Photobacterium leiognathi luciferase, gene coding photobacterium leiognathi luciferase, and applications of photobacterium leiognathi luciferase

A technology of luciferase gene and photobacillus sp. is applied in application, genetic engineering, plant genetic improvement and other directions, which can solve the problems of high cost and less preparation of enzyme solution.

Active Publication Date: 2015-08-19
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the dual-enzyme system of Photobacterium pachyrhiza is used for NADH d...

Method used

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  • Photobacterium leiognathi luciferase, gene coding photobacterium leiognathi luciferase, and applications of photobacterium leiognathi luciferase
  • Photobacterium leiognathi luciferase, gene coding photobacterium leiognathi luciferase, and applications of photobacterium leiognathi luciferase
  • Photobacterium leiognathi luciferase, gene coding photobacterium leiognathi luciferase, and applications of photobacterium leiognathi luciferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Cloning and expression of luciferase gene LuxAB in Escherichia coli RosettaDE3

[0022] PCR-amplified the LuxAB gene encoding luciferase in Photobacterium chinensis, and digested with restriction endonucleases BamHI and XhoI, and then recovered using an agarose gel DNA recovery kit (BPI); The final plasmid pET-28a(+) was subjected to a ligation reaction, and then the ligated product was introduced into the expression host Escherichia coli RosettaDE3. After the sequencing was correct, the expression was induced with 0.5mMIPTG for 10h, and purified by Ni Sepharose TM 6Fast Flow filler Obtain active recombinant luciferase protein.

Embodiment 2

[0023] Example 2 Detection and verification of FMN:NADH activity in Escherichia coli RosettaDE3

[0024] The crude enzyme solution extracted from Escherichia coli RosettaDE3 by sonication and centrifugation, 1ml of the enzyme solution was taken, and the substrates FMN-Na 0.5μL (10mM) and NADH 300μL (0.14mM) were quickly added at one time, and the absorbance value was measured. The absorbance value is measured by scanning with a UV fluorescence spectrophotometer. The detection conditions: set the wavelength to 340nm. After adding the solution or reagent, perform a time scan. The decrease in the absorbance value within 60s can reflect the activity of FMN:NADH oxidoreductase.

[0025] FMN:NADH oxidoreductase activity assay results in Escherichia coli rosettaDE3 are shown in Table 1.

[0026] Table 1

[0027]

[0028] In Table 1, the FMN reductase activity of Escherichia coli without the luciferase gene was almost equal to that of the recombinant Escherichia coli with the luci...

Embodiment 3

[0030] Example 3 Preparation of recombinant Escherichia coli RosettaDE3 in vitro dual enzyme crude enzyme preparation

[0031] At 24°C, 150r / min, induce 10h of recombinant Escherichia coli RosettaDE3300mL, collect the bacteria by centrifugation, and reconstitute the bacteria with 0.01mol / L PBS (pH 7.0, containing 10mmol / L EDTA, 1mmol / L DTT, the same below). Dissolving, ultrasonic crushing in an ice bath, ultrasonic time 15s, interval 15s, crushing 60 times, power 300W; 4°C refrigerated centrifugation (9000r / min, 30min), supernatant was salted out with 40% to 80% solid ammonium sulfate, centrifuged , the precipitate was redissolved in 30 mL of PBS, and dialyzed in PBS at 4°C for 24 hours to obtain the crude enzyme solution for the experiment.

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Abstract

The present invention relates to a photobacterium leiognathi luciferase, a gene coding the photobacterium leiognathi luciferase, and applications of the photobacterium leiognathi luciferase, and belongs to the technical field of biology. The present invention provides a photobacterium leiognathi luciferase gene, which can achieve efficient expression in escherichia coli and can be used for quantitative NADH detection so as to provide possibility for replacement of the luminescent bacteria double enzyme system detection. According to the present invention, with the introduction of the photobacterium leiognathi luciferase gene LuxAB, the conditions are optimized to make the exogenous luciferase protein achieve the efficient expression in the escherichia coli, the escherichia coli RosettaDE3 contains high activity FMN-NADH oxidoreductase so as to achieve the in vitro luminescence of the recombinant escherichia coli double enzyme body, and the obtained product is used for NADH detection.

Description

technical field [0001] The invention relates to a photobacterium luciferase luciferase, a gene encoding the enzyme and an application thereof, belonging to the field of biotechnology. Background technique [0002] Luminescent bacteria are a class of non-pathogenic bacteria that can emit visible light under normal conditions. Its luminescent mechanism belongs to the enzymatic oxidation reaction under the joint participation of luciferase and oxidoreductase. FMN-NADH oxidoreductase is specific for NADH and reduces FMN to FMNH in the presence of NADH 2 , providing a substrate for the luminescent enzymatic reaction, luciferase catalyzes FMNH 2 And long-chain fatty aldehydes (RCHO) with more than eight carbons undergo oxidation-reduction reactions, and at the same time release blue-green light with a maximum luminous intensity at a wavelength of 450-490nm. [0003] The luminescence intensity of bacterial luciferase: FMN-NADH oxidoreductase in vitro luminescent dual enzyme syst...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N1/21C12Q1/66C12R1/19
Inventor 王静雪林洪玄冠华
Owner OCEAN UNIV OF CHINA
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