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Preparation method of novel medicine source raw material of camptothecin

A camptothecin, a new type of technology, applied in botany equipment and methods, biochemical equipment and methods, angiosperms/flowering plants, etc., can solve the problems of insufficient expression and no significant increase in the yield of TIA metabolic end products

Inactive Publication Date: 2013-07-10
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It must be pointed out that transcription factors are not omnipotent. Although a single transformation of an ORCA3 gene can regulate the coordinated expression of multiple key enzyme genes such as AS, TDC, DXS, CPR and STR, some genes such as the key enzyme gene G10H are not affected. Regulation of ORCA3
In the ORCA3 gene overexpressed cells, due to the insufficient expression of the key enzyme gene G10H, the production of TIA metabolic end products such as vinblastine was not significantly improved.

Method used

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  • Preparation method of novel medicine source raw material of camptothecin
  • Preparation method of novel medicine source raw material of camptothecin
  • Preparation method of novel medicine source raw material of camptothecin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Obtaining of periwinkle CrORCA3 and CrG10H gene coding sequence

[0032] 1.1. Extraction of total RNA and synthesis of cDNA first strand from periwinkle

[0033] Total RNA was extracted from periwinkle seedlings using the RNA prep pure plant kit provided by TIANGEN (refer to the instructions in the kit for the extraction steps). The fresh weight of periwinkle seedlings used to extract total RNA is about 0.1 g, and the DNA in the sample has been removed with DNase working solution during the extraction process. Measure the relative absorbance value of the extracted RNA on a spectrophotometer, and calculate the purity and concentration of the extracted RNA. After calculation based on the concentration of different RNA samples, the first-strand cDNA was synthesized with reverse transcriptase XL (AMV) using 0.5 μg RNA as the initial amount (for the operation steps, refer to the relevant instructions provided by Promega).

[0034] 1.2. Design of specific pri...

Embodiment 2

[0036] Embodiment 2 Containing the construction of the plant expression vector of periwinkle CrORCA3 and CrG10H gene

[0037] 2.1. Intermediate vector pCAMBIA1304 + build

[0038] Using pBI121 and pCAMBIA1304 as materials, construct the plant expression vector pCAMBIA1304 + . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII / EcoRI; the pBI121-GUS expression cassette and the large fragment of pCAMBIA1304 were recovered; ligation transformation was carried out, and single clone colonies were picked to extract plasmids for digestion verification. The results showed that the plant expression vector pCAMBIA1304 + The build was successful.

[0039] 2.2. Plant expression vector pCAMBIA1304 + - Construction of CrG10H

[0040] The successful pCAMBIA1304 constructed above + Basically, replace the GUS gene on it with the CrG10H gene cloned from periwinkle. Specifically, BamHI / SacI double-digest pMD18T-CrG10H and pCAMBIA1304 + ; Recover CrG10H gene and pCAMBIA1304...

Embodiment 3

[0044] Example 3 Agrobacterium rhizogenes mediated genetic transformation of CrORCA3 and CrG10H genes to obtain transgenic hairy roots of Serpentis brevis

[0045] 3.1. Plant expression vector pCAMBIA1304 + -Acquisition of CrORCA3-CrG10H Agrobacterium rhizogenes Engineering Bacteria

[0046] The plant bivalent expression vector pCAMBIA1304 containing CrORCA3 and CrG10H gene in embodiment 2 + -CrORCA3-CrG10H was transferred into Agrobacterium rhizogenes C58C1, and a single clone colony was picked for PCR verification. The results showed that the plant expression vector containing CrORCA3 and CrG10H genes had been successfully constructed in Agrobacterium rhizogenes strain C58C1.

[0047] 3.2. Agrobacterium rhizogenes Mediated CrORCA3, CrG10H Gene Genetic Transformation of Serpentis brevis

[0048] 3.2.1. Preculture of explants

[0049] Cut the aseptic stems (1-3 cm) of Snakeroot short, inoculate them on the pre-cultivation medium (B5), and culture them in the dark at 28°...

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Abstract

The invention belongs to the technical field of bioengineering, and provides a method for improving camptothecin content in hairy roots of novel medicine source plant ophiorrhiza pumila of camptothecin. The method comprises the following steps of: cloning coder frame sequences of CrORCA3 and CrG10H genes from catharanthus roseus; constructing a bivalent efficient expression vector of a plant containing such genes; and carrying out genetic transformation to the ophiorrhiza pumila through the agrobacterium rhizogenes mediated transformation method, thus obtaining the hairy roots of the ophiorrhiza pumila containing transformed CrORCA3 and CrG10H genes. The camptothecin content in the obtained hairy roots of the transgenic ophiorrhiza pumila is obviously increased, the camptothecin output in each flask(2.62mg / flask) is averagely increased by 5.52 times in comparison with that in the contrast, and the average content (8.44mg / g DW) is increased by 4.04 times. The total camptothecin output of each flask, induced by plant hormones after culturing one strain for six weeks, is increased by 7.8 times. The preparation method provided by the invention is a novel method for producing an anti-cancer drug that is camptothecin under important clinical demand.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for increasing the content of camptothecin in the hairy roots of Snakeroot brevis, a novel drug source raw material for camptothecin, and provides a method for commercially producing the anticancer drug camptothecin. The invention discloses a preparation method of a novel drug source raw material. Background technique [0002] Camptothecin (CPT), a quinoline alkaloid, is a class of natural anticancer drugs originally extracted from Camptotheca acuminata. Camptothecin drugs have high efficiency, low toxicity and broad-spectrum anticancer It is one of the most effective natural anti-cancer drugs, and its anti-cancer mechanism is very unique. It is the only natural plant active ingredient discovered so far that exerts cytotoxicity by inhibiting topoisomerase Ⅰ. The discovery of the anti-tumor effect of camptothecin and its derivatives, paclitaxel and vitamin A compo...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29C12N15/53A01H5/06
Inventor 滕小娟开国银李姗姗郝小龙崔丽洁
Owner SHANGHAI NORMAL UNIVERSITY
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