Effects Of Recombinant Baculovirus AcMNPV-Bmk IT On Apoptosis Genes Expression And Actin Rearrangement In Sf9 Cell

AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) is used in most research and has high value as a kind of bioinsecticide. Its genome has about 130 Kbp and contains more than 150 open reading frames. Insect baculoviruses possess the advantage of high insecticidal potency, pests less likely producing resistance, highly host-species specific, safety to people and domestic animals, little pollution to environment or ecological balances, so they have been applied as new bioinsecticides around the world. A series of toxins generated from scorpion can interact specifically with various ion channels in excitable cell membrane. Especially, all kinds of insect-selective scorpion neurotoxins display no or less toxicity toward mammals but are specifically active toward insects. Therefore, they are widely used in recombinant biological pesticides. An excitatory insect toxin gene, BmK. IT, from Buthus martensii Karsch which is a sort of scorpion mainly distributed in China, was cloned previously in our lab.In our previous work, BmK IT gene was engineered into the genome of AcMNPV. The bioassay data indicated that the recombinant baculovirus AcMNPV-BmK IT (IE1) significantly enhanced the anti-insect efficacy. However, the anti-insect mechanism of AcMNPV-BmK IT is unknown at cell and molecular level.In this study, the experiments mainly contained three parts:In the first part, effects of recombinant baculovirus AcMNPV-BmK IT on the expression of apoptosis protein SfP53 in Sf9 cells were analyzed. Sfp53 gene was colned and expression plasmid pRSETc-Sfp53 was constructed to prepare the anti-SfP53 antibody. Sf9 cells were infected with AcMNPV and AcMNPV-BwK IT at the concentration of 2.28×1012 vp/mL (10 MOI) for 6-45 h, western blot was performed to analyze the expression level of SfP53. The results showed that the expression levels of SfP53 in AcMNPV-BmK IT treatment group were 4.16,3.50,4.10 and 2.56 times of these in AcMNPV treatment group at 12,21,27 and 33 hpi, respectively, which suggested that AcMNPV-BmK IT increased the expression level of SfP53 and promoted the cell apoptosis during this infection period. However, the expression levels of SfP53 in AcMNPV treatment group were 5.30 and 2.91 times of these in AcMNPV-BmK IT treatment group at 39 and 48 hpi, respectively.In the second part, analysis of recombinant baculovirus AcMNPV-BmK IT on the transcription level of virus anti-apoptosis genes iapl, iap2,p35 in Sf9 cells was performed. Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT at the concentration of 2.28×1012 vp/mL (10 MOI) for 6-72 h, respectively. qPCR was performed to analyze the transcription level of iap1, iap2 and p35. The results showed that AcMNPV-BmK IT treatment group obtained better transcription level of iapl. The transcription levels of iapl in AcMNPV-BmK IT treatment group were 20.15,6.88 and 2.35 times of these in AcMNPV treatment group at 36,48 and 72 hpi, respectively, which suggested that AcMNPV-BmK IT promoted the cell apoptosis and this action was favorable for the dissemination of progeny occlusion derived virions. The transcription levels of iap2 in AcMNPV treatment group were 15.22,12 and 3.7 times of these in AcMNPV-BwK IT treatment group at 36, 48 and 72 hpi, respectively, which suggested that AcMNPV-BmK IT suppressed the transcription level of iap2. The transcription level of p35 in AcMNPV-BmK IT treatment group were 15.47,22.94 and 2.61 times of these in AcMNPV treatment group at 36,48 and 72 hpi, respectively, which suggested that AcMNPV-BmK IT improved the transcription level of p35 and inhibited the cell apoptosis. This action may be a response to the increased expression of SfP53 during this period.In the third part, effects of recombinant baculovirus AcMNPV-BmK IT on the formation of early cables and nuclear polymerization of actin in Sf9 cells were analyzed. Firstly, Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT at the concentration of 2.28× 1012 vp/mL (10 MOI) for 2 h and 3 h, respectively. The results from immunofluorescence assay showed that coarse actin cables didn’t appear in uninfected cells. Actin cables appeared in AcMNPV-BmK IT treatment group were less than that in AcMNPV treatment group at 2-3 hpi. Then AcMNPV and AcMNPV-BwK IT infected Sf9 cells with the same concentration for 6-46 h, immunofluorescence and western blot were performed to analyze the nuclear polymerization of actin. The results showed that cells infected with AcMNPV and AcMNPV-BmK IT for 6-25 h, microfilaments were formed on the ventral surface and actin filaments appeared in nuclei in both treatment groups. At 31 and 37 hpi, the nuclear polymerization of actin in the AcMNPV treatment group was more than that in the AcMNPV-BwK IT treatment group. At 46 hpi, nuclear actin decreased in the AcMNPV-Bmî™' IT treatment group. These finding showed that in AcMMPV-BmK IT treatment group, the nuclear F-actin was formed more slowly and disappeared faster than these in the AcMNPV treatment group. The results from western blot analysis showed that, compared to these in AcMNPV treatment group, the polymerization of actin in nuclei in AcMNPV-BmK IT treatment group appeared slowly and disappeared faster, which consisted with the results from immunofluorescence assay.In this study, the effects of AcMNPV-BmK. IT on apoptosis-related genes and actin rearrangement in Sf9 cells were analyzed. These results provided the experimental basis for the development and mechanism analysis of recombinant virus biopesticides.