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A kind of coding gene and preparation method of firefly luciferase

A luciferase and gene-encoding technology, applied in the field of genetic engineering, can solve problems such as luciferase is expensive and protein production cannot meet actual needs

Active Publication Date: 2017-02-15
BEIJING ZHONGKEZIXIN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Or mutate the wild-type firefly luciferase gene through molecular biology techniques to improve its thermal stability. Although its thermal stability has been improved, its protein production still cannot meet the actual demand
In practical applications, commercialized luciferase is still relatively expensive and most of them are directly extracted from fireflies. For example, the price of firefly luciferase produced by SIGMA-ALDRICH is as high as 2371 yuan / mg

Method used

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  • A kind of coding gene and preparation method of firefly luciferase
  • A kind of coding gene and preparation method of firefly luciferase
  • A kind of coding gene and preparation method of firefly luciferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Highly expressed highly thermostable luciferase encoding gene

[0048] Using the firefly luciferase gene derived from Photinus pyralis as the original gene luc, a high-efficiency expression high thermostable luciferase coding gene was obtained by introducing the substitution of 372 nucleotide sites. The nucleotide sequence of the gene As shown in SEQ ID NO:1. The introduced nucleotide substitution sites are as attached figure 1 shown.

Embodiment 2

[0049] Embodiment 2 Construction of recombinant genetically engineered bacteria capable of efficiently expressing high thermostable luciferase

[0050] ⑴. Introducing restriction endonucleases EcoRI and HindIII enzyme cutting sites and corresponding protective bases respectively upstream and downstream of the nucleotide sequence shown in SEQ ID NO: 1 to obtain the nucleotide sequence shown in SEQ ID NO: 3 Nucleotide sequence, the nucleotide sequence is chemically synthesized and named hluc1;

[0051] (2) Digest the luciferase coding gene hluc1 and pET-28a (+) plasmids with restriction endonucleases EcoRI and HindIII described in step (1) of embodiment 2 with restriction sites and corresponding protective bases, and use DNA condensation Gel recovery kit was used to recover the digested product; the digested luciferase coding gene was ligated with the plasmid pET-28a(+) fragment after the same digestion at 16°C overnight to obtain the recombinant expression vector pET-28a(+)- h...

Embodiment 3

[0053] The preparation of the highly thermostable luciferase of embodiment 3 efficient expression

[0054] ⑴. With the recombinant genetically engineered bacterium constructed according to the method of Example 2 as the production strain, pick the recombinant genetically engineered bacterium E.coli BL21(DE3)-pET-28a(+)-hluc1 single bacterium colony, inoculate in 10mL containing final concentration In a sterilized seed medium containing 50 μg / mL kanamycin, cultivate at 37°C for 16 hours to obtain a seed liquid. The formula of the seed medium is as follows: 0.7 g of peptone, 1 g of yeast powder, 1 g of sodium chloride, 20% per 100 mL (v / v) 5 mL of glycerin solution, the balance being deionized water;

[0055] (2) Inoculate the seed liquid obtained in step (1) of Example 3 into 1000 mL of sterilized fermentation medium containing kanamycin with a final concentration of 50 μg / mL with an inoculation amount of 1% (v / v), cultivate at 37°C for 4 hours, and then add the final The conc...

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Abstract

The invention provides an efficiently-expressed high thermal stability luciferase encoding gene and a preparation method of the firefly luciferase. The gene is obtained through chemical synthesis after firefly luciferase genes derived from Photinuspyralis are subjected to molecular modification. At present, the efficiently-expressed high thermal stability luciferase encoding gene has not been reported, the luciferase expressed by the encoding gene in escherichia coli accounts for 72.1%-83.5% of soluble protein in whole cells, and the expression amount of the luciferase is 481-739 mg / 10 g of wet bacteria. According to the efficiently-expressed high thermal stability luciferase encoding gene, the expression product is a luciferase, the thermal stability of the luciferase is high, the temperature can be preserved for 20 minutes under the condition of 40 DEG C, and the relative activity of about 95% can still be kept.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to an encoding gene of firefly luciferase with highly expressed high thermal stability, and also relates to a preparation method of the firefly luciferase. Background technique [0002] Firefly luciferase (EC 1.13.12.5~7) belongs to the class of oxidoreductases, the most typical of which is North American firefly luciferase (EC 1.13.12.7). North American firefly luciferase consists of a single polypeptide chain, containing 511 amino acid residues, with a relative molecular weight of about 62KD, no cofactor and most of the amino acids are non-polar amino acids. The advanced structure of North American firefly luciferase contains two pairs of disulfide bonds and two tryptophans. Its emission wavelength ranges from 552 to 582nm and its maximum absorption wavelength is 562nm. [0003] Firefly luciferase in adenosine triphosphate (ATP), O 2 and Mg 2+ With the participati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/70C12N1/21C12N9/02C12R1/19
CPCC12N9/0069C12Y113/12007
Inventor 高静蔡亦梅吴超徐潇张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH
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