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Taxus chinensis cell strain with high-yield paclitaxel characteristic and application of taxus chinensis cell strain

A yew cell and paclitaxel technology, applied in plant cells, fermentation and other directions, can solve problems such as endangering yew resources, low paclitaxel content, damage to the environment, etc., to solve the problems of frequent felling and planting, simple extraction, separation and purification, and protect the earth. The effect of the environment

Inactive Publication Date: 2015-03-25
TIANJIN ACELBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, paclitaxel is extracted from the bark of the yew plant as raw material, which not only damages the environment and endangers yew resources, but also because the yew plant grows slowly and the content of paclitaxel is low, so the source of the drug is scarce and expensive
In addition, although the total chemical synthesis has been successful, it cannot realize the actual industrial production because of the high cost price.

Method used

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  • Taxus chinensis cell strain with high-yield paclitaxel characteristic and application of taxus chinensis cell strain
  • Taxus chinensis cell strain with high-yield paclitaxel characteristic and application of taxus chinensis cell strain
  • Taxus chinensis cell strain with high-yield paclitaxel characteristic and application of taxus chinensis cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Induction of a high-yielding cell line of paclitaxel, preservation number CGMCC No. 10002

[0049] The process of obtaining callus preservation number CGMCC No.10002 from the cell lines induced by the explants of Taxus chinensis var. mairei is: cleaning the explants, disinfecting with 75% alcohol, disinfecting with 0.1% mercuric chloride, inoculating and culturing [4] . See attached figure 1

[0050] 1. Explant disinfection

[0051] 1.1 Cleaning

[0052] Put the explants in a basin of clean water, add a small amount of detergent, gently rub the surface of the explants with your hands, then rinse with clean tap water 3 times, rinse with purified water 3 times, and finally rinse with ultrapure water 3 times. After washing, place it on clean filter paper, separate the leaves from the tender stems, place the leaves in a plate with clean filter paper, absorb excess water, and cover the lid. Cut the tender stems into small sections with scissors, and place them in a petri dish wit...

Embodiment 2

[0065] Subculture of callus of paclitaxel-producing cell line.

[0066] After the cells are successfully induced, they will be subcultured every 21 days. During the subculture process, the cells will brown and become dry. The problem of browning was solved by adjusting the subculture medium and adding anti-browning agents. After a series of experiments, VC, PVP, L-glutamine, etc. were added. After subculture adjustment, it was transferred to B5+2,4-D(1mg / l) + 6-BA (0.1mg / L) + L-glutamine (0.2928g / L) + VC (100mg / L), the cell status is stable and the browning is reduced; the cells are stable for passage. See attached image 3 .

Embodiment 3

[0068] Taxonomic identification of strains:

[0069] The identification method selects 18S rDNA ITS sequencing, and then conducts systematic evolution analysis based on the sequence, and conducts species identification from genetic biological information. Extraction of whole plant cell genome DNA: Operate according to the instructions of Shanghai Shenggong Company Ezup Column Type Plant Genomic DNA Extraction Kit SK8261; without liquid nitrogen, grind in the Buffer PCB solution heated directly in the material, and place the whole plant genome after TE dissolution. Store frozen at -20°C; PCR and nucleic acid electrophoresis: Primer pair: rDNA-18S-F sequence: 5'- GCG GTA GGA TCA TTG TCG-3'; ITS4-R sequence: 5'- TCC TCC GCT TAT TGA TAT GC- 3'Synthesized by Shanghai Shenggong Company, and the substrate is the extracted and purified plant cell DNA. Perform PCR according to the instructions of the Shanghai Biotech Taq PCR Master Mix Kit BS9293; after PCR, perform 1% w / v agarose gel el...

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Abstract

The invention discloses a taxus chinensis cell strain (Taxus wallichiana var.mairei) with the high-yield paclitaxel characteristic. The taxus chinensis cell strain is named taxus chinensis var.mairei plant cell system NO51-3 which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCCNo.10002. The invention further discloses a method for screening and establishing a paclitaxel high-yield cell system (Paclitaxel, CASNO:33069-62-4, with a trade name Taxol as a common name). The cell system with the high yield of paclitaxel is induced and screened from taxus chinensis explants, and the experiment result shows that the cell system is high in yield of taxane compounds such as secondary metabolite paclitaxel, and can be used as a raw material medicine for treating ovarian cancer, breast cancer and the like.

Description

Technical field [0001] The present invention belongs to the technical field of plant cell culture, and specifically relates to a yew cell line with the characteristics of high yield of taxol ( Taxus wallichiana var. mairei) And its application. Background technique [0002] Paclitaxel is an anti-cancer drug isolated from Taxus plants and has been proven to be effective in treating ovarian cancer, breast cancer and lung cancer. At present, taxol is extracted from the bark of Taxus plants as raw materials, which not only damages the environment and endangers the resources of Taxus, but also because of the slow growth of Taxus plants and the low content of taxol, the source of medicine is extremely scarce and expensive. In addition, although full chemical synthesis has been successful, actual industrial production cannot be realized because of the high cost. Therefore, it is necessary to find an alternative to chemical synthesis. The production of taxol by yew plant tissue and cell...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12P17/02
Inventor 张卫王丽丁靖志孙波吕建明
Owner TIANJIN ACELBIO BIOTECH
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