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Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycocyanobilin (PCB) and application thereof

A phycoerythrin and phycocyanin technology, which is applied to a molecularly designed phycoerythrin β subunit-like fluorescent protein combined with phycocyanin PCB and its application field, can solve complex technical procedures and high background. , the difficulty of quantitative determination by fluorescence immunoassay technology, etc., to achieve the effect of high fluorescence efficiency, easy purification, and good sensitivity

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 phycocyanobilin (PCB) and application thereof
  • Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycocyanobilin (PCB) and application thereof
  • Molecular design phycoerythrocyanin beta subunit fluorescent protein combining phycocyanobilin (PCB) and application thereof

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Experimental program
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Effect test

Embodiment 1

[0055]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. Phycocyanin is bound to the cysteine ​​residue at position 201 (corresponding to position 153 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as figure 1 As shown, the absorption peak is 595nm, and the fluorescence emission peak is 625nm.

Embodiment 2

[0057] 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. Phycocyanin is bound to the cysteine ​​residue at position 201 (corresponding to position 153 of the original phycoerythrocyanin beta subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 595nm, and the fluorescence emission peak is 625nm.

Embodiment 3

[0059] 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. Phycocyanin is bound to the cysteine ​​residue at position 329 (corresponding to position 153 of the original phycoerythrin beta subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 595nm, and the fluorescence emission peak is 625nm.

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Abstract

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

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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