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PROCESS FOR PREPARING A SERINE PROTEASE INHIBITOR FROM CELL CULTURES INDUCED FROM LINUM USITATISSIMUM
专利权人:
INSTITUTUL NATIONAL DE CERCETARE DEZVOLTARE CHIMICO-FARMACEUTICA - ICCF
发明人:
EREMIA MIHAELA CARMEN,EREMIA MIHAELA CARMEN,LUPESCU IRINA,LUPESCU IRINA,ROSU ANA,ROŞU ANA,TCACENCO LUMINITA,TCACENCO LUMINIŢA,SAVOIU GABRIELA,SAVOIU GABRIELA,SPIRIDON MARIA,SPIRIDON MARIA
申请号:
RO201101366
公开号:
RO128629A2
申请日:
2011.12.09
申请国别(地区):
RO
年份:
2013
代理人:
摘要:
The present invention relates to a process for preparing a serine protease inhibitor from cell cultures induced from flax (Linum usitatissimum) by sterilizing the biological material formed of cell cultures by using explants as a source, after sterilization, the explants are placed into Petri dishes, on the surface of a Murashige-Skoog growth medium, and are incubated in the growth chamber. Periodically, at an interval of 3 weeks, there takes place the transfer to a fresh growth medium, in compliance with the aseptic technique measures with the view of preventing accidental contamination with microorganims. The obtained somatic explants are processed in order to isolate the protease inhibitor by delipidation with acetone, extraction with acetate buffer solution 0.1M, pH=5 and fractional precipitation with ethanol, there resulting a protein precipitate with a specific protease inhibitory activity of 3129 IU/mg of protein, which is purified in phosphate buffer 0.05M, pH=7.5, on affinity chromatography column, on immobilized trypsin equilibrated with phosphate buffer. The non-bonded proteins are washed with the same buffer and by elution with phosphate buffer pH =7.5, in a concentration gradient from 0 to 0.3 M, in equal volumes, there resulting a serine protease inhibitor with a specific activity of 9681 IU/mg of protein.Prezenta invenţie se referă la un procedeu de obţinere a unui inhibitor serin-proteazic din culturi celulare induse de la in (), prin sterilizarea materialului biologic format din culturi celulare utilizând, ca sursă, explantule, după sterilizare, explantulele se plasează în cutii Petri, pe suprafaţa unui mediu de cultură Murashige-Skoog, şi se incubează în camera de creştere. Periodic, la interval de 3 săptămâni, se face transferul pe mediu de cultură proaspăt, cu respectarea măsurilor de asepsie, pentru a preveni contaminarea accidentală cu microorganisme. Explantele somatice obţinute se prelucrează în vederea izolării inhibitorului proteazic, pr
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