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Kelp carbohydrate sulfotransferase gene and application thereof

A carbohydrate and transferase technology, applied in the field of molecular biology, can solve the problems such as the need for further verification analysis, the large difference in the content of sulfuric acid groups, and the difficulty

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

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Problems solved by technology

Studies have shown that the biological activity of fucoidan sulfate is closely related to its sulfate group content, molecular weight and sulfonyl position. However, due to the large difference in the sulfate group content of fucoidan sulfate, the position of sulfonyl substitution is uncertain factors, which brings a certain degree of difficulty to the study of its structure-activity relationship, and affects the development and utilization of seaweed active substances.
[0003] At present, the biosynthetic pathway of fucoidan sulfate is still in the stage of theoretical speculation, and there is no complete experimental verification process to explain the related genes and functions in the synthetic pathway.
Moreover, although dozens of sulfotransferase sequences are predicted in the existing brown algae genome data, which sequences can actually catalyze the transfer of sulfate groups needs further verification and analysis

Method used

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  • Kelp carbohydrate sulfotransferase gene and application thereof

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Embodiment 1

[0018] The cloning method of kelp CHST gene of the present invention mainly comprises the following steps:

[0019] 1. Total RNA extraction and cDNA synthesis of young sporophytes of Laminaria;

[0020] 2. CHST gene-specific primer synthesis and PCR amplification product sequencing;

[0021] 3. CHST protein structure prediction analysis.

[0022] The specific operation is as follows:

[0023] 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.

[0024] 2. Synthesis of CHST gene-specif...

Embodiment 2

[0052] The method for expressing and purifying the kelp CHST recombinant protein of the present invention mainly includes the following steps:

[0053] 1. CHST recombinant plasmid construction and transformation;

[0054] 2. CHST recombinant protein expression;

[0055] 3. CHST recombinant protein soluble detection

[0056] The specific operation is as follows:

[0057] 1. Construction and transformation of CHST 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 sequence information of the target gene, Nde I and EcoR I were selected as double restriction sites, and amplification primers with restriction sites and protective bases were designed, namely CHST-F and CHST-R (Table 1). The expre...

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Abstract

The invention belongs to the technical field of molecular biology, and in particular to a kelp carbohydrate sulfotransferase gene and an application thereof. The sulfotransferase gene (CHST gene) is shown by the base in a sequence table SEDIQ No.1. According to the invention, a cDNA sequence of a carbohydrate sulfotransferase (CHST) gene with a high transcription level is cloned from kelp based onthe sequencing result of a kelp transcription group, the open reading frame (ORF) of the cDNA sequence is 1134bp, and a protein sequence containing 377 amino acids can be encoded. Expression of soluble recombinant protein can be successfully obtianed by utilizing an in-vitro prokaryotic expression system.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a kelp carbohydrate sulfotransferase gene and its application. Background technique [0002] Fucoidan sulfate, also known as fucoidan, fucoidan, and fucoidan sulfate, is the most studied non-mammalian source of biologically active sulfated polysaccharides. The main component is L-fucoidan-4 -Sulfate, mostly present in the cell wall of brown algae. Fucoidan sulfate has various biological activities such as anti-virus, anti-coagulation, and treatment of diabetic nephropathy, and has broad application prospects in the field of pharmaceutical development. Studies have shown that the biological activity of fucoidan sulfate is closely related to its sulfate group content, molecular weight and sulfonyl position. However, due to the large difference in the sulfate group content of fucoidan sulfate, the position of sulfonyl substitution is uncertain Factors have brought some d...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/70C12P19/04
CPCC12N9/13C12N15/70C12P19/04C12Y208/02
Inventor 段德麟卢畅邵展茹张朋艳
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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