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

A technology of phycoerythrin and phycoerythrin, which is applied to the phycoerythrin β subunit fluorescent protein combined with phycoerythrin PEB and its application field, can solve the problem of high background, complex technical procedures, fluorescent immunology Technical quantitative determination is difficult and other problems, to achieve the effect of good sensitivity, easy purification, high fluorescence efficiency

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

[0057] The amino acid sequence of the protein is shown in Sequence 1. The phycoerythrin beta subunit encoding gene is cloned into an expression plasmid, and the phycoerythrin beta subunit is expressed, and its N-terminus has a His-tag mark, which is not only beneficial to its Purification also helps to increase its solubility. Phycoerythrin is bound to the cysteine ​​residue at position 130 (equivalent to position 82 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 546nm, and the fluorescence emission peak is 566nm.

Embodiment 2

[0059] The amino acid sequence of the protein is shown in Sequence 2. The gene encoding the phycoerythrin beta subunit is cloned into an expression plasmid, and mutated by genetic engineering methods to obtain a phycoerythrin beta subunit mutant with an N-terminal band There is a His-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 phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 546nm, and the fluorescence emission peak is 566nm.

Embodiment 3

[0061] The amino acid sequence of the protein is shown in Sequence 3. The streptavidin encoding gene and the phycoerythrin beta subunit encoding gene were spliced ​​and cloned into the expression plasmid, and the streptavidin and phycoerythrin beta subunit were expressed. The fusion protein of the phycoerythrin beta subunit can be directly marked by streptavidin; and the N-terminal has a His-tag tag, which not only facilitates its purification, but also helps to improve its solubility. Phycoerythrobilin is bound to the cysteine ​​residue at position 258 (equivalent to position 82 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as Figure 4 As shown, the absorption peak is 546nm, and the fluorescence emission peak is 566nm.

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Abstract

The invention relates to phycocyanin and phycoerythrin beta subunits fluorescent protein combined with phycoerythrobilin PEB, fusion protein formed by phycocyanin and phycoerythrin 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 and phycoerythrin beta subunit conserved cysteine residue can be not only combined with phycocyanobilin PCB by a thioether bond, but the fact that the phycocyanin and phycoerythrin 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 and phycoerythrin, the spectroscopy of the protein is completely different from that of the phycocyanin and phycoerythrin 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 phycoerythrocyanin beta subunit-like fluorescent protein combined with phycoerythrin PEB and its application, belonging to the field of pigment protein materials in biotechnology, in particular to a phycoerythrocyanin beta subunit combined with phycoerythrin PEB The basic fluorescent protein, its fusion protein with streptavidin and its mutant, and a detection method for detecting soluble antigen or antibody by 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 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 ea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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