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Luciferase mutant and application thereof

A technology of luciferase and mutants, which is applied in the field of luciferase mutants and its applications, and can solve the problems of poor thermal stability of luciferase and the like

Active Publication Date: 2022-02-08
ZHENGZHOU IMMUNO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These reasons may be responsible for the poor thermal stability of luciferase

Method used

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  • Luciferase mutant and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Luciferase Example 1 embodiment combination optimization program constructs mutant

[0034] Pyralis containing mutation site (P.Pyralis) gene after the codon optimized synthesized from Suzhou Goldvision chi (SEQ ID NO: 3), using Nde I and Xho I digested pET28a vector is connected to a (commercially available from Novagen Corporation ), named luc-mut-pET28a.

[0035] Using universal T7 primer and T7t luc-mut-pET28a sequenced sequenced by Shanghai Sangon Biological Technology. Sequencing results showed that, luc-mut-pET28a inserted gene sequence SEQ ID NO: 3 shown in the same sequence.

Embodiment 2

[0036] EXAMPLE 2 Luciferase embodiment combination optimization expression of a gene mutation

[0037] In Example 1 the luc-mut-pET28a vector into BL21 (DE3) competent cells, an LB liquid medium at 1: 1000 kanamycin screening, single colonies were picked plate medium access 3mlLB placed desktop temperature oscillator 230 rpm, the value of the culture to 0.6-1.0 OD600 of 37 [deg.] C, 1ml of bacterial suspension was transferred to a 1.5ml microcentrifuge tube, add 1μl 1M IPTG, 3-4h induction culture, bacteria taking 20μl SDS-PAGE sample delivery detect the expression condition. Expression screening bacteria inoculated into 20L TB media at 16 deg.] C fermentor culture induced for 16 h, cells were harvested 360g.

Embodiment 3

[0038] Preparing purified muteins luciferase Example 3 Optimization combination thereof

[0039] (1) 200g cells taken from -20 deg.] C, 1:20 calculate the required volume of disrupted bacteria buffer (50mM Tris-HCl + 300Mm NaCl + 20mM imidazole pH7.4) and accurately weighed (e.g., 200g required amount of bacteria 4L50mM Tris-HCl + 300Mm NaCl + 20mM imidazole pH7.4).

[0040] (2) bacteria were resuspended: the cells were thawed on ice, was added in the cold breaking strain buffer (50mM Tris-HCl + 300Mm NaCl + 20mM imidazole pH 7.4), and poured into a 5L beaker, with the remaining buffer centrifuge tube was flushed, and poured into the same beaker, the sample was stirred under ice-cooling, and stirred on a magnetic stirrer fully 30min or no cells to a solid.

[0041] (3) Bacteria break; the bacterial suspension was poured into the cup high-pressure homogenizer, press the "Run" key to start running the sample out of the access to the beaker. Effluent effluent to be uniformly pressed ...

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Abstract

The invention relates to the technical field of biology, in particular to a luciferase mutant and application thereof. The luciferase mutant provided by the invention is a wild-type amino acid sequence as shown in SEQ ID NO: 1, comprises at least one of a mutation site L17R, a mutation site I47R, a mutation site F176R and a mutation site E354K, and can be efficiently expressed in a host, wherein the expression product has the characteristics of high activity and high thermal stability.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more particularly to fluorescein enzyme mismarries and their applications. Background technique [0002] The luciferase of the North American Fireflies (Photinus Pyralis) is composed of 2 subunits, and the enzymatic region is located between the base and the small subunit. The catalytic reaction process of luciferase is: in an environment in which oxygen is present, it provides energy by ATP, luciferase in mg. 2+ The oxidation of substrate fluorescein is catalyzed as a cofactor, and chemical can be converted into light energy. [0003] Luciferase is widely used in genetic engineering, which can track transcription, translation and expression in animals and flora, monitor biological processes in non-invasive forms while analyzing proteins and proteins. The fluorescein-fluorescene enzyme is high sensitivity, and the luminous intensity is proportional to the ATP content in the system, which can b...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/70C12N1/21C12Q1/66C12R1/19
CPCC12N9/0069C12Y113/12007C12N15/70C12Q1/66
Inventor 周同喜徐延伟姜婷婷张春鸽赵巧辉李桂林付光宇杨增利
Owner ZHENGZHOU IMMUNO BIOTECH
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