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Allophycocyanin subunits fluorescent protein combined with phycocyanobilin PCB and application thereof

A technology for allophycocyanin and phycocyanin, which is used in the detection field of soluble antigen or antibody detection, and can solve the problems of high background, non-specific staining problems that have not been completely solved, and difficulties in quantitative determination by fluorescent immunological technology.

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 phycocyanobilin PCB and application thereof
  • Allophycocyanin subunits fluorescent protein combined with phycocyanobilin PCB and application thereof
  • Allophycocyanin subunits fluorescent protein combined with phycocyanobilin PCB and application thereof

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Experimental program
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Effect test

Embodiment 1

[0079] 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. Phycocyanin is bound to the cysteine ​​residue at position 129 (equivalent to position 81 of the original allophycocyanin A subunit) through a thioether bond. Its spectrum is as figure 1 As shown, the absorption peak is 618nm, and the fluorescence emission peak is 641nm.

Embodiment 2

[0081] 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. Phycocyanin 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 2As shown, the absorption peak is 622nm, and the fluorescence emission peak is 641nm.

Embodiment 3

[0083] 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. Phycocyanin 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 613nm, and the fluorescence emission peak is 640nm.

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Abstract

The invention relates to allophycocyanin subunits fluorescent protein combined with phycocyanobilin PCB 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 phycocyanobilin PCB 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 phycocyanin PCB, which belongs to the field of pigment protein materials in biotechnology, in particular to an allophycocyanin subunit fluorescent protein combined with phycocyanin 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 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 夏坤佟顺刚
Owner GUANGZHOU TEBSUN BIO TECH DEV
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