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Firefly luciferase and encoding gene and obtaining method thereof

A technology of luciferase and firefly, which is applied in the field of encoding genes and the acquisition of the enzyme, can solve the problems of not being able to meet high temperature applications and poor thermal stability

Active Publication Date: 2014-12-10
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The first technical problem to be solved in the present invention is to overcome the poor thermal stability of the original North American firefly luciferase, which cannot meet the defects of high temperature application, and to mutate the original North American firefly luciferase to obtain a mutant enzyme with significantly improved thermal stability , to increase its application value

Method used

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  • Firefly luciferase and encoding gene and obtaining method thereof
  • Firefly luciferase and encoding gene and obtaining method thereof
  • Firefly luciferase and encoding gene and obtaining method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1 computer-aided design of highly stable mutation site

[0055] Download the coordinate file 1BA3 of luciferase in PDB format from the PDB database (http: / / www.rcsb.org / pdb / home / home.do), and use DeepView 4.1 software to process the 1BA3 file: complete missing residues and atoms to remove ligand molecules. Perform molecular dynamics simulation on the processed files. All simulations are carried out under the conditions of constant temperature (300K), constant pressure (atmospheric pressure), and force field using Gromos 96.1 (53A6), and the final simulation time is 10ns. Select the last 2ns (9-10ns) of the protein molecular dynamics trajectory, and calculate the RMSF (Root Mean Square Fluctuation) value of each amino acid of the protein. This value reflects the conformational status of the amino acid in the molecular dynamics simulation trajectory, and the higher the value, the more unstable the conformation. Through analysis, the region where the active si...

Embodiment 2

[0056] Embodiment 2 contains the construction of firefly luciferase gene expression vector

[0057] 1. Primer Design

[0058] With the nucleotide sequence of the plasmid pGL2-control vector (purchased from Promega) containing the North American firefly (P.Pyralis) luciferase gene, combine the multiple cloning site of the plasmid pET28a (purchased from Novagen) with the restriction of the Luc gene Design primers for endonuclease sites, as follows:

[0059] Primer luc+:5-CGGGATCCATGGAAGACGCCAAAAAC-3

[0060] Primer luc-:5-CCCAAGCTTTTACAATTTGGACTTTCCGC-3

[0061] Among them, CG in the upstream primer is the protective base, GGATCC in the upstream primer is the restriction site of BamHI, CCC in the downstream primer is the protective base, and AAGCTT is the Hind III endonuclease site. After the properties and parameters of the primers were qualified by oligo6 software, Beijing Aoke Dingsheng Biotechnology Co., Ltd. was entrusted to synthesize the primers.

[0062] 2. PCR ampli...

Embodiment 3Q

[0070] Example 3 Quick Change method site-directed mutagenesis firefly luciferase gene (Luc)

[0071] Rapid site-directed mutagenesis is to directly introduce mutations to amino acids in specific double-stranded DNA by PCR. It is completed in one step and does not require subcloning. This is currently the simplest site-directed mutagenesis method. The principle is: a circular plasmid is used as a template, and a pair of primers containing mutation points are used to amplify. The product is a complete unmethylated plasmid. Because the template plasmid is a methylated plasmid extracted from E. coli, it can The DpnⅠ enzyme digestion method was used to remove the template, and only the newly amplified mutant plasmid remained in the final product. The product could be directly transformed into competent cells of Escherichia coli, and positive clones were selected for sequencing verification. The process is as Figure 4 shown.

[0072] 1. Primer Design

[0073] The mutant primers o...

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Abstract

The invention relates to firefly luciferase and an encoding gene and an obtaining method thereof. The defect that the original firefly luciferase in North America is poor in heat stability and cannot be used at a high temperature is overcome; and the original firefly luciferase in North America is mutated, so that mutative enzyme of which the heat stability is obviously improved is obtained; and the application value is improved. An amino acid sequence is SEQ ID NO.1; and a protein deoxyribonucleic acid (DNA) sequence in encoding is SEQ ID NO.2. An encoding gene and a preparation method of firefly luciferase mutant in North America are provided. By the method, a recombinant expression vector containing a wild firefly luciferase gene is built; and the protein is purified by using an affinity chromatography method. The method is simple and feasible, simple to operate, and low in cost.

Description

technical field [0001] The invention relates to molecular enzymology and biotechnology, more specifically to a coding gene of firefly luciferase with high thermal stability and high activity and a method for obtaining the enzyme. Background technique [0002] In the presence of magnesium ions, ATP, and molecular oxygen, firefly luciferase catalyzes the oxidation of the substrate luciferin and emits fluorescence simultaneously. This reaction is extremely efficient. At present, firefly luciferase has appeared on the market as a new tool in the research field. It has many advantages such as non-radioactive, sensitive response, accurate results, wide application range, simple operation, and no toxic side effects, so it is very suitable for molecular biology. Science, life science, medicine, criminal investigation, pharmacology, microbial detection and other fields. For example, in medical treatment, it can be used to study cancer metastasis and detect the efficacy of anti-canc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12R1/19
Inventor 黄鹤于浩然王玥肖天雄
Owner TIANJIN UNIV
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