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Green fluorescent cyanobacterium phytochrome fluorescence indicator preparation method

A phytochrome, cyanobacteria technology, applied in bacterial peptides, chemical instruments and methods, antibody mimics/scaffolds, etc., can solve the problems of unstable expression, difficult to obtain qualified products with stable quality, etc. Strain stability and screening efficiency, the effect of reducing the number of genes

Inactive Publication Date: 2018-06-12
GUANGZHOU TEBSUN BIO TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the expression of fusion proteins is often limited by the design of the fusion gene and the fermentation process, and cannot be expressed stably, making it difficult to obtain qualified products with stable quality

Method used

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  • Green fluorescent cyanobacterium phytochrome fluorescence indicator preparation method
  • Green fluorescent cyanobacterium phytochrome fluorescence indicator preparation method
  • Green fluorescent cyanobacterium phytochrome fluorescence indicator preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Sequence Selection of Cyanobacterial Phytochrome GAF Domain

[0034] The cyanobacterial phytochrome gene sequence all3691 (algae species PCC7120) has two GAF domains with high homology. The GAF domain gene sequence selected in this program is the second domain gene sequence, hereinafter referred to as the gaf gene sequence. The amino acid sequence of the expressed protein is as follows:

[0035] ILFNVVNQMRQSLDLNAIFCVVTQNIRRILDVDRVGIYQFHLDVNYEYGEFVAEDVSPAFPSALAVKVQDHCFGENYANLYKQGRICAITDVQSSEILDCHRQILAQFHVRASLVVPIMQEEELWGLLCIHQCDRPRQWEPLEMQFAQQVGAQMGIALKQTDLLIQTQK (SEQ ID NO: 1).

[0036] Selection of transgenic plasmid combinations

[0037] This protocol uses the Duet series double plasmid combination, the sa::gaf fusion gene is inserted into the multiple cloning site 1 of pET Duet; the phycoerythrin synthase plasmid uses pACYC Duet-ho1-pebS.

[0038] The fusion design of streptavidin sa sequence, gaf sequence and 6his-taq affinity tag sequence was carried out, and th...

Embodiment 2

[0073] Get the monoclonal bacterial classification that the fermentation of embodiment 1 uses, carry out fermentation as follows.

[0074] 1) Preparation of primary seed solution: Take 20 μL of qualified monoclonal bacterial seed solution, insert it into 25 mL of LB medium, shake overnight at 37°C to a saturated concentration;

[0075] 2) Prepare the secondary seed solution: transfer 5mL of the primary seed solution into 1.5L LB medium, culture with medium-speed shaking at 37°C for 3-4h, OD 600 Control below 0.5;

[0076] 3) Inoculation: transfer 1.5L secondary seed solution to a 200L fermenter, medium 150L, ​​fermentation medium formula: peptone 1%, yeast powder 0.5%, sodium chloride 1%, glycerin 0.4%, blood red 0.004% element, the pH value was adjusted to 7.2 with NaOH solution;

[0077] 4) Cultivation: 37°C, 300rpm medium speed cultivation to the early growth stage, the duration is about 1h;

[0078] 5) Cool down: set the tank temperature to 20°C, reduce the rotation spe...

Embodiment 3

[0082] Get the monoclonal bacterial classification that the fermentation of embodiment 1 uses, carry out fermentation as follows.

[0083] 1) Preparation of primary seed solution: Take 20 μL of qualified monoclonal bacterial seed solution, insert it into 25 mL of LB medium, shake overnight at 37°C to a saturated concentration;

[0084] 2) Prepare the secondary seed solution: transfer 5mL of the primary seed solution into 1.5L LB medium, culture with medium-speed shaking at 37°C for 3-4h, OD 600 Control below 0.5;

[0085] 3) Inoculation: transfer 1.5L of secondary seed solution to a 200L fermenter, 150L of culture medium, fermentation medium formula: 1% peptone, 0.5% yeast powder, 1% sodium chloride, 0.4% glycerin, pH value NaOH solution adjusted to 7.2;

[0086] 4) Cultivation: 37°C, 300rpm medium speed cultivation to the early growth stage, the duration is about 1h;

[0087] 5) Cool down: set the tank temperature to 20°C, reduce the rotation speed to 150rpm, and culture a...

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Abstract

The invention discloses a green fluorescent cyanobacterium phytochrome fluorescence indicator preparation method. A fusion protein comprises a second GAF structural domain of cyanobacterium phytochrome protein all3691 and streptavidin. A sequence of the fusion protein can be easily and stably expressed in microorganisms, and defects of difficulty in acquisition of high-purity natural phycobiliprotein and cyanobacterium phytochrome, high preparation cost, chemical modifier consumption, polymerization morphological instability of natural phycobiliprotein and the like are avoided. The expressionmethod is free of phycobiliprotein lyase participation, transformational gene amount is decreased, and strain stability and screening efficiency are improved. In addition, by optimization of fermentation media and fermentation conditions, fusion protein yield is greatly increased.

Description

technical field [0001] The invention relates to the field of fusion protein expression, in particular to a method for preparing a phytochrome fluorescent marker. Background technique [0002] Fluorescent probes (or fluorescent markers) are important basic raw materials for fluorescent immunoassay technology, which are mainly obtained by combining the fluorescent substrate with the biotin-streptavidin system through chemical modification, which can further amplify the fluorescent signal and improve Sensitivity of immunoassay. [0003] Natural phycobiliprotein is one of the most common fluorescent substrates. Its natural structure is generally hexamer, and its subunits are composed of apoprotein and phycobilichrome, which are catalyzed by lyase. Phycobilichromes are formed from heme catalyzed by heme oxidase (HO1) and various biliverdin reductases. Common phycobilichromes include phycoerythrin (PEB), phycourobilin (PUB), Cholin (PCB) and Phycopurinoid (PVB). By combining di...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/70C12N15/62
CPCC07K14/195C07K2319/00C07K2319/21C12N15/62C12N15/70
Inventor 王小明宋建勋夏坤付卫雷佟顺刚
Owner GUANGZHOU TEBSUN BIO TECH DEV
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