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Small molecular double stranded RNA group prepared from yeast, preparation method and use thereof

A double-stranded ribonucleic acid and ribonuclease technology, which can be used in fermentation, pharmaceutical formulations, organic active ingredients and other directions, can solve the problems of high cost and cannot meet large-scale industrialization, and achieve the effects of easy operation and simple preparation method.

Active Publication Date: 2012-01-18
SHUIAI BIOTECH NANTONG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since dsRNA acts as a ligand to activate TLR3 with a broad spectrum (what sequence can be used as a ligand to activate TLR3), as long as it meets the functional length of 19-25bp, it can be used to activate TLR3 receptors (ME Kleinman, Nature 2008), but the raw material cost Expensive, tens of thousands of yuan per gram, currently unable to meet the requirements of large-scale industrialization

Method used

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  • Small molecular double stranded RNA group prepared from yeast, preparation method and use thereof
  • Small molecular double stranded RNA group prepared from yeast, preparation method and use thereof
  • Small molecular double stranded RNA group prepared from yeast, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Single and double-stranded RNA digestion test

[0069] The purpose of the experiment is to observe the digestion of three RNases (RNaseT1; RNaseI and RNaseA) with ssRNA and dsRNA and highly refolded natural yeast total RNA as substrates, and to select the best combined enzyme of the present invention.

[0070] 1 Main reagents and materials

[0071] 1.1 Main reagents: Three kinds of RNases were purchased from Fermentas Company (agent in China), among them: RNaseT1, 1000,000U / mL; RNaseI, 10,000U / mL; RNaseA, 10mg / mL. DNaseI (5000U / mL) was purchased from Takara Japan.

[0072] 1.2 Main material: small molecule total RNA of Candida tropicalis (Qiu Zhiyou Biotechnology; preservation number: CGMCC No.3558. Yeast total RNA has not been completely degraded into small molecules when it leaves the factory. This experiment is used to observe the effect of RNase on high renatured ssRNA and dsRNA digestion results); chemically synthesized RNA Oligo (Shanghai Sangon Biology), the sy...

Embodiment 2

[0083] 1. Saccharomyces culture: the saccharomyces cerevisiae (Saccharomyces cerevisiae AS21416 purchased from Chinese Academy of Sciences Institute of Microbiology) is inoculated in 3ml YPD liquid medium (10g yeast extract; 20g tryptone) by 1-3% concentration ; 20g glucose; add water to 1000mL), shake overnight at 33°C, and culture until late logarithmic growth. The bacterial suspension was inoculated in 100 mL of YPD liquid medium at a ratio of 1:50, cultured with shaking at 33°C for 2-3 hours until the OD600 reached 0.45-0.55, and the culture was stopped. Add 1% helicase (Beijing Huibao Lianhua Technology Co., Ltd.) to the culture medium, and shake and react at 30° C. for 1 h.

[0084] 2. Extraction of yeast total RNA: Centrifuge the yeast at 5000 g for 10 min, discard the supernatant and collect the bacterial sediment. Biomac RISO for yeast sediment TM RNA extract (Zhu Yuanyuan; Chinese Patent No. ZL01113523.9) was used to extract yeast total RNA, centrifuged (12000rpm)...

Embodiment 3

[0086] Digestion of yeast total RNA with DNaseI and RNase T1 mixture

[0087] 1. The mixed solution of RNaseT1 and DNase 1 (manufacturer is the same as in Example 1) is mixed at 1:0.001 (V:V). The reaction system for enzyme mixture digestion is as follows: 20 μL (20 μg) RNA in 200 μL solution; 10 μL RNaseT1 and DNaseI enzyme mixture; 20 μL 10XTE buffer, 150 μL sterile water. React at 37°C for 1 hour, add 20 μL EDTA (250 mM) to stop the reaction, and heat at 72°C for 20 minutes to inactivate the enzyme. Take 2X5μL electrophoresis analysis (repeated identification of two swimming lanes in parallel). Add 1 μL of yeast dsRNA to 99 μL of 1xTE buffer solution (100-fold dilution), and measure the ratio of OD260 and OD280 with an ultraviolet spectrophotometer (a value between 1.8 and 2.0 is double-stranded RNA).

[0088] 2. Electrophoresis analysis of digested products:

[0089] The electrophoresis results of digested products of S. cerevisiae total RNA treated with DNaseI and RNas...

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Abstract

The present invention discloses a small molecular double stranded RNA group prepared from yeast, a preparation method and a use thereof. According to the present invention, the small molecular double stranded RNA group is prepared through adopting a mixture and the total RNA of the yeast as substrates, wherein the mixture comprises at least two selected from ribonuclease T1, deoxyribonuclease I and ribonuclease III, the yeast is the yeast which kills plasmids. The small molecular double stranded RNA group provided by the present invention can be provided for preparing cancer prevention care products, anti-cancer health care products and anti-virus health care products. The preparation method provided by the present invention is simple, and is easy to operate.

Description

technical field [0001] The invention relates to a preparation method and application of a small molecule double-stranded ribonucleic acid group prepared from yeast. Background technique [0002] Toll-like receptors (Toll-like receptors, TLR) are an important class of protein molecules involved in nonspecific immunity (innate immunity), and are also a bridge connecting nonspecific immunity and specific immunity. TLRs are single transmembrane non-catalytic proteins that recognize molecules with conserved structures derived from microorganisms. When microorganisms break through the body's physical barriers, such as skin, mucous membranes, etc., TLR can recognize them and activate the body to generate immune cell responses. [0003] Currently, 11 members of the human TIRs family have been discovered in mammals and humans. According to chromosomal location, gene structure and amino acid sequence, human TLRs receptors can be divided into five subfamilies, namely TLR2, TLR3, TLR4...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12P19/34A61K48/00A61K31/713A61P35/00A61P31/12
Inventor 朱远源
Owner SHUIAI BIOTECH NANTONG
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