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Molecular design phycocyanin beta subunit fluorescent protein combining phycocyanobilin and application thereof

A technology of phycocyanin and phycocyanin, which is applied in the field of pigment protein materials in biotechnology, can solve the problems of high background, complicated technical procedures, and difficult quantitative determination of fluorescent immunoassays, and achieve good sensitivity and high fluorescence efficiency , easy to purify the 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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  • Molecular design phycocyanin beta subunit fluorescent protein combining phycocyanobilin and application thereof
  • Molecular design phycocyanin beta subunit fluorescent protein combining phycocyanobilin and application thereof
  • Molecular design phycocyanin beta subunit fluorescent protein combining phycocyanobilin and application thereof

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Effect test

Embodiment 1

[0057] 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 201 (corresponding to position 153 of the original phycocyanin beta subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 596nm, and the fluorescence emission peak is 626nm.

Embodiment 2

[0059] 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 201 (corresponding to position 153 of the original phycocyanin beta subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 596nm, and the fluorescence emission peak is 626nm.

Embodiment 3

[0061] 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 329 (corresponding to position 153 of the original phycocyanin beta subunit) through a thioether bond. Its spectrum is as Figure 4 As shown, the absorption peak is 596nm, and the fluorescence emission peak is 626nm.

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Abstract

The invention relates to a molecular design phycocyanin beta subunit fluorescent protein combining phycocyanobilin, fusion protein formed by the phycocyanin 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 phycocyanin beta subunit can be combined with the phycocyanobilin (PCB), and is prepared into beta subunit of fluorescent phycocyanin by escherichia coli of genetic engineering. The spectrum property of the phycocyanin is completely different from that of the natural phycocyanin, 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 phycocyanin beta subunit fluorescent protein combined with phycocyanin and its application, belonging to the field of pigment protein materials in biotechnology, in particular to phycocyanin beta subunit combined with phycocyanin PCB 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 each subun...

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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