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Tripterygium wilfordii pyrophosphate synthase twcps4 and its application in the preparation of abietane-type diterpenoids

A technology of pyrophosphate synthase and compound, applied in application, enzyme, isomerase and other directions, can solve the problem of unsuccessful preparation of triptolide

Active Publication Date: 2018-02-23
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-catalyzed preparation of tanshinone has been reported, but enzymatic preparation of triptolide has not been successful

Method used

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  • Tripterygium wilfordii pyrophosphate synthase twcps4 and its application in the preparation of abietane-type diterpenoids
  • Tripterygium wilfordii pyrophosphate synthase twcps4 and its application in the preparation of abietane-type diterpenoids
  • Tripterygium wilfordii pyrophosphate synthase twcps4 and its application in the preparation of abietane-type diterpenoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1. Analysis of induced expression of Tripterygium wilfordii TwCPS4

[0035] Primers were designed according to the nucleotide sequences of Tripterygium wilfordii housekeeping gene β-actin and Tripterygium wilfordii Twcps4 gene. After the target gene primers and housekeeping gene primers are tested to be qualified, real-time fluorescent quantitative PCR is carried out on the ABI 7500 real-time fluorescent quantitative PCR instrument. The equipment system is as follows:

[0036]

[0037] The PCR reaction conditions are:

[0038]

[0039] The real-time fluorescence quantitative PCR primer sequence is as follows:

[0040] After the reaction, the fluorescence value change curve and melting curve were analyzed. Each reaction was repeated three times, and the results were analyzed by the 2-ΔΔCT method.

[0041] According to the real-time fluorescence quantitative PCR experiment detection data, the relative quantitative expression analysis was carried out by us...

Embodiment 2

[0042] Example 2, Tripterygium wilfordii Twcps4 prokaryotic expression vector construction

[0043] The vector pMD-19-T-CPS4 plasmid containing the full-length cDNA of Tripterygium wilfordii Twcps4 gene was used as a template, and the gene coding region was amplified by PCR with primers containing restriction sites (see Table 3-1 for the primer sequences). 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 JET Gel Extraction Kit gel (method as follows).

[0044] Gene JET Gel Extraction Kit gel recovery steps:

[0045] (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 30-60min;

[0046] (2) Use a scalpel or razor blade to cut the gel containing DNA fragments as close as pos...

Embodiment 3

[0068] Example 3. Induced Expression of Recombinant Proteins

[0069] induced expression

[0070] 100mmol·L -1 IPTG: Weigh 238.3mg of IPTG with 10mL of ddH 2 Dissolve O, filter and store at -20°C; LB medium: Trytone 1.0%, Yeast Extract 0.5%, NaCl 1.0%, Agar 1.5%, pH 7.0.

[0071] Steps:

[0072] (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;

[0073] (2) Pick a monoclonal colony and transfer it to a cell containing 100 μg·mL -1 Amp in 2mL LB liquid medium, shake culture at 37℃ to OD 600 to 0.6-1.0;

[0074] (3) Take 1 mL of the bacterial solution 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 to 100 mL of LB+Amp liquid medium;

[0075...

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Abstract

The invention discloses novel common threewingnut root copalyl pyrophosphate synthase TwCPS4 and a coding gene thereof. TwCPS4 plays an importance role in inducing the initial synthesis of a common threewingnut root diterpene compound. Catalysis experiments show that by combining TwCPS4 with radix salviae miltiorrhizae SmkSL1 for use, an abietane diterpene precursor can be synthesized with GGPP as a substrate, and a secondary synthesis route of common threewingnut root diterpene with TwCPS4 involved is verified. The efficiency of the combination of TwCPS4 and radix salviae miltiorrhizae SmkSL1 is higher than that of the combination of SmCPS1 and SmkSL1 in abietane diterpene precursor synthesis.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a key enzyme for diterpene compound synthesis obtained through a genetic engineering method, in particular to tripterygium wilfordii diterpene synthase and its use in the preparation of abietane-type diterpene compounds. Background technique [0002] The medicinal plant Tripterygium wilfordii.Hook.f. is a Chinese herbal medicine, [0003] It is widely used in the treatment of rheumatoid arthritis and inflammation (Raphaela G M, Mildred W, Roy F, et al. Comparison of Tripterygium wilfordii Hook F Versus Sulfasalazine in the Treatment of Rheumatoid Arthritis: A Randomized Trial[J]. Annals of Internal Medicine, 2009, 151(4): 229-240. Tao X L, Lipsky P E. The Chinese anti-inflammatory and immunosuppressive herbal remedy Tripterygium wilfordii Hook F. [J]. Rheumatic Disease Clinics of North America, 2000, 26(1): 29 -50.). Terpenes are the main active ingredients of Tript...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12P15/00C12P17/18C12P17/04
CPCC12N9/90C12P15/00C12P17/04C12P17/181C12Y505/01012
Inventor 高伟黄璐琦苏平周家伟
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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