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Phycocyanin and phycoerythrin alpha subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof

A technology of phycoerythrin and phycoerythrin, which is applied in the field of phycoerythrin α subunit-like fluorescent proteins combined with phycoerythrin PEB, and can solve the problem of high background, complicated technical procedures, fluorescent immunoassays, etc. Technical quantitative determination is difficult and other problems, to achieve good sensitivity, high fluorescence efficiency, easy to purify the effect

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 alpha subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
  • Phycocyanin and phycoerythrin alpha subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof
  • Phycocyanin and phycoerythrin alpha subunits fluorescent protein combined with phycoerythrobilin PEB and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The amino acid sequence of the protein is shown in Sequence 1. The gene encoding phycoerythrocyanin alpha subunit is cloned into an expression plasmid, and the phycoerythrocyanin alpha 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. Phycoerythrin is bound to the cysteine ​​residue at position 132 (equivalent to position 84 of the original phycoerythrocyanin alpha subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 555nm, and the fluorescence emission peak is 570nm.

Embodiment 2

[0043] The amino acid sequence of the protein is shown in Sequence 2. The streptavidin encoding gene and the phycoerythrin alpha subunit encoding gene were spliced ​​and cloned into the expression plasmid, and the streptavidin and phycoerythrin alpha subunit were expressed. The fusion protein of the phycoerythrin alpha 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. Phycoerythrin is bound to the cysteine ​​residue at position 260 (equivalent to position 84 of the alpha subunit of the original phycoerythrocyanin) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 555nm, and the fluorescence emission peak is 570nm.

Embodiment 3

[0045] (1) Dilute the mouse monoclonal antibody to carcinoembryonic antigen CEA to 2 μg / mL with coating buffer (0.05M, pH9.6 carbonate buffer), take 150 μL and add it to a black 96-well microtiter plate, 4 °C Coating for 12 to 20 hours; pour off the antibody used for coating, wash the microtiter plate with washing buffer (pBS buffer containing 0.05% Tween-20, pH 7.2), wash 4 times, and dry the liquid; each well Add 300 μL of blocking buffer (pBS buffer containing 0.05% Tween-20 containing 1% bovine serum albumin) and block for 30 minutes at 37°C; pour off the blocking solution and wash with buffer (containing 0.05% Tween-20) 20 pH7.2 PBS buffer) to wash the microtiter plate, wash 4 times, dry the liquid, and set aside;

[0046] (2) Get carcinoembryonic antigen CEA, dilute to 50ng / mL with dilution buffer (pBS buffer solution containing 0.05% Tween-20 pH7.2 containing 0.1% bovine serum albumin), get 150 μ L and join step (1) In the microwells of the prepared microplate plate, m...

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Abstract

The invention relates to phycocyanin and phycoerythrin alpha subunits fluorescent protein combined with phycoerythrobilin PEB and fusion protein formed by phycocyanin and phycoerythrin alpha 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; phycocyanin and phycoerythrin alpha subunit conserved cysteine residue is combined with phycoviolobilin PVB by a thioether bond, the fact that the phycocyanin and phycoerythrin alpha subunit conserved cysteine residue can be combined with the phycoerythrobilin PEB by the thioether bond through the genetic engineering can be realized, so as to obtain the novel fluorescent phycocyanin and phycoerythrin, the spectroscopy of the protein is completely different from that of the phycocyanin and phycoerythrin alpha subunits fluorescent protein combined with PEB, and the protein has high fluorescence efficiency; 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 phycoerythrocyanin alpha subunit-like fluorescent protein of phycoerythrin PEB and an application thereof, belonging to the field of pigment protein materials in biotechnology, in particular to a phycoerythrocyanin alpha subunit combined with phycoerythrin PEB 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 subunit, ...

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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