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Method for acquiring new-type medicine source of camptothecin by adopting genetic co-transformation strategy

A technology of camptothecin and co-transformation, applied in the field of bioengineering

Inactive Publication Date: 2015-05-27
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the downstream synthetic pathway from oleandrin to camptothecin is not clear, and only a few intermediate products such as kaebebenoside / short snakeroot (3-S-Pumiloside) and deoxybebenoside ( 3-S-Deoxypumiloside) (Guan Shui et al 2004, Wang Lei et al 2008)

Method used

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  • Method for acquiring new-type medicine source of camptothecin by adopting genetic co-transformation strategy
  • Method for acquiring new-type medicine source of camptothecin by adopting genetic co-transformation strategy
  • Method for acquiring new-type medicine source of camptothecin by adopting genetic co-transformation strategy

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Obtaining of periwinkle CrSTR and CrG10H gene coding sequence

[0036] 1.1. Extraction of periwinkle total RNA and synthesis of cDNA first strand

[0037] Total RNA was extracted from periwinkle seedlings using the RNA prep pure plant kit provided by TIANGEN (see 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).

[0038] 1.2. Design of specific primers for CrSTR...

Embodiment 2

[0040] Embodiment 2 Contains the construction of the plant expression vector of periwinkle CrSTR and CrG10H gene

[0041] 2.1. Intermediate vector pCAMBIA1304 + build

[0042] 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.

[0043] 2.2. Plant expression vector pCAMBIA1304 + - Construction of CrG10H

[0044] The successful pCAMBIA1304 constructed above + Basically, replace the GUS gene on it with the CrG10H gene cloned from periwinkle. Specifically, BamHI / SacI double enzyme cut pMD18T-CrG10H and pCAMBIA1304 + ; Recover CrG10H gene and pCAMBIA1304 ...

Embodiment 3

[0048] Example 3 Agrobacterium rhizogenes mediates the genetic transformation of CrSTR and CrG10H genes to obtain transgenic hairy roots of Serpentis brevis

[0049] 3.1. Containing plant expression vector pCAMBIA1304 + -Acquisition of CrSTR-CrG10H Agrobacterium rhizogenes Engineering Bacteria

[0050] The plant bivalent expression vector pCAMBIA1304 containing CrSTR and CrG10H gene in embodiment 2 + -CrSTR-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 CrSTR and CrG10H genes had been successfully constructed in Agrobacterium rhizogenes strain C58C1.

[0051] 3.2. Agrobacterium rhizogenes Mediated CrSTR, CrG10H Gene Genetic Transformation of Serpentis brevis

[0052] 3.2.1. Preculture of explants

[0053] Cut aseptic short snakeroot stems (1-3cm) and inoculate them into the pre-culture medium (B 5 ), cultured in the dark at 28°C for 2 days...

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Abstract

The invention relates to the technical field of bioengineering and discloses a method for improving camptothecin content in hairy roots of the new-type medicine source plant ophiorrhiza pumila of camptothecin. The method disclosed by the invention comprises the following steps of: cloning coding frame sequences of genes of ophiorrhiza japonica strictosidine synthase and geraniol-10-hydroxylase in catharanthus roseus to build a plant bivalent efficient expression vector containing the genes, carrying out genetic transformation on ophiorrhiza pumila via an agrobacterium rhizogenes mediated method to acquire the hairy roots of the ophiorrhiza pumila for transforming CrSTR and CrG10H genes; and inducing and treating high-yield camptothecin strains by adopting plant hormones to acquire high-yield hairy roots with the camptothecin content of 4.703mg / g DW. The MTT (Methyl Thiazolyl Tetrazolium) detection proves that camptothecin crude extract acquired via a transgenosis manner is good in biological activity and the lethality to cancer cells reaches 35.9%. By adopting the method disclosed by the invention, a new medicine source for acquiring the camptothecin is provided and a new method for producing anti-cancer medicine camptothecin in important clinical demand is provided.

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 by using a gene co-transformation strategy, and provides a new drug source for the commercial production of the anticancer drug camptothecin Method of raw material preparation. Background technique [0002] Camptothecin (CPT), a quinoline alkaloid, is a class of natural anticancer drugs originally extracted from Camptotheca acuminata. 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 compounds was hailed as "the three major discoveries of anti-cancer drugs in the 1990s". At present, a variety of camptothecin d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/60C12N15/53A01H5/06
Inventor 滕小娟开国银季倩时敏崔丽洁
Owner SHANGHAI NORMAL UNIVERSITY
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