Inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase as well as encoding gene and application of inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase

A technology of sulfatase and encoding gene, which is applied in the field of genetic engineering to achieve the effects of repairing brain nerve damage, high endosulfatase activity and regulating metabolism

Active Publication Date: 2014-07-30
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no reports on the cloning, expression and characterization of endo-CS / DS sulfatase, so finding and identifying endo-CS / DS sulfatase has important scientific and application value

Method used

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  • Inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase as well as encoding gene and application of inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase
  • Inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase as well as encoding gene and application of inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase
  • Inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase as well as encoding gene and application of inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1, the acquisition of chondroitin sulfate / dermatan sulfate sulfatase

[0045] Pick the Vibrio sp. FC509 strain and inoculate it into 100 mL of liquid medium, and culture it on a shaking table for 12 hours at a temperature of 28° C. and a rotation speed of 200 rpm. Add chondroitin sulfate to make the mass concentration reach 0.01%. Continue to cultivate for 48 hours; the culture solution is centrifuged to collect the supernatant medium, add ammonium sulfate to the supernatant medium to a mass concentration of 80%, collect the precipitate, and the precipitate is dialyzed with PBS buffer solution to remove ammonium sulfate to obtain chondroitin sulfate / Dermatan sulfate sulfatase.

[0046] The above-mentioned Vibrio sp. FC509 was deposited in the General Microbiology Center of the China Committee for the Collection of Microorganisms on March 12, 2014, with the preservation number CGMCC NO.8913, and address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District...

Embodiment 2

[0050] Embodiment 2, the extraction of vibrio (Vibrio sp.) FC509 bacterial strain genomic DNA

[0051] Inoculate the Vibrio sp. FC509 strain into the liquid medium (same as in Example 1), and culture it with shaking until OD at 30°C and 200rpm 600 =0.8; Take 40 mL of the cultured bacteria, centrifuge at 12,000rmp for 25min, collect the bacterial pellet, wash with 20mL of lysozyme buffer (10mM Tris-HCl pH8.0), centrifuge at 12,000rmp for 25min, collect the bacteria Body precipitation;

[0052] Add 12.0mL of lysozyme buffer solution to each tube to obtain about 14.0mL of bacterial solution in the above bacteria precipitation, add 560μL of lysozyme with a concentration of 20mg / mL respectively, and the final concentration is about 800μg / mL; after 1.0h in ice bath , incubate at 37°C for 2.0h until the solution is viscous; add 10wt% SDS 0.82mL, 100mg / mL proteinase K solution 60μL, bathe in 52°C for 1.0h; add Tris-balanced phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) 15...

Embodiment 3

[0053] Example 3 Genome scanning and sequence analysis of Vibrio sp. FC509 strain.

[0054] The large-molecular-weight genomic DNA prepared in Example 2 was sequenced (Meiji Biotechnology Co., Ltd.). The sequencing results were analyzed with the software on NCBI (National Center for Biotechnology Information, http: / / www.ncb1.nlm.nih.gov / ). The NCBI analysis software used is Open Reading Frame Finder (ORF Finder, http: / / www.ncb1.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast. ncb1.nlm.nih.gov / Blast.cgi).

[0055] NCBI analysis results show that Vibrio sp.FC509 strain genome carries a CS / DS endo-sulfatase gene, the gene coding region is 1566bp long, and its nucleotide sequence is shown in SEQ ID NO.1. It has 86% homology with 448 amino acids of the sulfatase gene in the whole genome sequence of Photobacterium profundum SS9 (NCBI accession number: YP_131812.10).

[0056] The CS / DS endosulfatase 4-O-Sulfatase encoded by 4-O-sulfatase cons...

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Abstract

The invention relates to inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase as well as an encoding gene and an application of the inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase. The invention firstly discloses an amino acid sequence of the inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase and a nucleotide sequence of the encoding gene of the inscribe chondroitin sulfate/dermatan sulfate 4-O-sulfatase. Found through detection, the enzyme has relatively high inscribe sulfatase activity, plays a role in removing sulfate radicals at the terminal and the inside of polysaccharide and can be effectively acted on polysaccharide, used for modifying and regulating the structure inside polysaccharide and applied to preparation of drugs for restoring cranial nerve involvement, regulating metabolism and treating inflammation and cancer.

Description

technical field [0001] The invention relates to an endo-type chondroitin sulfate / dermatan sulfate 4-O-sulfatase and its coding gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Chondroitin Sulfate (CS) is a straight disaccharide unit composed of D-glucuronic acid and N-acetylgalactosamine via α-1,3-glycosidic bonds and repeated β-1,4-glycosidic bonds. chain polysaccharides. In the process of sugar chain synthesis, the D-glucuronic acid of chondroitin sulfate is usually converted into L-iduronic acid under the action of C5-epimerase, thus forming dermatan sulfate (Dermatan Sulfate, DS ), so chondroitin sulfate and dermatan sulfate regions often exist alternately in the sugar chain, forming a complex hybrid structure CS / DS. One of the main reasons for the highly complex structure of CS / DS is due to the sulfation of hydroxyl groups (-OH) at different positions in the sugar chain. CS synthesized in organisms in nature ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/16C12N15/55A61K38/46A61P25/00A61P3/00A61P29/00A61P35/00C12R1/63
CPCA61K38/00C12N9/16
Inventor 李福川王文爽郑小煜蔡幸雅韩文君蔡晓娟
Owner SHANDONG UNIV
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