Application of methyl-beta-cyclodextrins to expansion of silkworm bombyx mori nuclear polyhedrosis virus host range

Abstract

The invention discloses application of methyl-beta-cyclodextrins (MbetaCD) to expansion of a silkworm bombyx mori nuclear polyhedrosis virus (BmNPV) host range. After MbetaCD with a certain concentration is used for processing BmNPV non-host cells, the efficiency at which BmNPV infects non-host insect cells can be remarkably improved, BmNPV can be duplicated and amplified in the non-host cells, infectious virus particles and polyhedral bodies are produced, contrast cells BmNPV can not be duplicated at all, and no polyhedral bodies can be observed. The problem that the BmNPV host range is narrow is solved, and BmNPV can be duplicated in the non-host cells and efficiently infects the non-host cells to produce polyhedral bodies. Meanwhile, the defects that baculovirus is narrow in host range, low in insect killing speed and the like when serving as a biological insecticide can be overcome, and the development of research on the interaction of insect baculovirus and host cells, the mechanism of baculovirus receptors and baculovirus invading a host and the like can also be promoted.

Description

technical field

[0001] The invention belongs to the field of biopesticides and baculovirology, and relates to the use of methyl-β-cyclodextrin (Methyl-β-cyclodextrin, MβCD for short, also expressed as MBCD, Me-βCD, and MeBCD) in expanding the polyangular karyotype of silkworm. applications in the host domain of body viruses. Background technique

[0002] Baculovirus is the largest group of known insect viruses. It is the earliest discovered insect virus with the most research and great practical significance. It is an enveloped virus with closed circular double-stranded DNA. The genome size is about 80-180kb . Since baculovirus is also the control factor of major pests in agriculture and forestry, it is also widely used in biopesticides.

[0003] The vast majority of baculoviruses are isolated from insects, and all have relatively narrow host domains. Autographa californica nucleopolyhedrovirus AcMNPV and Bombyx mori nucleopolyhedrovirus (BmNPV) are the two most widely st...

Examples

Embodiment 1

[0032] Example 1: The final concentration is 0.25mM MβCD incubation, the improvement of BmNPV infection non-host cell efficiency

[0033] Inoculate approximately 5×10 cells in two 35mm cell culture dishes 5 After the sf21 cells per plate adhered to the wall, take the prepared MβCD storage solution and add it to the cell culture medium of one of the plates, so that the final concentration of MβCD was 0.25mM respectively, and incubated with 0mM MβCD (that is, the corresponding volume of 1×PBS). Add another dish as a control, incubate at 27°C for 30 min, then remove 1×PBS and MβCD incubation solution, wash the cells gently with serum-free TC-100 medium twice, then use MOI=10 to carry the green fluorescent protein gene and The BmBac-egfp-ph virus of the polyhedron gene was used to infect cells with different treatments. After infecting at 27°C for 2 hours, lightly wash twice with serum-free TC-100 medium, and add conventional TC-100 containing 10% fetal bovine serum. The culture ...

Embodiment 2

[0034] Example 2: After treatment with MβCD with a final concentration of 0.125, 0.25, 0.5, 0.75, 1, 1.5, and 2 mM respectively, the changes in virus infection efficiency were counted by flow cytometry

[0035] Inoculate approximately 1 × 10 cells in each 24-well cell culture plate 5 sf21 per well, adhered to the wall for 24 hours, respectively took 1.25 μL, 2.5 μL, 5 μL, 7.5 μL, 10 μL, 15 μL, and 20 μL of the prepared MβCD storage solution and added them to the cell culture medium (the total volume of the cell culture medium was 500 μL / well) ), so that the final concentrations of MβCD were 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2mM, respectively, and 20 μL 1×PBS was added to the BmNPV control and AcMNPV control, incubated at 27°C for 30 minutes, then removed 1×PBS and incubated with different concentrations of MβCD Wash the cells gently twice with serum-free TC-100 medium, then infect 0.125, 0.25, 0.5, 0.75, 1 , 1.5, 2mMMβCD and PBS-incubated cells, and AcMNPV with MOI = 1 to infec...

