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

A phycoerythrin and phycocyanin technology, which is applied to a phycoerythrin β subunit-like fluorescent protein combined with phycocyanin PCB and its application field, can solve complex technical procedures, high background, fluorescent immunity technical quantitative determination difficulties, 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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  • Phycocyanin and phycoerythrin beta subunits fluorescent protein combined with phycocyanobilin PCB and application thereof
  • Phycocyanin and phycoerythrin beta subunits fluorescent protein combined with phycocyanobilin PCB and application thereof
  • Phycocyanin and phycoerythrin 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

[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 130 (equivalent to position 82 of the original phycoerythrin beta subunit) through a thioether bond. Its spectrum is as figure 1 As shown, the absorption peak is 602nm, and the fluorescence emission peak is 629nm.

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 130 (equivalent to position 82 of the original phycoerythrin beta subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 602nm, and the fluorescence emission peak is 629nm.

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 258 (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 602nm, and the fluorescence emission peak is 629nm.

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Abstract

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

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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