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Preparation method of phytochrome-derived yellow fluorescent label

A phytochrome and streptavidin technology, which is applied in the field of preparation of phytochrome fluorescent markers, can solve problems such as unstable expression and difficulty in obtaining qualified products with stable quality, so as to increase production and improve strain stability and screening efficiency, the effect of reducing the number of genes

Inactive Publication Date: 2018-06-15
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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  • Preparation method of phytochrome-derived yellow fluorescent label
  • Preparation method of phytochrome-derived yellow fluorescent label
  • Preparation method of phytochrome-derived yellow fluorescent label

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Sequence Selection of Cyanobacterial Phytochrome GAF Domain

[0034] The cyanobacterial phytochrome gene sequence slr (algae species PCC6803) has three GAF domains with high homology. The GAF domain gene sequence selected in this program is the third domain gene sequence, which is hereinafter referred to as the gaf gene sequence. The amino acid sequence of the expressed protein is as follows:

[0035] LQNIFRATSDEVRHLLSCDRVLVYRFNPDWSGEFIHESVAQMWEPLKDLQNNFPLWQDTYLQENEGGRYRNHESLAVGDVETAGFTDCHLDNLRRFEIRAFLTVPVFVGEQLWGLLGAYQNGAPRHWQAREIHLLHQIANQLGVAVYQAQLLARFQE (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 the gene...

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 preparation method of a phytochrome-derived yellow fluorescent label. According to the preparation method, a fusion protein comprises the 3th GAG domain of cyanobacteria phytochrome protein slr1393 and streptavidin. Stable expression of the fusion protein sequence can be realized in microorganisms; defects such as acquisition difficulty of high purity natural phycobiliprotein and cyanobacteria phytochrome, high preparation cost, applications of chemical modifying agents, and unstable natural phycobiliprotein polymeric species are avoided. In the expression method, nophycobiliprotein lyase is needed, the number of converted genes is reduced, bacterial strain stability and screening efficiency are increased; and optimization of fermentation culture medium and fermentation conditions is capable of increasing fusion protein yield greatly.

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): C07K19/00C12N15/62C12N15/70
CPCC07K14/195C07K2319/00C07K2319/21C12N15/62C12N15/70
Inventor 吴明卢艳华陈彦蓉夏坤佟顺刚
Owner GUANGZHOU TEBSUN BIO TECH DEV
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