Application of reovirus dsRNA in preparation of medical endogenous interferon inducer

A reovirus, interferon-induced technology, applied in antiviral agents, pharmaceutical formulations, allergic diseases, etc., can solve the problems of lack of function, can not completely terminate the TLRs signal, etc., achieve high safety, high efficiency, convenient The effect of industrialized production, treatment of diseases and enhancement of physical fitness

Inactive Publication Date: 2014-12-03
武汉星垣生物技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

MyD88 molecule is an adapter molecule in most TLRs signal transduction, ...

Method used

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  • Application of reovirus dsRNA in preparation of medical endogenous interferon inducer
  • Application of reovirus dsRNA in preparation of medical endogenous interferon inducer
  • Application of reovirus dsRNA in preparation of medical endogenous interferon inducer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] Embodiment 1. The resuscitation of reovirus (Reovirus) amplification and the cultivation and passage of vero cells and other sensitive cells and the culture and passage of cell lines (taking vero cells as an example)

[0111] 1.1 Vero cell culture and proliferation

[0112] Use DMEM medium, add 10% calf serum, adjust the pH to 7.0-7.4, and store at 37°C, 5% CO 2 Under the condition of cultivating and proliferating Vero cells, the steps are as follows.

[0113] 1.1.1 Take out the cryopreservation tube of African green monkey kidney cells (Vero cells) from the liquid nitrogen tank, quickly place it in a 37°C water bath with a pre-adjusted temperature, and shake the cryopreservation tube gently to make the cell suspension in the cryopreservation tube Melts rapidly and completely within 1 min.

[0114] 1.1.2 After thawing, centrifuge the cryopreservation tube, put the cryopreservation tube into the centrifuge, 800-1000rpm, 10-20min, so that the cells are completely precip...

Embodiment 2

[0123] Embodiment 2.Reovirus (see figure 1 , figure 2 )proliferation

[0124] 2.1 Select Vero cells in good condition. When the cells reach 80%-90% confluence, aspirate and discard the culture medium; use 1×PBS with pH 7.4 (NaCl: 8g, KCl: 0.2g, NaCl: 2 HPO 4 : 1.44g, KH 2 PO 4 : 0.24g, H 2 O: 800ml; adjust pH to 7.4 with hydrochloric acid, add H 2 (0 to 1000ml) wash the adherent cells 2-3 times.

[0125] 2.2 Inoculate the cultured cells with Reovirus according to 2-4 MOI (multiplicity of infection); after 1-2 hours of absorbing the virus at 37°C, discard the virus liquid, add DMEM medium containing 2% fetal bovine serum, and keep at 37°C , 5% CO 2 The culture was maintained under (v / v) conditions.

[0126] 2.3 Then observe under the light microscope every 12 hours until the cells have obvious lesion effect, that is, when the CPE reaches 90%, harvest the virus-infected adherent cell flasks and store them at -20°C for later use.

Embodiment 3

[0127] Example 3. Reovirus virulence assay

[0128] 3.1 Take out the spare Reovirus-infected cells and store them at -20°C, freeze and thaw the virus 3 times repeatedly, and release the virus into a Reovirus suspension.

[0129] 3.2 Use 1×PBS (pH7.4) to make a 10× gradient dilution of the Reovirus suspension, that is, the gradient starts from 10 0 -10 -8 , Pay attention when diluting, replace the pipette tip after each gradient dilution is completed.

[0130] 3.3 After trypsinizing the Reovirus, add 15ml of cell culture medium to disperse the cells.

[0131] 3.4 Cell count on hemocytometer (cell number in 1ml = average cell number per grid x 4 x 10 6 ×dilution factor) the cell volume was 3.86×10 8 / ml, add 100 μl of cell suspension to each well.

[0132] 3.5 Place the 96-well plate at 37°C, 5% CO 2 After incubating under (v / v) conditions for 12 hours, the culture medium was sucked off, and the virus solution of gradient dilution was added, 6 wells per gradient, 100 μl per ...

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Abstract

The invention discloses application of reovirus dsRNA in preparation of a medical endogenous interferon inducer. Reov-dsRNAEnII is prepared by using purified reovirus dsRNA as a material, directly acts on a body to induce and generate various endogenous interferons and endows a human body with functions of virus resistance, tumor resistance and constitution enhancement. The reovirus has the advantages that: (A) the reovirus cannot cause remarkable diseases to a human body, does not have tumor derivation, and is remarkably safe; (B) the interferon generated by inducing the human body is complete in types and high in in-vivo concentration, is highly in accordance with the species specificity in human-sourced virus and eukaryotic molecules, does not have immunological rejection or dependence, and has extremely strong bioactivity and pharmacologic effect and high drug effect on a human body; (C) the dsRNA biomaterial is natural and green, and eco-friendly; (D) the resource is endlessly renewable, and is inexhaustible; (E) the special virus dsRNA material can realize high-throughput proliferation, extraction and modern preparation, and is low in cost.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid pharmacy. The application of reovirus (Reovirus) double-stranded RNA nucleic acid (dsRNA) in the preparation of medical endogenous interferon inducer (Endo-Interferon Inducer, EnII) refers to the use of modern biotechnology in vitro propagation of natural, infinitely reproducible , the remarkably safe green resource of Reovirus, its dsRNA molecule is extracted with high purity, and finally prepared into a nucleic acid preparation for in vivo use, that is, the medical Reovirus dsRNA endogenous interferon inducer (Reov-dsRNA EnII). The EnII is directly used in the human body to induce a large number of various related cells in the human body, such as leukocytes, fibroblasts, immune lymphocytes and other related cells, to produce a full range of highly species-specific, non-immune Endogenous interferons (Endo-Interferon, ENIs), which are highly potent in biological activity and pharmacological ef...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61P31/12A61P31/22A61P35/00A61P37/04
Inventor 董长垣陈冬娥聂志平邓庚粮刘军
Owner 武汉星垣生物技术有限公司
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