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METHOD OF PRODUCTION OF DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA) FROM CELLS OF YEAST Saccharomyces cerevisiae

机译：从酵母菌的细胞生产双链核糖核酸（dsRNA）的方法

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FIELD: biotechnology.SUBSTANCE: method of production of double-stranded ribonucleic acid (dsRNA) from cells of strain Saccharomyces cerevisiae RNCIM Y-448 comprises breaking the yeast cells in a buffer with pH 7.4, containing 10 mM Tris, 20 mM EDTA and 0.5 M NaCl, processing with sodium dodecyl sulphate at a concentration from 0.5 to 1.0% for 20-25 minutes at 20°C and with chloroform at a concentration of up to 25% for 20-25 minutes at 20°C. The resulting mixture is centrifuged at 6000 rev/min for 20 minutes. Concentration of dsRNA is carried out in 7-8% solution of PEG 6000 for at least 5 hours at 6°C followed by centrifugation at 6000 rev/min for 20 minutes and dissolving the resulting precipitated concentrate in water. SsRNA is separated from dsRNA in the 2 mol/l solution of LiCl for 5 h at 6°C, followed by centrifugation of the mixture at 6000 rev/min for 20 minutes, and collecting the aqueous phase. DsRNA is precipitated from the aqueous phase in 3.5 mol/l solution of LiCl for 5 h at 6°C followed by centrifugation at 6000 rev/min for 20 minutes to obtain a precipitate and dissolving it in water. Purification and precipitation of dsRNA from the solution is carried out with 55% ethanol solution. Yield of dsRNA is at least 90%, and the interferon titer in blood serum using the preparation based on the solution of dsRNA is after 24 hours 1280±60 u/ml.EFFECT: increased yield of dsRNA higher purity and interferon-inducing activity.2 cl, 5 dwg, 3 tbl, 6 ex

机译：由酿酒酵母RNCIM Y-448菌株的细胞生产双链核糖核酸（dsRNA）的方法包括在pH 7.4的缓冲液中破坏酵母细胞，该缓冲液含有10 mM Tris，20 mM EDTA和0.5 M NaCl，在20°C下用浓度为0.5％至1.0％的十二烷基硫酸钠处理20-25分钟，在20°C下用浓度最高为25％的氯仿处理20-25分钟。将所得混合物以6000转/分钟离心20分钟。 dsRNA的浓缩在6°C下于PEG 6000的7-8％溶液中进行至少5小时，然后以6000转/分钟的速度离心20分钟，然后将所得沉淀的浓缩物溶解在水中。在2 mol / l的LiCl溶液中于6°C下5个小时将SsRNA与dsRNA分离，然后将混合物以6000转/分钟的速度离心20分钟，并收集水相。在6°C下于3.5 mol / l的LiCl溶液中从水相中沉淀DsRNA 5小时，然后在6000转/分钟的条件下离心20分钟，以获得沉淀并将其溶解在水中。用55％乙醇溶液从溶液中纯化和沉淀dsRNA。 dsRNA的产率至少为90％，使用dsRNA溶液制备的制剂在血清中的干扰素效价为1280±60 u / ml，在24小时后。效果：提高dsRNA的产率具有更高的纯度和诱导干扰素的活性。 2 cl，5 dwg，3 tbl，6 ex

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公开/公告号RU2558256C1

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公开/公告日2015-07-27

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申请/专利权人 FEDERALNOE BJUDZHETNOE UCHREZHDENIE NAUKI GOSUDARSTVENNYJ NAUCHNYJ TSENTR VIRUSOLOGII I BIOTEKHNOLOGII VEKTOR (FBUN GNTS VB VEKTOR);
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申请/专利号RU20140119351
发明设计人 LEBEDEV LEONID RUDOLFOVICH;AZAEV MAMEDJAR SHAKIROVICH;ALIKIN JURIJ SERAFIMOVICH;PODGORNYJ VLADIMIR FEDOROVICH;
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申请日2014-05-13
分类号C12P19/34;C12N1/16;
国家 RU
入库时间 2022-08-21 14:56:13

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