Method for preparing firefly luciferase

The technology of luciferase and luciferase gene is applied in the field of preparing firefly luciferase, which can solve the problems of high breeding cost, long production cycle, complex enzyme composition and the like, and achieve the effects of easy purification, low cost and simple process

Inactive Publication Date: 2008-11-12
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As the raw material for FL production, the artificial capture or cultivation of fireflies is limited by regions a

Method used

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  • Method for preparing firefly luciferase
  • Method for preparing firefly luciferase
  • Method for preparing firefly luciferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the construction of the recombinant expression vector containing firefly luciferase gene (luc)

[0021] 1. Primer design

[0022] According to the nucleotide sequence of the vector pGEM-luc (purchased from Promega) containing the North American firefly (P. The multiple cloning site of pET28a and the restriction endonuclease site of luc gene were used to design primers. The NdeI restriction site was introduced at the 5' end of the upstream primer, and the XhooI restriction site was introduced at the 5' end of the downstream primer. The primers were synthesized by Shanghai Sangong Company. The specific sequences are as follows:

[0023] Upstream primer luc-R: 5'-GGCCGG CATATG GAAGACGCCAAAAAC-3'

[0024] Downstream primer luc-F: 5'-GGCC CTCGAG TTACAATTTGGACTTTCC-3'

[0025] 2. PCR amplification of luciferase gene (luc)

[0026] Using the vector pGEM-luc (purchased from Promega) containing the luc gene as a template, PCR amplification was carried out, ...

Embodiment 2

[0046] Embodiment 2, induced expression of luciferase

[0047]The recombinant expression vector pET28a-luc of the above-mentioned embodiment 1 is transferred in the competent cell of Escherichia coli BL21 (DE3); Pick a single colony and inoculate it in 2 ml of LB culture fluid containing kanamycin with a final concentration of 50 μg / ml , 37 ° C 200 rpm / min shaking culture overnight. Expand the culture of the bacterial suspension at a volume ratio of 1:100, when the OD of the bacterial liquid 600 When the value reaches 0.8, take 0.5ml of bacterial solution in different concentrations of isopropyl-β-D-thiogalactoside (IPTG) (0.5, 1, 1.5, 2mmol / L), different temperatures (22 ° C, 37 ° C ) and at different times (2, 4, 6, 12 and 18h) to optimize the induction conditions. At the same time, Escherichia coli BL21 (DE3) transfected with pET28a and not induced with IPTG were used as controls.

[0048] Take the Escherichia coli BL21(DE3) transformed into the recombinant expression ve...

Embodiment 3

[0050] Embodiment 3, separation and purification of luciferase

[0051] Since the recombinant expression vector pET28a-luc constructed in the above-mentioned Example 1 has a fusion tag (His·Tag) gene sequence, the expressed target protein also has a His·Tag tag. Therefore, purification of the target protein can be performed by affinity chromatography using Ni-complexed agarose particles (90 μm). Affinity chromatography columns were purchased from GE Healthcare.

[0052] The Escherichia coli BL21(DE3) transformed into the recombinant expression vector pET28a-luc was cultured in LB medium containing kanamycin, and after the expression was induced with IPTG according to the method of the above-mentioned Example 2, it was harvested by centrifugation at 5000rpm / min for 10min Bacteria precipitation; re-dissolve the bacterial precipitation with BindingBuffer (20mM sodium phosphate, 0.5M NaCl, 20mM imidazole, pH7.4) containing a final concentration of 0.2mg / ml lysozyme, and ultrasoni...

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Abstract

The invention discloses a method for making firefly luciferase, comprising the following steps of: inserting the firefly luciferase gene into multiple cloning sites of a prokaryotic expression vector; obtaining a recombinant expression vector containing the firefly luciferase gene; introducing the recombinant expression vector into a colon bacillus cell to obtain a recombinant cell containing the recombinant expression vector; culturing the recombinant cell to obtain the firefly luciferase from the expression. The method for making the firefly luciferase has the advantages of simple process, short growth period, low cost, easy purification, high enzyme activity and stable performance and so on. The method can relieve a situation that the firefly luciferase mainly depends on import, and can promote the development of a fluorescent microorganism rapid detection system and the application of the firefly luciferase in medicine and environment science and so on, thereby providing excellent enzyme preparation for the rapid detection and applications in other aspects of ATP bioluminescence microorganism.

Description

technical field [0001] The invention relates to a method for preparing firefly luciferase. Background technique [0002] Luciferase (luciferase) is a kind of high-efficiency biocatalyst that can convert chemical energy into light energy. o 2 as substrate, in Mg 2+ When it exists, it converts chemical energy into light energy and emits photons. The reaction formula is as follows: [0003] ATP is not only an essential substrate for luciferase to catalyze luminescence, but also an energy source for all biological life activities. In the case that other reaction substrates are present in excess, the value of luciferase-catalyzed emission fluorescence has a linear relationship with the amount of ATP. Taking advantage of this characteristic, McELroy et al. used FL to determine ATP as early as 1947. Since then, luciferase has been continuously developed in the analysis and application of biochemistry and biotechnology. Especially in the aspect of bacterial detection, since t...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70
Inventor 林金明肖勤
Owner TSINGHUA UNIV
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