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Phycocyanin beta subunits fluorescent protein combined with phycocyanobilin PCB and application thereof

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

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 phycocyanobilin PCB and application thereof
  • Phycocyanin beta subunits fluorescent protein combined with phycocyanobilin PCB and application thereof
  • Phycocyanin beta subunits fluorescent protein combined with phycocyanobilin PCB 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. Phycocyanin 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 618nm, and the fluorescence emission peak is 644nm.

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. Phycocyanin 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 618nm, and the fluorescence emission peak is 644nm.

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. Phycocyanin 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 618nm, and the fluorescence emission peak is 644nm.

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Abstract

The invention relates to phycocyanin alpha subunits fluorescent protein combined with phycocyanobilin PCB and fusion protein formed by phycocyanin beta subunits fluorescent protein and streptavidin, 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 combined with phycocyanobilin PCB by a thioether bond, and the beta subunit of the fluorescent phycocyanin can be obtained by utilizing escherichia coli through genetic engineering, the spectroscopy of the protein is completely different from that of natural phycocyanin; 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 phycocyanin PCB and its application, belonging to the field of pigment protein materials in biotechnology, in particular to phycocyanin beta subunits combined with phycocyanin PCB 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) ​​is ...

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 赵开弘周明佟顺刚夏坤
Owner GUANGZHOU TEBSUN BIO TECH DEV
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