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A kind of breviscapine glycosyltransferase, preparation method and application thereof

A technology of breviscapine sugar and radical transfer, applied in the biological field, can solve the problems of difficulty in separation and identification of unknown glycosylases, large differences in glycosylases, etc.

Active Publication Date: 2017-10-27
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Glycosylases from different species differ greatly in protein primary structure, and it is difficult to isolate and identify unknown glycosylases

Method used

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  • A kind of breviscapine glycosyltransferase, preparation method and application thereof
  • A kind of breviscapine glycosyltransferase, preparation method and application thereof
  • A kind of breviscapine glycosyltransferase, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The acquisition of the coding nucleotide of embodiment 1 F7GAT

[0053] According to the nucleotide sequence information published in the present invention, one of the following two methods can be used to obtain the coding nucleotide of F7GAT.

[0054] Method 1: artificially synthesize the coding nucleotide of F7GAT according to the information of sequence SEQ ID NO: 1, or code the nucleotide sequence for the target host cell (such as Escherichia coli, etc.) according to the amino acid sequence of SEQ ID NO: 2 Artificial synthesis after sub-optimization.

[0055] The second method is to obtain by amplifying or isolating the genome or transcriptome of Erigeron breviscapus. Specifically, conventional methods or kits can be used to extract DNA or RNA from the plant scutellaria breviscapus, thereby establishing a scutellaria breviscapus DNA sequence library or cDNA library (SEQ ID NO.3), using it as a template, using primers F7GAT-F1( SEQ ID NO.4) and F7GAT-R1 (SEQ ID NO....

Embodiment 2

[0056] Example 2 Cloning of the CDS region of the F7GAT coding gene

[0057] Different vectors were selected for cloning according to different host cells expressing F7GAT.

[0058] 1) Cloning into E. coli expression vector pET28-a(+):

[0059] Use primers F7GAT-F1 (SEQ ID NO.4) and F7GAT-R1 (SEQ ID NO.5) as primers, and carry out conventional PCR with DNA or RNA containing the F7GAT coding nucleotide sequence as a template to obtain the coding nucleoside of F7GAT acid.

[0060] The 5' ends of the primers F7GAT-F1 and F7GAT-R1 have NdeI and XhoI restriction site sequences, and the two ends of the PCR products also have NdeI and XhoI restriction sites respectively.

[0061] PCR product purification.

[0062] The vector pET-28a(+) and PCR purified product were digested with restriction endonucleases NdeI and XhoI.

[0063] Inactivate the endonuclease according to the endonuclease instructions.

[0064] Mix the digested vector and the PCR fragment, and use T4 DNA ligase to l...

Embodiment 3

[0077] Example 3 Expression of F7GAT by recombinant Escherichia coli, and isolation and purification of F7GAT

[0078] 1. Gene expression

[0079] (1) The Escherichia coli expression-type recombinant plasmid 28a-F7GAT (the construction method is as in Example 2a)) was transformed into E. coliBL21(DE3) strain to obtain recombinant bacteria. Positive clone selection (Kan+, 100 mg / mL) was carried out on a kanamycin-resistant plate, and cultured overnight at 37°C;

[0080] (2) Put monoclonal PET-28a-31047 into 5 mL LB liquid medium (Kan+, 100 mg / mL), and culture at 37°C and 220 rpm until OD600 is 0.6-0.8. Transfer the bacterial liquid in 5 mL LB medium to 800 mL 2YT medium (Kan+, 100 mg / mL), culture at 37 °C and 220 rpm until the OD600 is 0.6-0.8, cool down to 16 °C, add a final concentration of 0.5 mMIPTG, and induce Express 16h;

[0081] (3) The above-mentioned cultured bacteria liquid is collected in the bacteria collection bottle, and centrifuged at 5500rpm for 15min;

[0...

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Abstract

The invention discloses an erigeron breviscapus. The erigeron breviscapus comprises polypeptides selected from the following (a)-(c): (a) a polypeptide composed of an amino acid sequence represented by SEQ ID NO.2; (b) a polypeptide which is formed by substituting, deleting or adding one or more amino acids in the amino acid sequence in the (a) and provided with a polysaccharide transfer function on a flavone compound or flavonoid 7-hydroxy and derived from the (a); and (c) a polypeptide which has the amino acid sequence having more than 95% identity with the amino acid in the (a) and is provided with a polysaccharide transfer function on a flavone compound or flavonoid 7-hydroxy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a polypeptide derived from Erigeron breviscapus and having the function of transferring sugar on the 7-hydroxyl group of flavonoid compounds or flavonoids, as well as its preparation and application. Background technique [0002] Breviscapus [Erigeron breviscapus (Vant.) Hand.-Mazz.] is one of the "five natural series" medicines developed in Yunnan. Breviscapus was first recorded in the "Southern Yunnan Materia Medica" in the Ming Dynasty. It is a traditional medicinal plant of the Miao people in Yunnan. Its medicinal properties are pungent, slightly bitter, warm, heart-guiding, and liver meridian. For anemofrigid-damp arthralgia, apoplexy paralysis, chest pain and heartache, toothache, colds and other diseases. Erigeron breviscapus is a perennial plant of the family Asteraceae. With its total flavonoids, scutellarin (flavonoids) and caffeoylquinic acid compounds as active ingredients...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/11C12P19/60
CPCC12N9/1048C12P19/60
Inventor 江会锋丁文涛刘莹卢丽娜董扬马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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