Bacterial glutamine synthetase and preparation method thereof

A technology of glutamine and synthetase, which is applied in the field of molecular biology, can solve the problems of low yield and cumbersome cultivation, and achieve the effect of simple method and easy operation

Inactive Publication Date: 2018-02-27
HUBEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But the main source of producing glutamine synthetase is plant and fungus at present, the productive rate of the glutamine synthetase from plant and fungus is low, and it is loaded down with trivial details to cultivate

Method used

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  • Bacterial glutamine synthetase and preparation method thereof
  • Bacterial glutamine synthetase and preparation method thereof
  • Bacterial glutamine synthetase and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Acquisition of Bacterial Glutamine Synthetase Gene

[0023] 1) Obtain the strain containing the target gene

[0024] The present invention utilizes a strain of the genus Cryobacterium, which is screened from the soil of Changbai Mountain. The specific screening process is like the Chinese invention patent with the patent number 201710034491.5. The strain is named: Cryobacteriumbaishanse 02, and it is preserved in the China Type Culture Collection Center , deposit number CCTCC NO: M2016604. The date of deposit is October 31, 2016, and the deposit address is Wuhan University, Wuhan, China.

[0025] 2) Genome extraction

[0026] The genome of the Cryobacterium baishanse 02 strain was extracted using a DNA extraction kit (TAKARA Dalian) as a template, and the extracted genome samples were stored at -20°C until use.

[0027] 3) PCR amplification of the target gene

[0028] Design primers: the forward primer is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ...

Embodiment 2

[0036] Construction of Expression Vector of Bacterial Glutamine Synthetase

[0037] The PCR amplified product obtained in Example 1 was carried out to gel recovery, and the PCR amplified product was carried out double digestion reaction with restriction endonuclease BamHI and HindIII; Carrier pET21a (+ ) for double enzyme digestion reaction, and then catalyzed by high-efficiency DNA ligase High Ligation (TOYOBO) to connect the pET21a(+) and PCR amplification product after the double enzyme digestion reaction; construct the expression vector pET21a- GS, the expression vector pET21a-GS was constructed as figure 2 shown.

Embodiment 3

[0039] Expression and purification of bacterial glutamine synthetase protein

[0040] The expression vector pET21a-GS obtained in Example 2 is transformed into the competent thallus of Escherichia coli BL21, and the preparation of the competent state of Escherichia coli BL21 adopts CaCl 2 method, CaCl 2 The competent method for preparing Escherichia coli BL21 is a routine experimental method in the laboratory, so it will not be repeated here. The resulting expression vector pET21a-GS was transformed into Escherichia coli BL21 to obtain recombinant strain Ecoli BL21-pET21a-GS. Expand Ecoli BL21-pET21a-GS to OD 600 After = 0.6, add 1mM IPTG, induce culture and expression of bacterial glutamine synthetase protein at 28°C, collect the bacteria after the induction culture is over, break the bacteria and purify with Ni-NTA affinity chromatography column to obtain the purified bacteria Glutamine synthetase proteins, such as image 3 As shown, the protein molecular weight of the o...

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Abstract

The invention relates to the field of molecular biology, in particular to a bacterial glutamine synthetase and a preparation method thereof. The preparation method of bacterial glutamine synthetase comprises the following steps of: 1) amplifying a base sequence shown in SEQ ID NO.2 by using a PCR (polymerase chain reaction) technology; 2) cloning the base sequence shown in SEQ ID NO.2 into a vector plasmid to construct an expression vector of bacterial glutamine synthetase; and 3) transforming the expression vector into competent thalli of Escherichia coli to obtain recombinant thalli, and obtaining bacterial glutamine synthetase protein through the sequential processes of expanding culture, inducing expression, collecting thalli, crushing the thalli and purifying protein. The method for inducing and expressing bacterial glutamine synthetase protein by using an Ecoli BL21-pET21a-GS recombinant strain is simple and easy to operate, and finds a novel glutamine synthetase gene sequence, thereby providing a new research direction for the research of glutamine synthetase.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a bacterial glutamine synthetase and a preparation method thereof. Background technique [0002] Glutamine, Chinese name: 2-amino-4-carbamoylbutyric acid, English name: Glutamine (Gln). Glutamine is used as a nutritional supplement and flavoring supplement in food processing. And L-Glutamine is an important nutritional supplement for bodybuilders and bodybuilders. Glutamine is used medically to improve the brain function of children with mental retardation and mental disorders, alcoholism, and epilepsy. It can also be used to treat peptic ulcers (gastric ulcers, duodenal ulcers), acute and chronic gastritis, and as Brain function improver and treatment of alcoholism. [0003] At present, domestic research on the production of glutamine mainly stays on the optimization of mutagenesis breeding and fermentation process. Although some glutamine-producing strains have been obtaine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/70
CPCC12N9/93C12N15/70C12Y603/01002
Inventor 宫春杰胡征张抒杨杨波王毅孔萌萌
Owner HUBEI UNIV OF TECH
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