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Allophycocyanin subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof

A technology of allophycocyanin and phycoerythrin, applied in the field of allophycocyanin subunit fluorescent proteins combined with phycoerythrin PEB and its application, can solve the problem of non-specific staining that has not been completely solved, fluorescence immunology technology Difficult quantitative determination, high background, etc.

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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  • Allophycocyanin subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
  • Allophycocyanin subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
  • Allophycocyanin subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof

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Experimental program
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Effect test

Embodiment 1

[0082] The amino acid sequence of the protein is shown in Sequence 1. The gene encoding the allophycocyanin A subunit is cloned into an expression plasmid, and the allophycocyanin A 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. Phycoerythrobilin is bound to the cysteine ​​residue at position 129 (equivalent to position 81 of subunit A of the original allophycocyanin) through a thioether bond. Its spectrum is as figure 1 As shown, the absorption peak is 551nm, and the fluorescence emission peak is 568nm.

Embodiment 2

[0084] The amino acid sequence of the protein is shown in Sequence 2. The gene encoding the allophycocyanin A2 subunit is cloned into the expression plasmid, and the allophycocyanin A2 subunit is expressed, and its N-terminal 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 129 (equivalent to position 81 of the original allophycocyanin A2 subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 552nm, and the fluorescence emission peak is 568nm.

Embodiment 3

[0086] The amino acid sequence of the protein is shown in Sequence 3. The gene encoding the allophycocyanin B subunit is cloned into an expression plasmid, and the allophycocyanin B 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. Phycoerythrobilin is bound to the cysteine ​​residue at position 130 (equivalent to position 82 of the original allophycocyanin B subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 554nm, and the fluorescence emission peak is 571nm.

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Abstract

The invention relates to allophycocyanin subunits fluorescent protein combined with phycoerythrobilin PEB and fusion protein formed by allophycocyanin subunits fluorescent protein and streptavidin, the sequences are from sequence 1 to sequence 10; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; allophycocyanin subunit conserved cysteine residue is combined with the phycoerythrobilin PEB by a thioether bond, and the subunit of the fluorescent allophycocyanin can be obtained by utilizing escherichia coli through genetic engineering, the spectroscopy of the protein carries His-tag label, therefore, 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 an allophycocyanin subunit fluorescent protein combined with phycoerythrin PEB and its application, belonging to the field of pigment protein materials in biotechnology, and in particular to allophycocyanin subunits combined with phycoerythrin PEB 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...

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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