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Preparation method of orange-fluorescence cyanobacteria phytochrome fluorescence indicator

A phytochrome, cyanobacteria technology, applied in bacterial peptides, chemical instruments and methods, polypeptides containing affinity tags, etc., can solve the problems of unstable expression, difficult to obtain qualified products with stable quality, etc. Improve strain stability and screening efficiency, and avoid the effect of difficulty in obtaining

Inactive Publication Date: 2018-07-06
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 orange-fluorescence cyanobacteria phytochrome fluorescence indicator
  • Preparation method of orange-fluorescence cyanobacteria phytochrome fluorescence indicator
  • Preparation method of orange-fluorescence cyanobacteria phytochrome fluorescence indicator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Sequence Selection of Cyanobacterial Phytochrome GAF Domain

[0034] The cyanobacterial phytochrome gene sequence all2699 (algae species PCC7120) has 3 GAF domains with high homology. The GAF domain gene sequence selected in this program is the first domain gene sequence, hereinafter referred to as the gaf gene sequence. The amino acid sequence of the expressed protein is as follows:

[0035] LELEDIITATTAEVRALLGTDRVMIYKFHPDGSGQVIAESIYENRLPSLLGLNFPADDIPPQARELLVKSKVRSIVDVATGMIGQSPVHDLETGELISEDICYRPVDSCHVEYLTAMGVKSSVVAPIFCQDELWGLLVSHHSENRTVSEDELEAMQMIVDQLAVAIAQSH (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 ...

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 an orange-fluorescence cyanobacteria phytochrome fluorescence indicator. A fusion protein comprises a first structural protein and streptavidin of a GAFstructural domain of a cyanobacteria phytochrome protein all2699. The sequence of the fusion protein of the invention is easily stably expressed in a microorganism, so that shortcomings of the difficulty in obtaining a high-purity natural phycobiliprotein and cyanobacteria phytochrome, relatively high preparation cost, use of a chemical modifier, an instable polymerization shape of the natural phycobiliprotein and the like are avoided. According to an expression method disclosed by the invention, no participation of phycobiliprotein lactase is needed, the number of transformed genes is reduced, and the strain stability and the screening efficiency are improved. Meanwhile, by optimizing a fermentation culture medium and a fermentation condition, the yield of the fusion protein 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): C07K19/00C12N15/62C12N15/70
CPCC07K14/195C07K2319/20C07K2319/21C12N15/62C12N15/70
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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