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Gene of kelp mannuronic acid C5-isomerase and application of gene

A technology of mannuronic acid and isomerase, which is applied in the field of molecular biology, can solve the problems of low expression, insoluble expressed protein, etc.

Inactive Publication Date: 2020-06-02
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In brown algae, mannuronate C5-isomerase can specifically catalyze the transformation of M residues to G residues. At present, studies have begun to analyze the mannuronate C5-isomerase of brown algae, but in the protein In the in vitro production process, there are problems such as low expression level and insolubility of the expressed protein. At present, no highly active in vitro kelp mannuronate C5-isomerase has been obtained.

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  • Gene of kelp mannuronic acid C5-isomerase and application of gene
  • Gene of kelp mannuronic acid C5-isomerase and application of gene
  • Gene of kelp mannuronic acid C5-isomerase and application of gene

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Embodiment 1

[0020] The cloning method of kelp MC5E gene of the present invention mainly comprises the following steps:

[0021] 1. Total RNA extraction and cDNA synthesis of young sporophytes of Laminaria;

[0022] 2. MC5E gene-specific primer synthesis and PCR amplification product sequencing;

[0023] 3. MC5E protein structure prediction analysis.

[0024] The specific operation is as follows:

[0025] 1. Extraction of total RNA from kelp young sporophytes: Take an appropriate amount of kelp sample (≤100 mg), grind it in a liquid nitrogen precooled mortar, repeat 3 to 5 times, and take the ground powder for RNA extraction. Laminaria total RNA was extracted according to the steps of Qiagen's Plant RNA Extraction Kit (RNeasy Plant Mini Kit). cDNA was synthesized by reverse transcription using the Transcriptor FirstStrand cDNA Synthesis Kit from Roche for gene amplification. For detailed operation, please refer to the instructions of each kit.

[0026] 2. MC5E gene-specific primer syn...

Embodiment 2

[0036] The kelp MC5E recombinant protein expression and purification method of the present invention mainly comprises the following steps:

[0037] 1. Construction and transformation of MC5E recombinant plasmid;

[0038] 2. MC5E recombinant protein expression;

[0039] 3. Purification of MC5E recombinant protein.

[0040] The specific operation is as follows:

[0041] 1. Construction and transformation of MC5E recombinant plasmid: In order to realize the soluble expression of the target protein, the cold shock protein expression vector pColdⅡ was selected for the expression of the target protein. The N-terminus of the protein expressed by this vector has a His tag, which can be used for subsequent fusion protein purification . According to the target gene sequence information, design amplification primers, the gene expression amplification sequence does not contain the signal peptide sequence, select Nde I and EcoR I as double restriction sites, and add linearized cloning v...

Embodiment 3

[0045] The kelp MC5E recombinant protease activity analysis method of the present invention mainly comprises the following steps:

[0046] 1. Enzymatic reaction: prepare a 10ml reaction system according to the following components: 10mM MOPS, 0.1M NaCl, 4mM CaCl 2 , 5 μg MC5E, 0.25% polyM. At the same time, a control reaction system was prepared, no MC5E was added to the reaction system, and other components were the same.

[0047] 2. Determination of the M / G ratio of alginic acid before and after the reaction: take 3ml of the reaction solution and the same control reaction solution, heat at 95°C for 10min, and centrifuge at high speed (12000rpm) to remove the denatured protein; add 4 times the volume of anhydrous ethanol to the supernatant, and vortex. Spin and mix, centrifuge at 12,000 rpm for 15 min, discard the supernatant; follow the above steps, wash the precipitate with absolute ethanol three times; after the obtained precipitate is air-dried, add 1 ml of D 2 O, repea...

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Abstract

The invention belongs to the technical field of molecular biology and relates to cloning, expression and enzymatic analysis of a kelp MC5E gene sequence, and in particular to a gene of kelp mannuronicacid C5-isomerase and an application of the gene. The gene has a base sequence in a sequence table SED IQ No.1. The protein encoded by the MC5E gene is an amino acid sequence in a sequence table SEDIQ No.2. The recombinant protein of the gene can convert mannuronic acid (M) into guluronic acid (G) in the presence of Ca<2+>, raise the content of G in algin, lower the M / G of algin, and increase viscosity of algin, so that the physical and chemical properties of algin are improved.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology and relates to the cloning, expression and enzymatic analysis of kelp MC5E gene sequence. Specifically, it is a gene of kelp mannuronic acid C5-isomerase and its application. Background technique: [0002] Alginic acid (C 6 h 8 o 6 ) n It is a heteropolysaccharide formed by linear polymerization of β-D-mannuronic acid (M) and α-L-guluronic acid (G) through β-1,4 glycosidic bonds. Alginic acid can be combined with more than 2 valent metal ions, and the forms of water-insoluble alginic acid and water-soluble alkali metal salts such as sodium and potassium alginate are collectively called alginate. Different alginate molecules have different M and G ratios and sequences, resulting in differences in the physical and chemical properties of alginic acid: alginates with high M / G ratios have low gel strength, while alginates with low M / G ratios have high gel strength. At present, industri...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/90C12P19/24C12P19/02
CPCC12N9/90C12Y501/03C12P19/24C12P19/02
Inventor 段德麟张朋艳邵展茹卢畅
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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