Embodiment 3

[0036] Example 3: When the final concentration of MβCD was 0.25mM, the virus growth curve was measured after BmNPV infection

[0037] Inoculate approximately 5×10 cells in 6-well cell culture plates 5 sf21 cells / well, after adherence, take 10 μL of the prepared MβCD storage solution and add it to the cell culture medium of adherent cells sf21, so that the final concentration of MβCD is 0.25 mM (total volume is 2 mL), incubate at 27 ° C for 30 min, 10 μL of 1×PBS incubation solution was used as a control. After the incubation time is up, remove the 1×PBS and MβCD incubation solution, wash the cells gently twice with serum-free TC-100 medium, and then use MOI=20 to carry the green fluorescent protein gene and polyhedrin gene BmBac-egfp- The ph virus was used to infect cells with different treatments. After infecting at 27°C for 1 hour, gently wash twice with serum-free TC-100 medium, add 2 mL of normal cell culture medium (this time was set as 0 h), and culture at 27°C. 10 μL ...

technical field

[0001] The invention belongs to the field of biopesticides and baculovirology, and relates to the use of methyl-β-cyclodextrin (Methyl-β-cyclodextrin, MβCD for short, also expressed as MBCD, Me-βCD, and MeBCD) in expanding the polyangular karyotype of silkworm. applications in the host domain of body viruses. Background technique

[0002] Baculovirus is the largest group of known insect viruses. It is the earliest discovered insect virus with the most research and great practical significance. It is an enveloped virus with closed circular double-stranded DNA. The genome size is about 80-180kb . Since baculovirus is also the control factor of major pests in agriculture and forestry, it is also widely used in biopesticides.

[0003] The vast majority of baculoviruses are isolated from insects, and all have relatively narrow host domains. Autographa californica nucleopolyhedrovirus AcMNPV and Bombyx mori nucleopolyhedrovirus (BmNPV) are the two most widely st...

Examples

Embodiment 1

[0032] Example 1: The final concentration is 0.25mM MβCD incubation, the improvement of BmNPV infection non-host cell efficiency

[0033] Inoculate approximately 5×10 cells in two 35mm cell culture dishes 5 After the sf21 cells per plate adhered to the wall, take the prepared MβCD storage solution and add it to the cell culture medium of one of the plates, so that the final concentration of MβCD was 0.25mM respectively, and incubated with 0mM MβCD (that is, the corresponding volume of 1×PBS). Add another dish as a control, incubate at 27°C for 30 min, then remove 1×PBS and MβCD incubation solution, wash the cells gently with serum-free TC-100 medium twice, then use MOI=10 to carry the green fluorescent protein gene and The BmBac-egfp-ph virus of the polyhedron gene was used to infect cells with different treatments. After infecting at 27°C for 2 hours, lightly wash twice with serum-free TC-100 medium, and add conventional TC-100 containing 10% fetal bovine serum. The culture ...

Embodiment 2

[0034] Example 2: After treatment with MβCD with a final concentration of 0.125, 0.25, 0.5, 0.75, 1, 1.5, and 2 mM respectively, the changes in virus infection efficiency were counted by flow cytometry

[0035] Inoculate approximately 1 × 10 cells in each 24-well cell culture plate 5 sf21 per well, adhered to the wall for 24 hours, respectively took 1.25 μL, 2.5 μL, 5 μL, 7.5 μL, 10 μL, 15 μL, and 20 μL of the prepared MβCD storage solution and added them to the cell culture medium (the total volume of the cell culture medium was 500 μL / well) ), so that the final concentrations of MβCD were 0.125, 0.25, 0.5, 0.75, 1, 1.5, 2mM, respectively, and 20 μL 1×PBS was added to the BmNPV control and AcMNPV control, incubated at 27°C for 30 minutes, then removed 1×PBS and incubated with different concentrations of MβCD Wash the cells gently twice with serum-free TC-100 medium, then infect 0.125, 0.25, 0.5, 0.75, 1 , 1.5, 2mMMβCD and PBS-incubated cells, and AcMNPV with MOI = 1 to infec...

Embodiment 3

[0036] Example 3: When the final concentration of MβCD was 0.25mM, the virus growth curve was measured after BmNPV infection

[0037] Inoculate approximately 5×10 cells in 6-well cell culture plates 5 sf21 cells / well, after adherence, take 10 μL of the prepared MβCD storage solution and add it to the cell culture medium of adherent cells sf21, so that the final concentration of MβCD is 0.25 mM (total volume is 2 mL), incubate at 27 ° C for 30 min, 10 μL of 1×PBS incubation solution was used as a control. After the incubation time is up, remove the 1×PBS and MβCD incubation solution, wash the cells gently twice with serum-free TC-100 medium, and then use MOI=20 to carry the green fluorescent protein gene and polyhedrin gene BmBac-egfp- The ph virus was used to infect cells with different treatments. After infecting at 27°C for 1 hour, gently wash twice with serum-free TC-100 medium, add 2 mL of normal cell culture medium (this time was set as 0 h), and culture at 27°C. 10 μL ...

Images