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Alisma orientale squalene cyclooxygenase and application thereof

A technology of ene epoxidase and Alisma shark, which is applied in the field of molecular biology, can solve the problems of low content, narrow distribution of resources, and inability to meet utilization needs

Active Publication Date: 2019-06-21
NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Alisma orientale (Alisma orientale) is a plant belonging to the family Alismaceae. Its main medicinal ingredients are proterane-type tetracyclic triterpenoids, which have obvious anti-hyperlipidemia, hypotension, and anti-HIV1 effects, especially It has remarkable anticancer activity and broad prospects for its development and application. However, its resource distribution is narrow and its content is low. It only exists in a few plant groups such as Alisma genus, which cannot meet the needs of utilization. Bioengineering is one of the effective ways to increase the yield of medicinal ingredients

Method used

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  • Alisma orientale squalene cyclooxygenase and application thereof
  • Alisma orientale squalene cyclooxygenase and application thereof
  • Alisma orientale squalene cyclooxygenase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This example specifically illustrates the full-length cloning method of Alisma squalene epoxidase of the present invention.

[0039] Alisma is collected from the planting base of Jian'ou, Fujian Province, and it is identified as Alisma orientale (Sam.) Juzep, a plant of the Alismaceae family, by Professor Gu Wei of Nanjing University of Traditional Chinese Medicine. After collection, transplant them to the greenhouse of Nanjing University of Traditional Chinese Medicine. After the plants grow stably, take fresh leaves and pack them into cryopreservation tubes, and store them in liquid nitrogen for later use.

[0040] 1.1 Extraction and detection of Alisma total RNA

[0041] The extraction of total RNA was carried out according to the operation steps of Tiangen Company Total RNA Extraction Kit (batch number: DP419):

[0042] ①Take 100 mg of plant tissue, transfer it to a pre-cooled mortar, and grind it into powder in liquid nitrogen;

[0043] ② Add 1mL of lysate to the...

Embodiment 2

[0067] This example illustrates the prokaryotic expression method of the gene of the present invention.

[0068] 2.1 Vector construction: According to the sequences of Alisma AoSE1 and AoSE2, the primers (SF5, SR5; SF6, SR6) shown in Table 2 were designed respectively, and SE was cloned into the pGHn vector. The reaction system and procedures were as follows:

[0069] Table 2 List of primer sequences in the text

[0070]

[0071] The reaction system is:

[0072]

[0073]

[0074] Reaction program: 94°C for 5min; 94°C for 30sec, 54°C for 30sec, 72°C for 1min, 30cycles; 72°C for 1min.

[0075] The PCR product was mixed with Loading Buffer and loaded on a 1% agarose gel, and the target band was detected in a gel imaging system. Recovery, ligation, PCR identification, and cloning and sequencing of target fragments. The gene AoSE ORF was cloned between NdeI and XhoI of the pCzn1 expression vector, and the target gene was fused with 6X his tag protein to obtain the pCzn1...

Embodiment 3

[0097] This example illustrates the functional validation of the AoSE1 and AoSE2 enzymes.

[0098] In order to test the catalytic function of AoSE1 and AoSE2, an in vitro enzymatic reaction system was established.

[0099] The reaction system is 500μl: 0.1mM DTT, 25mM MgCl 2, 490μM squalene, 0.14mM NADPH, 0.5% Triton X-100, 1mM FAD, 10μL target protein, add Tris-HCl (pH 7.4) to 500μL reaction system, incubate at 25℃ for 20h, add 15 %KOH / MeOH was used to terminate the experimental reaction, and an equal volume of n-hexane was extracted twice for content determination. Negative controls were performed under the same conditions, but using extracts of E. coli carrying empty vectors.

[0100] Metabolites were qualitatively analyzed by HPLC, chromatographic conditions: YMC C 18 Chromatography column (5μm, 4.6×250mm). Mobile phase: (A phase: water, B phase: acetonitrile); Elution program: 0–20min, 25%–65%B; 20–40min, 65%–85%B; 40–50min, 85%B; 50 –60min, 85-95% B; detector: UV de...

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Abstract

The invention discloses alisma orientale squalene cyclooxygenase and an application thereof. The alisma orientale squalene cyclooxygenase has an amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4. The invention clones the full length of alisma orientale squalene cyclooxygenase gene for the first time, provides alisma orientale squalene cyclooxygenase, a prokaryotic expression method of the enzyme gene, prepares a polyclonal antibody of the enzyme protein, provides an ELISA detection method and a Western Blot detection method of the protein, and can be used for improving the biosynthesis efficiency of alisma orientale prototerpane triterpene and monitoring the dynamic accumulation process of pharmacodynamic components.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to Alisma squalene cyclooxygenase and its application. Background technique [0002] Alisma orientale (Alisma orientale) is a plant belonging to the family Alismaceae. Its main medicinal ingredients are proterane-type tetracyclic triterpenoids, which have obvious anti-hyperlipidemia, hypotension, and anti-HIV1 effects, especially It has remarkable anticancer activity and broad prospects for its development and application. However, its resource distribution is narrow and its content is low. It only exists in a few plant groups such as Alisma genus, which cannot meet the needs of utilization. Bioengineering is one of the effective ways to increase the yield of medicinal ingredients . Squaleneepoxidase (SE) is a key enzyme in the biosynthesis of protane-type triterpenes, which can be used to improve the biosynthesis efficiency of protane-type triterpenes to meet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/70C07K16/40C07K16/06G01N33/573
Inventor 谷巍吴启南巢建国张阿琴徐飞李思蒙田方
Owner NANJING UNIVERSITY OF TRADITIONAL CHINESE MEDICINE
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