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Application of anti-gliotoxin self-protection gene GliK in assisting host cells in resisting gliotoxin

A technology of gliotoxin and gene protection, applied in the field of genetic engineering, can solve problems such as redox reaction imbalance, induction of cell apoptosis, and reduction of immune activity

Active Publication Date: 2021-04-20
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The toxicity of gliotoxin to the host mainly includes: inducing apoptosis; causing redox reaction imbalance; inhibiting proteasome activity, leading to the inhibition of NF-κB activity, reducing immune activity, etc.

Method used

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  • Application of anti-gliotoxin self-protection gene GliK in assisting host cells in resisting gliotoxin
  • Application of anti-gliotoxin self-protection gene GliK in assisting host cells in resisting gliotoxin
  • Application of anti-gliotoxin self-protection gene GliK in assisting host cells in resisting gliotoxin

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Example 1 Obtaining the self-protection gene sequence of the deep-sea fungus Geosmithiapallida FS140 against gliotoxin

[0021] Amplification of the gene GliK: Inoculate the deep-sea fungus Geosmithiapallida FS140 on a YPD medium plate, culture at 37°C for 72 hours, pick fresh mycelium, use the fungal RNA extraction kit to extract RNA, and then use the All-in-oneRTMaster Kit to reverse The cDNA was recorded. According to the results of transcriptome sequencing, the sequence of the anti-trichothecene self-protection gene GliK was predicted, and specific primers were designed upstream and downstream of it. The primer sequence was GliK-F:5'-ATGACCATACAACTCCCTCACAC-3'; GliK-R:5' -AGCGGAGGGCTGGGCGTAGC-3', using the cDNA library as template amplification to obtain PCR product ( figure 2). The product was recovered and cloned by TA with pEASY-T1 kit, transformed into Escherichia coli competent cells, spread on the ampicillin resistance plate to screen out positive clones, a...

Embodiment 2

[0022] Example 2 Functional verification of anti-gliotoxin self-protection gene GliK

[0023] The gene GliK was inserted into the yeast vector YEp352-TEF1-CYC1 by homologous recombination (YEp352-TEF1-CYC1 is an early constructed plasmid, carrying a constitutive promoter TEF1 and a terminator CYC1, see the vector map image 3 A, known products in the prior art: Xiaodan Ouyang, Yaping Cha, Wen Li, Chaoyi Zhu, Muzi Zhu, ShuangLi, Min Zhuo, Shaobin Huang and Jianjun Li. Stepwise engineering of Saccharomyces cerevisiae to produce(+)-valencene and its related sequiterpenes, RSC Adv., 2019, 9, 30171, DOI: 10.1039 / c9ra05558d). First design the upstream and downstream primers YEp352-GliK-F and YEp352-GliK-R for the amplification of gene GliK (SEQ ID NO.1), the primer sequence of which is YEp352-GliK-F:5'- AGCAATCTAATCTAAGTCTAGA ATGACCATACAACTCCCTCACAC-3'; YEp352-GliK-R:5'- TTACATGATGCGGCCCGTCGAC AGCGGAGGGCTGGGCGTAGC-3' (the underlined sequence is the homology arm fragment), the cD...

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Abstract

The invention discloses an application of an anti-gliotoxin self-protection gene GliK in assisting host cells in resisting gliotoxin. The nucleotide sequence of the anti-gliotoxin self-protection gene GliK is as shown in SEQ ID NO.1. Therefore, an anti-gliotoxin self-protection gene GliK sequence is obtained from a cDNA library of deep sea fungi FS140 and is successfully introduced into saccharomyces cerevisiae S.cerevisiae BJ5464 for anti-toxic function verification, so that the anti-gliotoxin capability of the saccharomyces cerevisiae is improved in the later period, the heterologous expression level of the gliotoxin is improved, and a molecular biological foundation is laid for obtaining the novel gliotoxin.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to the application of an anti-gliotoxin self-protection gene GliK in assisting host cells to resist gliotoxin. Background technique [0002] In the early stage, our research group isolated more than 20 gliotoxin compounds from the deep-sea fungus Geosmithia pallida FS140, including gliotoxin dimer compounds with rare structures, most of which have obvious antitumor activity . [0003] Gliotoxin (GT) is a kind of diketopiperazine (epipolythiodioxopiperazine, ETP), ETP as an important virulence factor, it produces specific toxicity to various cells through different pathways, in invasive aspergillosis play an important role in synergy. The toxic and side effects of ETP are mainly through the cross-linking of disulfide bonds and sulfhydryl groups with the target protein to inactivate the protein activity, and can generate toxic reactive oxygen species (reactive oxygen sp...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N15/80C12N1/19C12N1/15C12R1/865C12R1/645
Inventor 李赛妮章卫民叶伟张维阳
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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