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GDP-mannose-4,6-dehydratase-encoding gene in Sacchrina japonica, and protein and application thereof

A mannose and protein technology, applied in genetic engineering, plant genetic improvement, enzymes, etc., to achieve the effect of excellent genetic resources and enzyme resources

Active Publication Date: 2017-12-22
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Microorganisms can be used as another source of GDP-fucose, but the production of GDP-fucose by microbial fermentation is affected by many factors

Method used

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  • GDP-mannose-4,6-dehydratase-encoding gene in Sacchrina japonica, and protein and application thereof
  • GDP-mannose-4,6-dehydratase-encoding gene in Sacchrina japonica, and protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The acquisition of SjaGM46D1 gene in the sea-tangle of embodiment 1

[0027] The kelp was collected from Rongcheng City, Shandong Province, and the total RNA of the female gametophyte of kelp was extracted by Trizol method, and PrimeScript II 1 from TAKARA Company was used st The Strand cDNA Synthesis kit was used to reverse transcribe the total RNA of Laminaria gametophytes to obtain the first-strand cDNA. Using the cDNA as a template, the CDS full-length sequence of the Laminaria SjaGM46D1 gene was amplified, and EcoRI and SalI restriction sites were introduced at both ends (the amplification primers were 5'-GAATTCATGGCTGAGCCGGAGACG-3' and 5'-GTCGACTTAGGCGTGCTCGTTGCC-3' ). The PCR amplification program was: 94°C for 3min; 30 cycles of 94°C for 30s, 59°C for 30s, 72°C for 2min; 72°C for 10min. After the PCR products were detected by 1% agarose gel electrophoresis, the gel blocks containing the target bands were cut under ultraviolet light, and the target fragments w...

Embodiment 2

[0028] Cloning expression of embodiment 2 SjaGM46D1 gene

[0029] The SjaGM46D1 gene fragment obtained in Example 1 was connected between the EcoRI and SalI sites of the pGEX-6p-1 vector (purchased from GE Healthcare) to obtain the pGEX-SjaGM46D1 recombinant vector; the obtained pGEX-SjaGM46D1 recombinant vector was transformed into large intestine Bacillus E.coli BL21 (DE3) competent cells (purchased from Takara Company), spread on LB solid culture plates containing 100 μg / mL ampicillin, and cultivate overnight at 37°C; In ampicillin-based LB liquid medium, cultivate at 37°C until the OD600 of the bacterial liquid is 0.4, add a final concentration of 0.1mMIPTG, and induce the expression of the target protein for 16h under the induction conditions of 16°C and 160rpm.

Embodiment 3

[0030] The separation and purification of embodiment 3 SjaGM46D1 protein

[0031] Collect the bacterial cells obtained by inducing expression in Example 2, resuspend each liter of bacterial liquid with 50ml PBS, add 1% Triton X-100, 1% β-mercaptoethanol; ultrasonically break the bacterial cells on ice, centrifuge at 12000rpm for 10min Take the supernatant, add an appropriate amount of GST-beads to the supernatant, shake gently to make it adsorb protein for 1h; centrifuge at 5000rpm for 3min and discard the supernatant; add at least 10 times the volume of PBS, shake gently until the beads are suspended in the solution, 5000rpm for 3min Discard the supernatant by centrifugation and repeat this step twice; add 1mL GST Elution Buffer and shake gently for 10min; centrifuge at 5000rpm for 3min to collect the supernatant; repeat the first two steps twice to obtain the purified protein, which is encoded by the SjaGM46D1 gene The protein whose amino acid sequence is shown in SEQ ID NO:...

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Abstract

The invention specifically provides a GDP-mannose-4,6-dehydratase-encoding gene in Sacchrina japonica, and a protein and application thereof, belonging to the field of genetic engineering technology. The gene is derived from Sacchrina japonica and is named gene SjaGM46D1. The nucleotide sequence of the gene is a sequence shown as SEQ ID NO. 1. An amino acid sequence of the gene encoding the protein is a sequence as shown in SEQ ID NO. 2, and the protein is GDP-mannose-4,6-dehydratase. According to the invention, the gene sequence is cloned by using gene cloning technology, and a prokaryotic expression vector is constructed. The results of enzyme activity assay of the recombinant protein show that the recombinant protein is capable of catalyzing the conversion of GDP-mannose into GDP-4-keto-6-deoxymanose, can be applied to the synthesis of GDP fucose, and has significantly higher activity compared with other GDP-mannose-4,6-dehydratases disclosed in the prior art.

Description

technical field [0001] The invention belongs to the field of biotechnology, specifically to the field of genetic engineering, and more specifically relates to a gene encoding GDP-mannose-4,6-dehydratase in kelp (Sacchrina japonica), its encoded protein and its application. Background technique [0002] Polysaccharides play an important role in the post-translational modification of lipids and proteins. For example, it is well known that the structure of oligosaccharides will change in the malignant metastasis of tumors. The biological behavior of tumor cells is accompanied by glycoproteins and glycolipids on the cell surface. The remodeling of glycoproteins and glycolipids is achieved by changing the structure of the polysaccharides bound to them. Fucose, as an important component of polysaccharides, is especially associated with cancer and inflammation. [0003] Fucose is a deoxyhexose sugar that widely exists in different organisms. Fucose is mainly involved in the compo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12P19/32
CPCC12N9/88C12P19/32C12Y402/01047
Inventor 刘涛池姗刘翠
Owner OCEAN UNIV OF CHINA
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