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Application of heterogeneous bluetongue virus dsRNA (double-strand ribonucleic acid) in preparation of endogenic interferon inducer

A technology of interferon induction and bluetongue virus, which is applied in the field of biopharmaceuticals, can solve problems such as market and clinical application restrictions, and achieve the effect of low cost, complete variety, and physical fitness enhancement

Inactive Publication Date: 2012-02-15
陈冬娥
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As mentioned earlier, Poly[I:C] has a series of defects, and the market and clinical application are greatly limited

Method used

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  • Application of heterogeneous bluetongue virus dsRNA (double-strand ribonucleic acid) in preparation of endogenic interferon inducer
  • Application of heterogeneous bluetongue virus dsRNA (double-strand ribonucleic acid) in preparation of endogenic interferon inducer
  • Application of heterogeneous bluetongue virus dsRNA (double-strand ribonucleic acid) in preparation of endogenic interferon inducer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1. Vero cell culture

[0113] 1.1 Recovery and culture of passaged cell lines

[0114] 1.1.1 Take out the Vero cell cryopreservation tube of African green monkey kidney cells from the liquid nitrogen tank, immediately put it into the pre-adjusted temperature of 37°C water, shake gently, so that the cell suspension in the cryopreservation tube melts rapidly within 1 min.

[0115] 1.1.2 After thawing, centrifuge the cryovial at 1000r / min for 15min; discard the supernatant after centrifugation, add 1.5mL DMEM culture medium, and pipette to resuspend the precipitate.

[0116] 1.1.3 Centrifuge the cryovial again at 1000r / min for 15min; discard the supernatant after centrifugation, and dissolve the cell pellet with DMEM medium containing 10% (v / v) calf serum to make the final concentration 1× 10 3-4 Cells / mL; pipette the cell solution to re-suspend the pellet; inoculate culture bottles with 8-12mL cell suspension per bottle, place in a 37°C, 5% (v / v) carbon dioxide ...

Embodiment 2

[0121] Example 2. BTV Proliferation

[0122] 2.1 Using DMEM medium containing 10% (v / v) calf serum, at 37°C, 5% (v / v) CO 2 Vero cells were cultured under these conditions.

[0123] 2.2 Within 24 hours after subculture, when the cells grow to 80-90% confluence, discard the culture medium; use 1×PBS with pH 7.4 (NaCl: 8g, KCl: 0.2g, NaCl: 0.2g, 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 (2 to 1000ml) wash adherent cells 1-2 times; inoculate BTV in cultured cells by 2-4MOI (multiplicity of infection).

[0124] 2. After absorbing the inoculated virus at 337°C for 1-2 hours, discard the virus liquid; use DMEM medium containing 2% (v / v) calf serum, 37°C, 5% (v / v) CO 2 Maintain the culture under the conditions; then observe under the light microscope every 12 hours, until the cells have obvious lesion effect, that is, the virus is harvested when the CPE reaches 90%.

[0125] 2.4 Collect the activated virus suspension, de...

Embodiment 3

[0126] Example 3.BTV dsRNA extraction

[0127] 3.1. Extract and purify BTV according to the method in our authorized patent - "reverse co-immunoprecipitation technology", and put it into a dialysis bag;

[0128] 3.2. PEG 6000 concentrated virus, buffer A (10mM Tris [pH 8.0], 2mM MgCl 2 , 4% sucrose reverse dialysis, the dsRNA virus reached 1 × 10 13 VP / ml (viral particles / ml);

[0129] 3.3. Add 3 times the volume of 0.5% NP-40 lysate (50mM Tris-Cl pH 6.0, 15mM NaCl, 5mM EDTA, 0.5% NP-40, 1mM PMSF, prepared immediately) to lyse BTV;

[0130] 3.4. Add 10 μL of mixed magnetic beads per ml of virus lysate, vortex for 20 seconds, and then stand at room temperature for 3 minutes;

[0131] 3.5. Put it on the magnetic rack, let it stand for 20 seconds, and carefully aspirate and discard the supernatant;

[0132] 3.6. Add 4-5 times the volume of buffer B (50mM Tris-Cl pH 6.0, 15mM NaCl) to repeatedly wash the magnetic beads that adsorb dsRNA;

[0133] 3.7. Add 6-8 volumes of buffe...

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Abstract

The invention discloses application of a heterogeneous bluetongue virus double-strand RNA (ribonucleic acid) in the preparation of an inducer as an endogenic interferon. The inducer is prepared by extracting naked dsRNA (double-strand ribonucleic acid) molecules from an animal bluetongue virus, is directly used for a human body, induces the human body to generate the endogenic interferon, and gives an antiviral function, an anticancer function and wide disease prevention and treatment and health invigoration functions to the human body. The application has the main advantages that: (A), the human body is directly induced to generate the ENIs (endogenic interferons) with complete varieties, the in-vivo concentration is high, the antiviral activity and the antitumor activity are strongest, the immunogenicity of a heterogeneous protein is free, the dependence is free, the purification is not necessary, and the manufacturing cost is low; (B), the infectivity is free, the heterogeneous bluetongue virus is a dsRNA virus, the tumor origin is free, the heterogeneous bluetongue virus is a natural virus, and the ecological equilibrium is not influenced; (C), the heterogeneous bluetongue virus is a brand-new natural green biomaterial which is safe to people, is inexhaustible, can be regenerated infinitely, and is convenient for in-vitro proliferation, purification and preparation; and (D), the species specificity is highly accorded, and the pharmacological action in the human body is strongest.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals. It specifically relates to a heterologous bluetongue virus double-stranded RNA (dsRNA) endogenous interferon inducer (BTV NdsRNA EnII) directly used in the human body to induce the human body to produce various endogenous interferons (ENIs), endowing the human body with antiviral , anti-tumor and a wide range of disease prevention, treatment and physical enhancement functions. Background technique [0002] Interferon (Interferon, IFN) (Edited by Dong Changyuan, "Modern Molecular Virology", Wuhan University Press, October 1996), which has a wide range of disease prevention and treatment functions, is the best type of cytokine discovered by humans so far; Also because endogenous interferon (Endo-Interferon, ENI) has incomparable advantages compared with exogenous interferon (Exo-Interferon, EXI), and human beings have not yet obtained an ideal endogenous interferon inducer (Endo-Interfe...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K31/713C12N15/113C12N15/10A61P31/12A61P35/00A61P37/04
Inventor 陈冬娥董长垣
Owner 陈冬娥
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