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Fungal cell wall synthesis gene

A technology of fungal cell walls and fungi, which can be used in antifungal agents, genetic engineering, plant genetic improvement, etc., and can solve the problem of few effective therapeutic drugs

Inactive Publication Date: 2006-12-06
EISIA R&D MANAGEMENT CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although strategies for the control of infectious diseases (including the problem of bacterial resistance) will be one of the crucial issues in the coming aging society, relatively few effective therapeutic drugs currently exist

Method used

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  • Fungal cell wall synthesis gene
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  • Fungal cell wall synthesis gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment A

[0275] The present invention will be described in detail below with reference to examples, but the present invention is not limited by these examples.

Embodiment A1

[0276] Example A1: Construction of a reporter gene and its introduction into Saccharomyces cerevisiae

[0277] (1) Construction of lysozyme as the reporter gene of the reporter enzyme

[0278] Use the pESH plasmid (IchikawaK et al, Biosci. Biotech. Biochem., 57 (10), 1686-1690, 1993) comprising the ENO1 promoter + secretion signal + lysozyme gene as a template, with SEQ ID NO: 8 and SEQ ID The oligonucleotide NO:9 was used as a primer, and the lysozyme gene containing the promoter sequence was amplified by PCR and subcloned into the SalI-EcoRI site of pCR-Script SK (+) (a). In addition, using Saccharomyces cerevisiae chromosomal DNA as a template and oligonucleotides of SEQ ID NO: 10 and SEQ ID NO: 11 as primers, the CWP2 gene was amplified by PCR and subcloned into the EcoRI-HindIII site of pUC19 (b). Similarly, using pYES2 (INVITROGEN) as a template and using oligonucleotides of SEQ ID NO: 12 and SEQ ID NO: 13 as primers, the CYC1 terminator was amplified by PCR and subclo...

Embodiment A2

[0286] Example A2: Screening Drugs by the Saccharomyces cerevisiae Reporter System

[0287] Since the sensitivity of the enzymatic reaction using cephalosporinase was higher than that of using lysozyme, Saccharomyces cerevisiae (S. cerevisiae CW63 strain) into which pRCW63T was introduced was used for screening of compounds.

[0288] After statically culturing in YPD liquid medium at 30°C for 48 hours, the yeast cell culture was diluted 100 times with YPD liquid medium (3~5×10 5 cells / ml), and inoculate them in 75 μl / well aliquots in V-bottom 96-well plates (containing 25 μl / well diluted samples), and culture statically at 30° C. for 48 hours. After centrifuging the plate, 25 μl of the supernatant was sampled and placed in a flat-bottomed 96-well plate as the culture supernatant fraction.

[0289] The precipitated cells were suspended, and 75 µl / well of an enzymatic enzyme (Seikagaku Corporation) solution prepared with 2.4 M sorbitol was added in aliquots, and reacted at 30°C...

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PUM

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Abstract

A reporter system reflecting the transport process that transports GPI-anchored proteins to the cell wall was constructed and compounds inhibiting this process were discovered. Further, genes conferring resistance to the above compounds were identified and methods of screening for compounds that inhibit the activity of the proteins encoded by these genes were developed. Therefore, through the novel compounds, the present invention showed that antifungal agents having a novel mechanism, i.e. inhibiting the process that transports GPI-anchored proteins to the cell wall, could be achieved.

Description

[0001] This application is based on the divisional application of the invention application whose application number is 200610004398.1, the application date is July 6, 2001, and the invention title is "Fungus Cell Wall Synthesis Gene". technical field [0002] The present invention relates to DNA encoding proteins involved in fungal cell wall synthesis, proteins encoded by the DNA, methods of examining whether certain compounds have an effect on the transport process involved in the transport of GPI-anchored proteins to the cell wall, and the effects on GPI-anchored proteins. Antifungal agents that affect the transport process involved in the transport of anchored proteins to the cell wall. Background technique [0003] In recent years, management of opportunistic infections has become more important than ever due to an increase in the number of elderly people and immunocompromised patients due to intensive chemotherapy and the like. Deep fungal infections caused by Candida,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/31C07K14/37C07K16/14G01N33/15G01N33/50A61K39/395A61K45/00A61K31/435A61K31/436A61K31/437A61K31/44A61K31/472A61K31/537A61P31/10C07D213/16C07D213/61C07D213/65C07D213/69C07D213/74C07D217/18C07D217/20C07D401/10C07D401/12C07D405/06C07D405/10C07D405/12C07D471/04C07D491/048C07D491/056A61K31/00A61K31/4355A61K31/4365A61K31/4725A61K31/5377A61K38/00C07K14/38C07K14/39C07K14/395C07K14/40
CPCC07K2319/02A61K31/437C07K14/40A61K31/4365A61K31/5377A61K31/44C07K14/395C07K14/39A61K31/4725A61K31/472A61K31/00C07K14/38C07K14/37G01N2500/00A61K38/00A61K31/4355
Inventor 塚原克平畑桂相根康司中本和孝土谷满美子渡边直彰大场史记塚田格上田教博田中圭悟甲斐纯子
Owner EISIA R&D MANAGEMENT CO LTD
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