Firefly luciferase gene and application thereof

Abstract

The invention discloses a firebug luciferase gene and applications thereof, the gene of the luciferase gene is a DNA molecule shown in sequence 1 of the sequence list. A recombination expression vector can be established by using the firebug luciferase gene, and then is transduced into host cells, thus obtaining the firebug luciferase. Compared with the present production methods of firebug luciferase, the method of the invention is characterized by being easy purified, high yield and purity, and good preserved activity; the invention is a high effective production method of firebug luciferase and suitable for being applied in industrial production.

Description

technical field [0001] The present invention relates to a novel firefly luciferase gene, in particular to the application of the gene in the production of firefly luciferase. Background technique [0002] Firefly luciferase is an efficient biocatalyst that can convert chemical energy into light energy. 2+ and O 2 In the presence of fluorescein, the oxidation reaction is carried out with fluorescein as the substrate. The chemical reaction is as follows: fluorescein + ATP + O 2 = Oxyfluorescein+AMP+PPi+CO 2 + light. The quantum yield of this reaction is about 0.88. In the presence of excess substrate and excess enzyme, the emitted light intensity is proportional to the ATP in the reaction system, so this luminescent property enables firefly luciferase to be widely used in a wide variety of research and production assays for ATP application. [0003] In 1985, Jeffrey et al. constructed the recombinant vector pkw101 containing the luciferase gene. Matuda et al. also const...

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Problems solved by technology

However, in the production of firefly luciferase using the recombinant vector pkw101 constructed by Jeffrey, in the process of bacterial culture, it needs to be treated with heat induction at 45°C for 30 minutes, and then transferred to 37°C for cultivation to produce enzyme, which affects the stability of the enzyme; The recombinant produced by Matuda produces firefly luciferase, which has the disadvantages of cumbersome extraction method and complex process

In 1989, the applicant of the Japanese patent (PCT / JP89 / 00811) obtained the luciferase gene from marine crustaceans and constructed a recombinant DNA, but the same problem still existed during the cultivation of E. coli. In 1993, Jin Zhen Hua et al. constructed a recombinant luciferase DNA with a secretion signal peptide, which can be secreted into the periplasmic space. However, when the bacteria are broken, the purification of luciferase also has separation difficulties, low purity, and low yield. Serious live loss and other problems (patent number: 93117156)

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