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Phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof

A technology of phycocyanin and phycoerythrin, applied in application, algae/moss peptides, hybrid peptides, etc., can solve the problems of complicated technical procedures, high background, difficult quantitative determination by fluorescence immunology technology, etc., and achieve fluorescence efficiency. High, easy to purify, good sensitivity effect

Inactive Publication Date: 2010-06-30
GUANGZHOU TEBSUN BIO TECH DEV +1
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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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  • Phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
  • Phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
  • Phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof

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Experimental program
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Effect test

Embodiment 1

[0056] The amino acid sequence of the protein is shown in Sequence 1. The gene encoding the phycocyanin beta subunit is cloned into an expression plasmid, and the phycocyanin beta subunit is expressed, and its N-terminal has a His-tag mark, which is not only conducive to its purification , also helps to improve its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 130 (equivalent to position 82 of the original phycocyanin beta subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 556nm, and the fluorescence emission peak is 572nm.

Embodiment 2

[0058] The amino acid sequence of the protein is shown in Sequence 2. The gene encoding the phycocyanin beta subunit is cloned into an expression plasmid, and mutated by genetic engineering methods to obtain a phycocyanin beta subunit mutant with His at its N-terminus. -tag mark, which not only facilitates its purification, but also helps to improve its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 130 (equivalent to position 82 of the original phycocyanin beta subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 556nm, and the fluorescence emission peak is 572nm.

Embodiment 3

[0060] The amino acid sequence of the protein is shown in Sequence 3. The streptavidin coding gene and the phycocyanin beta subunit coding gene are spliced ​​and cloned into the expression plasmid, and the fusion of streptavidin and phycocyanin beta subunit is expressed protein, directly achieve streptavidin labeling of phycocyanin beta subunit; and the N-terminal is marked with His-tag, which not only facilitates its purification, but also helps to improve its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 258 (equivalent to position 82 of the original phycocyanin beta subunit) through a thioether bond. Its spectrum is as Figure 4 As shown, the absorption peak is 556nm, and the fluorescence emission peak is 572nm.

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Abstract

The invention relates to phycocyanin beta subunits fluorescent protein combined with phycoerythrobilin PEB, fusion protein formed by phycocyanin beta subunits fluorescent protein and streptavidin and a mutant thereof, the sequences include sequence 1, sequence 2, sequence 3 and sequence 4; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; phycocyanin beta subunit conserved cysteine residue can be not only combined with phycocyanobilin PCB by a thioether bond, but the fact that the phycocyanin beta subunit conserved cysteine residue can be combined with the phycoerythrobilin PEB by the thioether bond through the genetic engineering can be realized, so as to obtain the novel fluorescent phycocyanin, the spectroscopy of the protein is completely different from that of the phycocyanin beta subunits fluorescent protein combined with PCB, and the protein has high fluorescence efficiency; in addition, as the protein carries His-tag label, purification is not only convenient, but also the dissolubility of the protein can be improved; the protein is combined with the streptavidin to form the fusion protein for fluorescent immunological detection directly, thereby being beneficial to the application of the protein in all kinds of the field.

Description

technical field [0001] The invention relates to a phycocyanin beta subunit fluorescent protein combined with phycoerythrin PEB and its application, belonging to the field of pigment protein materials in biotechnology, in particular to phycocyanin beta subunits combined with phycoerythrin PEB Fluorescent protein, its fusion protein with streptavidin and its mutant, and a detection method using the fusion protein to detect soluble antigen or antibody. 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 for short), phycoerythrocyanin (PEC for short), phycocyanin (CPC for short) and variable phycocyanin. (allophycocyanin, referred to as APC). CPE, PEC, CPC, and APC contain alpha and beta subunits, and in each subunit, phycobilin (phycobilin) ​...

Claims

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Application Information

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IPC IPC(8): C07K14/405C07K19/00C12N15/31C12N15/62C12N15/63G01N33/52G01N33/543
Inventor 赵开弘周明佟顺刚夏坤
Owner GUANGZHOU TEBSUN BIO TECH DEV
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