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Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells

A technology of cyclodextrin and methyl group, applied in the directions of pharmaceutical formulations, medical preparations containing active ingredients, organic active ingredients, etc., can solve the problems of non-patent and literature reports, etc.

Inactive Publication Date: 2014-09-10
JIANGSU UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no patents and literature reports on the application of MβCD in the prevention and treatment of viruses and BmNPV

Method used

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  • Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells
  • Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells
  • Application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Construction of BmNPV carrying egfp reporter gene

[0025] The hsp70-egfp fragment was cloned into the BamH I (Takara) and Not I (Takara) sites of pFastBacDual (Invitrogen), the donor plasmid pFastBacDual-hsp70-egfp was constructed, and BmBac-egfp was obtained by transposition (see schematic diagram figure 1 ), after the BmBac-egfp DNA was transfected into BmN (bombyx mori ovary cells) cells, the expression of green fluorescence could be observed under a fluorescent microscope. The cell supernatant was used to infect BmN cells, and the virus was harvested 72 hours after infection, and the virus titer was determined by the terminal dilution method, and the virus was stored in a refrigerator at 4°C in the dark.

[0026] 2. Inoculation of cells and preparation of stock solution of MβCD (Sigma)

[0027] (1) Inoculate about 10 cells in a 35mm cell culture dish (Corning) respectively. 5 Add 2 mL of 10% FBS TC100 insect cell culture medium to each cell dish of BmN cells. ...

Embodiment 2

[0037] When the final concentration of MβCD is 4mM, the prevention and treatment of BmNPV infection carrying egfp reporter gene:

[0038] (1) Take 10 of each inoculation 5 3 culture dishes of cells, remove the old culture medium, add 105.2 μL MβCD storage solution and 894.8 μL 10% FBS TC100 medium to the 3 cell culture dishes respectively, so that the final concentration of MβCD in the culture dish is 4 mM, 27 °C After culturing for 30 minutes, remove the medium containing MβCD, wash twice with serum-free TC100 medium, and shake gently for 3 minutes each time.

[0039] (2) After washing the cells, remove the washing solution, add BmBac-egfp virus with MOI=10, and infect the cells at 27° C. for 1 hour.

[0040] (3) After 1 hour, wash twice with serum-free TC100 medium, shake gently for 3 minutes each time, remove the washing solution, add 2 mL of 10% FBS TC100 medium, and incubate at 27°C for 6 hours.

[0041] (4) After 6 hours, place 3 dishes in a fluorescent confocal micros...

Embodiment 3

[0043] When the final concentration of MβCD is 6mM, the prevention and treatment of BmNPV infection carrying egfp reporter gene:

[0044] (1) Take 10 of each inoculation 5 3 culture dishes of cells, remove the old medium, add 157.8 μL MβCD storage solution and 842.2 μL 10% FBS TC100 medium to the 3 cell culture dishes respectively, so that the final concentration of MβCD in the culture dish is 6 mM, 27 Incubate at ℃ for 30 minutes, remove the medium containing MβCD, wash twice with serum-free TC100 medium, shake gently, each time for 3 minutes.

[0045] (2) After washing the cells, remove the washing solution, add BmBac-egfp virus with MOI=10, and infect the cells at 27° C. for 1 hour.

[0046] (3) After 1 hour of infection, wash twice with serum-free TC100 medium, shake gently for 3 minutes each time, remove the washing solution, add 2 mL of 10% FBS TC100 medium, and incubate at 27°C for 6 hours.

[0047] (4) After 6 hours, place 3 dishes in a fluorescent confocal microscop...

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Abstract

The invention discloses application of methyl-beta-cyclodextrin in preparation of medicines for preventing and controlling BmNPV from infecting silkworm cells and provides a medicine which takes MbetaCD as an active ingredient and is used for preventing and controlling BmNPV from infecting cells. The medicine has significance in intensive research on virus invasion mechanisms and the research aspect of receptors of hosts infected by baculovirus. When the MbetaCD concentration is over 8mM, the virus infection can be completely inhibited. The medicine has important application and popularization values in research and development and production of silkworm medicines for controlling grasserie.

Description

technical field [0001] The invention belongs to the field of molecular biology and virology, and relates to the use of methyl-β-cyclodextrin (Methyl-β-cyclodextrin, MβCD for short, also expressed as MBCD, Me-βCD, and MeBCD) in the preparation and prevention of BmNPV-infected silkworm cell medicine Applications. Background technique [0002] Bombyx mori blood type pus disease is the most serious disease among the three major virus diseases in sericulture. It is highly contagious and has occurred more commonly in sericulture production in recent years. production situation. Blood pyogenic disease is caused by Bombyx mori nucleopolyhedrovirus (BmNPV), which belongs to the baculoviridae family and the genus Nucleopolyhedrovirus, and is a double-stranded DNA virus with an envelope. During the virus replication process, two kinds of virions will be formed, namely inclusion body-derived virions and budding virions. The former enters the midgut cells of silkworm midgut with food, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/724A61P31/20
Inventor 黄金山郝碧芳沈兴家程晨梁飞
Owner JIANGSU UNIV OF SCI & TECH
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