Beta subunit fluorescent protein, its fusion protein with streptavidin and its mutants, and a detection method for detecting soluble antigen or antibody using the fusion protein. Background technique [0002] Phycobiliproteins are functional components of photosynthetic light-harvesting complexes in cyanobacteria and red algae. According to their absorption spectrum and fluorescence spectrum characteristics, phycobiliproteins can be divided into phycoerythrin (CPE), phycoerythrocyanin (PEC), phycocyanin (CPC) and metaphycocyanin. (allophycocyanin, referred to as APC). CPE, PEC, CPC and APC contain alpha and beta subunits, in each subunit phycobilin is linked to the ...

Claims

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Application Information

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IPC IPC(8): C07K14/405C07K19/00C12N15/31C12N15/62C12N15/63G01N33/52G01N33/543
Inventor 夏坤佟顺刚
Owner GUANGZHOU TEBSUN BIO TECH DEV
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