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Phycocyanin alpha subunits fluorescent protein combined with phycocyanobilin PCB and application thereof

A technology of phycocyanin and phycocyanin, which is applied in the fields of application, algae/bryopeptide, hybrid peptide, etc., can solve the problems of complicated technical procedures, high background, and difficult quantitative determination of fluorescence immunoassay, and achieve fluorescence efficiency High, good sensitivity, easy to purify the effect

Inactive Publication Date: 2010-06-30
GUANGZHOU TEBSUN BIO TECH DEV +1
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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 alpha subunits fluorescent protein combined with phycocyanobilin PCB and application thereof
  • Phycocyanin alpha subunits fluorescent protein combined with phycocyanobilin PCB and application thereof
  • Phycocyanin alpha subunits fluorescent protein combined with phycocyanobilin PCB and application thereof

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Experimental program
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Effect test

Embodiment 1

[0041] The amino acid sequence of the protein is shown in Sequence 1. The gene encoding the phycocyanin alpha subunit is cloned into an expression plasmid, and the phycocyanin alpha subunit is expressed, and its N-terminus is marked with a His-tag, which is not only conducive to its purification , also helps to improve its solubility. Phycocyanin is bound to the cysteine ​​residue at position 133 (equivalent to position 85 of the original phycocyanin alpha subunit) through a thioether bond. Its spectrum is as figure 2 As shown, the absorption peak is 619nm, and the fluorescence emission peak is 641nm.

Embodiment 2

[0043] The amino acid sequence of the protein is shown in Sequence 2. The streptavidin coding gene and the phycocyanin alpha subunit coding gene are spliced ​​and cloned into the expression plasmid, and the fusion of streptavidin and phycocyanin alpha subunit is expressed protein, directly achieve streptavidin labeling of the phycocyanin alpha subunit; 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 261 (equivalent to position 85 of the original phycocyanin alpha subunit) through a thioether bond. Its spectrum is as image 3 As shown, the absorption peak is 619nm, and the fluorescence emission peak is 641nm.

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 alpha subunits fluorescent protein combined with phycocyanobilin PCB and fusion protein formed by phycocyanin alpha subunits fluorescent protein and streptavidin, the sequences include sequence 1 and sequence 2; furthermore, the invention discloses a method of the fluorescent protein fused with streptavidin for fluorescent immunological detection directly; phycocyanin alpha subunit conserved cysteine residue can be combined with phycocyanobilin PCB by a thioether bond, and the alpha subunit of the fluorescent phycocyanin can be obtained by utilizing escherichia coli through genetic engineering, the spectroscopy of the protein is completely different from that of natural phycocyanin; 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 present invention relates to a phycocyanin alpha subunit fluorescent protein combined with phycocyanin PCB and its application, belonging to the field of pigment protein materials in biotechnology, in particular to phycocyanin alpha subunit combined with phycocyanin PCB A basic fluorescent protein and its fusion protein formed with streptavidin, 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, phycobilin...

Claims

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Application Information

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IPC IPC(8): C07K14/405C07K19/00C12N15/31C12N15/62C12N15/63G01N33/52G01N33/543
Inventor 赵开弘周明佟顺刚夏坤
Owner GUANGZHOU TEBSUN BIO TECH DEV
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