Taxus chinensis cell strain with high yield of 10-DAB and application thereof

A technology of 10-DAB and yew cells, applied in the field of yew cell lines, can solve the problems that docetaxel cannot be extracted, and achieve the effects of frequent felling and planting, slow and stable growth, and simple extraction, separation and purification

Active Publication Date: 2015-03-11
安赛搏(重庆)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, at present, docetaxel cannot be extracted from natural plants, and can only be produced by chemical physical synthesis after the production of 10-DAB

Method used

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  • Taxus chinensis cell strain with high yield of 10-DAB and application thereof
  • Taxus chinensis cell strain with high yield of 10-DAB and application thereof
  • Taxus chinensis cell strain with high yield of 10-DAB and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Induction of 10-DAB-producing high-yield cell line preservation number CGMCC No.10001

[0049] (1) The callus preservation number of the cell line induced by explants of Taxus chinensis CGMCC No.10001

[0050] The explants were cleaned, sterilized with 75% alcohol and 0.1% mercuric chloride, inoculated and cultured.

[0051] 1. Disinfection of explants

[0052] 1.1 Cleaning

[0053] Put the explants into a clear water basin, add a small amount of detergent, gently scrub the surface of the explants with your hands, then rinse with clean tap water 3 times, with purified water for 3 times, and finally with ultrapure water for 3 times. After washing, put it on clean filter paper, separate the leaves and tender stems, put the leaves in a plate with clean filter paper, absorb excess water, and cover with a lid. Cut the tender stems into small pieces with scissors, and put them in a plate with clean filter paper and cover them for disinfection.

[0054] 1.2 Disinfection

...

Embodiment 2

[0068] Subculture of callus producing 10-DAB cell line.

[0069] After the cells were successfully induced, they were subcultured every 21 days. During the subculture process, the cells appeared brown and dry. The browning problem was solved by adjusting the subculture medium and adding anti-browning agents. After a series of experiments, VC, PVP, L-glutamine, etc. were added, and after subculture adjustment, it was transferred to B5+2,4-D (1mg After / l)+6-BA (0.1mg / l)+L-glutamine (0.2928g / l)+VC (100mg / l), the cell state was stable and the browning was alleviated; the cells were stably subcultured

[0070] The callus was subcultured for 13 generations, among which 7 times were subcultured in WR medium, and then transferred to B5-15, which was labeled as B5-15 (12#WR stem was transferred for 5 generations), and the callus was subcultured. See attached Figure 5 .

Embodiment 3

[0072] Taxonomic identification of strains:

[0073] (1) The identification method is 18S DNA ITS sequencing, and then the phylogenetic analysis is carried out according to the sequence.

[0074] Species identification from genetic biological information. Whole-genome DNA extraction from plant cells: operate according to the instructions of Ezup Column Plant Genome DNA Extraction Kit SK8261 of Shanghai Shenggong Company; without liquid nitrogen, grind with Buffer PCB solution directly heated in the material, dissolve the whole genome of plants in TE, and place in Freeze 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' was synthesized by Shanghai Shenggong Co., Ltd., and the substrate is the extracted and purified whole gene DNA of plant cells. Perform PCR according to the instructions of Taq PCR Master Mix Kit BS9293 from Shanghai Sangon Company; after PCR, pe...

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Abstract

The invention discloses a taxus chinensis cell strain with high yield of 10-DAB, is named taxuswallichianavar. mairei plant cell line N12#, and is preserved in CGMCC, and the preservation number is CGMCC No.10001. The invention further discloses screening and establishment of a 10-DAB high-yield cell line (10-DeacetylbaccatinIII, CASNO: 32981-86-5). A high-yield 10-DAB cell line is induced and screened from taxuswallichianavar. mairei explant. Experimental results prove that the cell line can highly produce taxane diterpenoids such as secondary metabolites 10-DAB and can be used for production of crude drug precursors of paclitaxel and docetaxel.

Description

technical field [0001] The invention belongs to the technical field of plant cell culture, in particular to a yew cell line ( Taxus wallichiana var. mairei) and its application. Background technique [0002] 10-deacetyl baccatin Ⅲ (10-deacetyl baccatin Ⅲ, abbreviated as 10-DAB) is a taxane diterpenoid compound isolated and extracted from the short branches of Taxus chinensis. It not only has anticancer activity itself, but also It is an important precursor compound for biosynthesis and chemical semisynthesis of paclitaxel and docetaxel, and plays an irreplaceable role in solving the problem of drug sources of paclitaxel and docetaxel for clinical treatment and scientific research. Scholars at home and abroad have done a lot of basic research work on the production of paclitaxel by yew cell suspension culture, but there is still a long way to go from industrial production. There are few reports on the production of 10-DAB by yew cell culture. The present invention produces...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04C12P17/02C12R1/91
Inventor 王丽朱瑞雪刘晓月王海燕张桂才张卫
Owner 安赛搏(重庆)生物技术有限公司
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