Sesquiterpene synthase from eupatorium adenophorum spreng, gene, vector, engineering bacterium and application of sesquiterpene synthase

A technology of genetically engineered bacteria and Eupatorium adenogenum, which is applied in the fields of genetic engineering, application, plant gene improvement, etc., can solve the problems of low content, complex components, high requirements and difficult extraction process of elemene, etc.

Active Publication Date: 2017-03-08
JINGANG PHARMA DALIAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the development and clinical application of elemene series of anti-tumor herbal medicines are in full swing, and the market share is also expanding, there are still many technical difficulties in the extraction and separation of elemene from the natural plant curcuma curcuma: first, the natural plant only contains Elemene, and its content is extremely low, accounting for about 1/1000 to 2/1000; at the same time, the content of elemene is easily affected by many factors such as variety, root quality, planting environment, etc.
Second, the components in the volatile oil are complex. Elemene is mixed with m

Method used

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  • Sesquiterpene synthase from eupatorium adenophorum spreng, gene, vector, engineering bacterium and application of sesquiterpene synthase
  • Sesquiterpene synthase from eupatorium adenophorum spreng, gene, vector, engineering bacterium and application of sesquiterpene synthase
  • Sesquiterpene synthase from eupatorium adenophorum spreng, gene, vector, engineering bacterium and application of sesquiterpene synthase

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Experimental program
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Effect test

Embodiment 1

[0028] 1. Cloning of the full-length EaSQS gene

[0029]The present invention first uses the conserved amino acid sequence of the plant-derived sesquiterpene synthase gene to design a merger primer, and uses Eupatorium adenophorum cDNA as a template to perform PCR amplification to obtain the partial sequence of Eupatorium adenophorum sesquiterpene synthase, Then RACE technology was used to amplify the full-length sequence. The coding region of the nucleotide sequence of the sesquiterpene synthase EaSQS gene derived from Eupatorium adenophorum was determined by comparative analysis by means of bioinformatics.

[0030] RNA was extracted from the tender leaves of Eupatorium adenophorum by Trizol method, reverse-transcribed and sent to Shanghai Jierui Biotechnology Co., Ltd. for sequencing, and bioinformatics BLAST analysis method was used to compare with the Genbank database to obtain a possible sesquiterpene compound Enzyme gene, according to the nucleotide sequence of the gene...

Embodiment 2

[0036] Catalytic property analysis of embodiment 2 recombinant enzyme EaSQS

[0037] 1. Enzyme amount

[0038] The EaSQS recombinant enzyme solution prepared by the method of Example 1 (concentration is 68.8 mg / L, see Table 1 for volume) is used as a catalyst, and pH7.0 Tris-HCL buffer solution, 2 μg FPP, 500 mM MgCl are added 2 20 μL of solution, 2 μL of 500 mM DTT, and 100 μL of dilute glycerol (1,2,3-propanetriol) constituted a reaction system of 1 ml, stirred and reacted fully at 30°C, and at the same time, the headspace-solid phase microextraction technology was used to absorb the reaction product. After 60 minutes of reaction When the extraction head is taken out and injected into the gas chromatograph, the product is analyzed quantitatively and qualitatively. The catalytic properties of the sesquiterpene enzyme studied in this experiment were judged by analyzing the size of the chromatographic peak area with β-elemene standard substance as a control. Chromatographic c...

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Abstract

The invention discloses sesquiterpene synthase from eupatorium adenophorum spreng, a gene, a vector, an engineering bacterium and application of the sesquiterpene synthase. Amino acid sequences of the sesquiterpene synthase are shown as SEQ ID NO.2. The sesquiterpene synthase, the gene, the vector, the engineering bacterium and the application have the advantages that catalysts come from the eupatorium adenophorum spreng which is a high-yield medicinal plant for beta-elemene, as proved by in-vitro activity, substrates FPP (Farnesyl pyrophosphate) can be catalyzed by the catalysts to generate the beta-elemene, and the final yield of the beta-elemene can ultimately reach 7.499 micro-grams/mL.

Description

[0001] (1) Technical field [0002] The invention relates to the preparation of β-elemene, in particular to a sesquiterpene synthase derived from Eupatorium adenophorum, a gene, a carrier, an engineering bacterium and an application thereof for preparing β-elemene. [0003] (2) Background technology [0004] Elemene (Elemene) is an active ingredient extracted from turmeric, a plant of Zingiberaceae, which has anti-cancer effects. It is a new class of sesquiterpene drugs independently developed in my country. In clinical application, the drug is highly effective and has few side effects; it can improve microcirculation and help chemotherapy and radiotherapy drugs enter cancer tissues to play a role; it has dual effects of selectively inhibiting tumor cell proliferation and improving immune function; it can also directly act on cell membrane, causing tumor cells to rupture and die. At present, elemene is being widely used in the treatment of malignant tumors such as lung cancer,...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12P5/00
CPCC12N9/88C12P5/002
Inventor 谢恬殷晓浦谌容
Owner JINGANG PHARMA DALIAN
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