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Application of Tripterygium wilfordii twks and twcps3 in the preparation of kaurane-type diterpenoids

A kaurane-type, kaurane technology, applied in the field of 16α-kaurane, can solve the problems of restricted development, slow plant growth, low content, etc.

Active Publication Date: 2018-04-13
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Developing new drugs from the active ingredients of traditional Chinese medicine is a very promising way, but due to the slow growth of plants and the low content of these active ingredients in plants, its development is greatly limited

Method used

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  • Application of Tripterygium wilfordii twks and twcps3 in the preparation of kaurane-type diterpenoids
  • Application of Tripterygium wilfordii twks and twcps3 in the preparation of kaurane-type diterpenoids
  • Application of Tripterygium wilfordii twks and twcps3 in the preparation of kaurane-type diterpenoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment one, Tripterygium wilfordii Twcps3 and Twks prokaryotic expression vector construction ( figure 1 )

[0032] Using the vector pMD-19-TPS plasmid containing the full-length cDNA of Tripterygium wilfordii Twcps3 and Twks genes as templates, PCR amplification of the gene coding region was carried out with primers containing restriction sites (see Table 1 for the primer sequences), and inserted into the prokaryotic expression vector middle. The DNA polymerase uses high-fidelity DNA polymerase (PrimeSTAR HS DNA Polymerase). The PCR parameters were 98°C for 3min, 1 cycle; 98°C for 10s, 60°C for 10s, 72°C for 2min30s, 30 cycles; 72°C for 7min; 4°C for maintenance. The amplified product was recovered by Gene JETGel Extraction Kit gel (method as follows).

[0033] Gene JET Gel Extraction Kit gel recovery steps:

[0034] (1) Take the PCR product and pre-mix it with 6×loading buffer, and electrophoresis on 1.5% agarose gel with low voltage (about 5Vcm-1) for 30-60mi...

Embodiment 2

[0055] Example 2. Induced Expression of Recombinant Proteins

[0056] induced expression

[0057] 100mmol·L-1IPTG: Weigh 238.3mg of IPTG, dissolve in 10mL of ddH2O, filter and store at -20°C; LB medium: Trytone 1.0%, Yeast Extract 0.5%, NaCl 1.0%, Agar1.5%, pH 7.0.

[0058] Steps:

[0059] (1) Take 1 μL of the recombinant plasmid containing the target gene that has been correctly identified by enzyme digestion and has been verified by sequencing, and transform it into 50 μl TransB (DE3) competent cells, smear LB+Amp (ampicillin sodium) solid plate, and incubate at 37°C 12-16h;

[0060] (2) Pick out monoclonal colonies and transfer them to 2mL LB liquid medium containing 100μg·mL-1Amp after enzyme digestion and identification, and shake culture at 37°C until OD600 reaches 0.6-1.0;

[0061] (3) Take 1 mL of bacterial liquid at 5000 g and centrifuge at 4°C for 5 minutes to collect the bacterial cells, suspend the bacterial cells with fresh LB+Amp liquid medium, and transfer t...

Embodiment 3

[0068] Embodiment three, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( figure 2 )

[0069] Reagent preparation

[0070] 30% acrylamide stock solution (neurotoxicity, wear a mask and gloves during operation): In a fume hood, weigh 29.2g of acrylamide and 0.8g of methylenebisacrylamide, add ddH2O to dissolve, and dilute to 100mL. After filtering with a syringe filter, put it in a brown bottle and store it at 4°C;

[0071] pH 8.8 Tris-HCl separating gel buffer: prepare 1.5M Tris-HCl, adjust the pH to 8.8, and store at 4°C;

[0072] pH 6.8 Tris-HCl stacking gel buffer: prepare 1M Tris-HCl, adjust the pH to 6.8, and store at 4°C;

[0073] 10% SDS: Weigh 1.0g of SDS, dissolve in 10mL of distilled water, store at 4°C;

[0074] 10% ammonium persulfate (APS): Take 1.0g of APS, dissolve in 10mL of distilled water, and store at 4°C;

[0075] TEMED (tetraethylethylenediamine) stock solution;

[0076]5×sample buffer (10mL): 0.6mL 1mol L-1 Tris-HCl (pH 6.8), ...

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Abstract

The invention relates to application of common threewingnut root copalyl pyrophosphate synthase TwCPS3 and common threewingnut root kaurene synthase TwKS to preparation of kaurane diterpene. Through recombination expression of the common threewingnut root copalyl pyrophosphate synthase gene TwCPS3 and the common threewingnut root kaurene synthase gene TwKS, the precursor derivative kaurane diterpene compound of gibberellin is successfully synthesized with GGPP as the substrate under the catalysis of recombinant protein. The common threewingnut root copalyl pyrophosphate synthase TwCPS3 and the common threewingnut root kaurene synthase TwKS are of great significance in biosynthesis of gibberellin and synthesis regulation of diterpene compounds like triptolide.

Description

technical field [0001] The present invention successfully synthesizes 16α-kaurane, which is related to the biosynthesis of gibberellins, by constructing TwCPS3 and TwKS eukaryotic and prokaryotic expression vectors, and through in vitro enzymatic and yeast fermentation methods. The invention relates to the regulation of the growth and development of tripterygium wilfordii and the synthesis of active components, and belongs to the field of genetic engineering of medicinal plants. Background technique [0002] The medicinal plant Tripterygium wilfordii. Hook.f. is a Chinese herbal medicine widely used in the treatment of rheumatoid arthritis and inflammation. Terpenes are the main active ingredients of Tripterygium wilfordii, including triptolide, triptophenolide and celastrol. Existing studies have shown that kaurane-type diterpenes are the main components of the terpenoid natural product family, and these molecules usually exhibit various important biological activities suc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/18C12N15/81
CPCC12N9/88C12N9/90C12N15/81C12N2800/102C12P17/18C12Y402/03019C12Y505/01013
Inventor 高伟黄璐琦苏平周家伟
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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