Preparation method of heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer

A technology of interferon induction and bluetongue virus, which is applied in the field of preparation of heterologous bluetongue virus double-stranded RNA endogenous interferon inducers, can solve problems such as market and clinical application limitations, and achieves the need for no purification, complete varieties, The effect of enhancing physical fitness

Inactive Publication Date: 2012-02-15
WUHAN UNIV
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  • Claims
  • Application Information

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Problems solved by technology

As mentioned earlier, Poly[I:C] has a series of defects, a

Method used

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  • Preparation method of heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer
  • Preparation method of heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer
  • Preparation method of heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1V

[0113] Embodiment 1Vero cell culture

[0114] 1.1 Recovery and culture of passaged cell lines

[0115] 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.

[0116] 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.

[0117] 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× 103-4 cells / mL; pipette the cell solution to resuspend the pellet; inoculate the culture flask with 8-12mL cell suspension per bottle, and place it in a 37°C, 5% (v / v) carbon d...

Embodiment 2B

[0122] Embodiment 2BTV proliferation

[0123] 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.

[0124] 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).

[0125] 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%.

[0126] 2.4 Collect the activated virus suspension, d...

Embodiment 3B

[0127] Example 3 BTVdsRNA Extraction

[0128] 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;

[0129] 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);

[0130] 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;

[0131] 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;

[0132] 3.5. Put it on the magnetic rack, let it stand for 20 seconds, and carefully aspirate and discard the supernatant;

[0133] 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;

[0134] 3.7. Add 6-8 volumes of buffer...

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Abstract

The invention discloses a preparation method of a heterologous bluetongue virus double-strand RNA (ribonucleic acid) endogenous interferon inducer. The inducer is prepared by extracting naked double-strand ribonucleic acid (dsRNA) from animal bluetongue virus, and can be directly applied in a human body to induce the human body to generate endogenous interferon, thereby endowing the human body with the functions of resisting virus, resisting cancer, preventing diseases, treating diseases and enhancing physique. The inducer has the main advantages that: A) the inducer can directly induce the human body to generate various endogenous interferons (ENI), and has high in-vivo concentration, strongest antiviral and anticancer activities, no immunogenicity of heterologous protein, no dependence, no need of purification and low cost; B) the inducer has no infectivity, is dsRNA virus, has no tumor origin, is natural virus and has no influence on ecological balance; C) the inducer is a fully new natural and green biological material, which is safe to human, is never used up, can be regenerated infinitely and is favorable for in-vitro proliferation and purification preparation; and D) the inducer highly meets the species specificity and has a strongest pharmacological action in the human body.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a preparation method of a heterologous bluetongue virus double-stranded RNA endogenous interferon inducer, which can be directly used in human body to induce human body to produce various endogenous interferon (ENIs), endow the human body with anti-virus, anti-tumor and extensive functions of disease prevention, treatment and physical enhancement. 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-Interferon). -Interf...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N7/00C12N15/113A61P31/12A61P35/00
Inventor 董长垣陈冬娥刘军邓庚粮方兰杨继红
Owner WUHAN UNIV
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