Firefly luciferase gene and application thereof

A luciferase gene and luciferase technology, applied in the application field of the gene in the production of firefly luciferase, can solve the problems of loss of enzyme activity, affecting enzyme stability, low purity, etc., and achieve good enzyme activity, Fast purification speed and high purity effect

Active Publication Date: 2008-02-20
河南华智营养科技有限公司
View PDF0 Cites 9 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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)

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Firefly luciferase gene and application thereof
  • Firefly luciferase gene and application thereof
  • Firefly luciferase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1. Acquisition of firefly luciferase gene and construction of its recombinant expression vector

[0033] 1. Preparation of firefly luciferase gene

[0034] According to the firefly luciferase gene sequence, without changing its amino acid sequence, the codons were optimized, and the gene was directly synthesized artificially (Aoke Bio Inc.), named mutLuc. The gene was then ligated into pGEM T vector (Promega) and sequenced (Aoke Bio). The sequencing results showed that the obtained nucleotide sequence of the mutLuc gene was sequence 1 in the sequence listing, and its open reading frame (ORF) was deoxyribonucleotides from the 1st to 1671th positions from the 5' end of sequence 1 in the sequence listing, encoding Firefly luciferase of sequence 2 in the sequence listing. The recombinant vector containing mutLuc was named pGEM-mutLuc.

[0035] 2. Construction of firefly luciferase gene recombinant expression vector pQE-mutLuc

[0036] Take 3ug of pGEM-mutLuc pla...

Embodiment 2

[0037] Example 2. Preparation of firefly luciferase and detection of its characteristics

[0038] 1. Obtainment of engineered strains containing firefly luciferase gene and induction of protein expression

[0039] 1) Obtaining engineering strains

[0040] Competent cells of Escherichia coli BL21(DE3) (Tiangen Biotechnology Co., Ltd.) were prepared, the pQE-mutLuc plasmid was transformed into Escherichia coli BL21(DE3), LB medium plates containing 50 mg / L ampicillin were coated, and ampicillin-resistant ( Amp R ) transformant; extract plasmid DNA, and carry out enzyme digestion, sequencing identification, and obtain the recombinant expression genetic engineering strain containing the firefly luciferase gene whose nucleotide sequence is sequence 1 in the sequence table, which is named pQE-mutLuc-E1 .

[0041] 2) Inducible expression of firefly luciferase

[0042] The pQE-mutLuc-E1 strain was inoculated into 20 mL of LB medium, and cultured overnight at 37°C with shaking. Usi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N5/10C12N1/21
Inventor 王磊李成伟高飞
Owner 河南华智营养科技有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap