Effect And Mechanism Analysis Of AcMNPV-mediated Expression Of BmK IT On Sf9 Cells

Insect baculoviruses are enveloped, double-stranded DNA viruses which named from the bacilliform shape of virus particles. Their hosts mostly include the insects of lepidopteron, hymenopteran, dipters. From the end of last century, baculoviruses have been applied in the pesticide prevention as new bioinsecticides. According to the morphology of inclusion and distribution of pathogenic microorganisms in cells, baculoviruses can be divided into nucleopolyhedravirus (NPV) and granulovirus (GV). Autographa californicamultiple nuclear polyhedrosis virus (AcMNPV) is an important species of baculoviruses. Its genome has about 130 Kbp and contains more than 150 open reading frames. The vcath protein of baculoviruses exists in the nucleocapsid and envelope. It can degrade the actin protein in the host cells specifically. This may directly lead to the collapse of the cytoskeleton. The genes in baculoviruses have many promoters, such as IE1, PH, P10, P6.9 and ETL which are often used to regulate the expression of recombinant foreign gene. BmK IT is an insect toxin from Buthus martensi Karsch, which interacts with sodium channels of insectile cell membrane and causes the repeated action potential distribution of sodium channel and allows the insect to generate excitement palsy. These neurotoxins can interact specifically with sodium channel in excitable cell membrane and disorder the metabolism of insects. The high toxicity and strict insect selectivity of these scorpion toxins make it a promising biological insecticide. Based on our previous work, the experiment intends to investigate the mechanism underlying the AcMNPV mediated insecticidal activity of BmK. IT. BmK IT was regulated by IE1, PH and P10 promoters in this study.This experiment mainly divided into three parts. (1) AcMNPV-mediated expression of BmK IT on the proliferation of Sf9 cells and replication of AcMNPV; (2) Effect of BmK IT under three promoters of AcMNPV on the proliferation of Sf9 cells; (3) Effect of AcMNPV mediated BmK IT and vcath synergism expression on the proliferation of Sf9 cells.In the first part, results from the MTT assay, TUNEL assay, analysis of the expression level of apoptosis-related proteins of Sf9 cells, the transcription level of key genes of AcMNPV, and viral propagation assay demonstrated that AcMNPV-mediated expression of BmK IT promoted the apoptosis of Sf9 cells and replication of AcMNPV during the early infection period. Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(IE1) at different concentrations (1.52×1010 vp/mL(0.6 MOI),1.9×1010 vp/mL(0.8 MOI),2.5x1010 vp/mL(1 MOI) and 3.8×1010 vp/mL (1.5 MOI)) for 24 h, respectively. MTT test showed that the inhibition rates of Sf9 cells in the AcMNPV-K IT(IE1) treatment group were 2.8,2.1,2.0 and 2.4 times of these in AcMNPV treatment group under each concentration. Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(IE1) at the concentration of 1.29×1010 vp/mL (0.5 MOI) for 24 h. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of fragmented DNA (TUNEL) assay showed that AcMNPV and AcMNPV-BmK IT(IE1) could induce the apoptosis of Sf9 cells. Compared with that in the AcMNPV treatment group, the number of apoptotic cells was increased significantly in the AcMNPV-BmK IT(IE1) treatment group. Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(IE1) at the concentration of 0.56×1010 vp/mL (0.2 MOI) for 24 h. Western blot analysis showed that AcMNPV-BmK IT(IE1) elevated the expression of c-Myc, cleaved-Caspase3 and Bax, but it decreased the stability of Bcl-2 in Sf9 cells. Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(IE1) at the concentration of 0.56×1010 vp/mL(0.2 MOI) for 24 h. The transcription level of key genes of AcMNPV was analyzed by quantitative RT-PCR. The transcription level of 38K, C42, P78 and F genes in the AcMNPV-BmK IT(IE1) treatment group were 2.1,1.5,1.6 and 1.8 times of these in AcMNPV treatment group. Sf9 cells were infected with AcMNPV and AcMNPV-BmK IT(IE1) at different concentrations (1.52×1010 vp/mL (0.6 MOI),1.9×1010 vp/mL (0.8 MOI), 2.5×1010 vp/mL (1 MOI) and 3.8×1010 vp/mL (1.5 MOI)) for 24 h, respectively. The titers of virus in AcMNPV-BmK IT(IEl) treatment group had been increased by 58.3%,66.7%,70.6% and 42.9%, respectively, compared with these in AcMNPV treatment group. These results demonstrated AcMNPV-mediated expression of BmK. IT promoted the apoptosis of Sf9 cells and replication of AcMNPV during the early infection period.In the second part, AcMNPV, AcMNPV-K IT (IE1), AcMNPV-K IT(P10) and AcMNPV-K IT(PH) were used. Sf9 cells were infected with these four viruses at the concentration of 3.8×1010 vp/mL (1.5 MOI) for 12 h、24 h、36 h and 48 h. Proliferation assay was performed by MTT test. The results showed that the inhibition rate of AcMNPV-BmK IT(IE1), AcMNPV-BmK IT(P10), AcMNPV-K IT(PH) and AcMNPV was ranged from high to low at 12 h and 24 h. And the inhibition rate of AcMNPV-BmK IT(PH), AcMNPV-BmK IT(P10), AcMNPV-BmK IT(IE1) and AcMNPV was ranged from high to low at 36 h and 48 h. Sf9 cells were infected with these four viruses at the concentration of 0.56×1010 vp/mL(0.2 MOI) for 12 h and 48 h. Western blot analysis showed that, compared with these in AcMNPV-BmK IT(P10) and AcMNPV-BmK IT(PH) treatment group, AcMNPV-BmK IT(IE1) elevated the expression level of c-Myc, cleaved-Caspase3 and Bax, but it decreased the stability of Bcl-2 in Sf9 cells at 12h. However, these results were opposite at 48 h.In the third part, AcMNPV, AcMNPV-BmK IT(P10), AcMNPV-vcath(PH) and AcMNPV-BmK IT(P10)-vcath(PH) were used. Sf9 cells were infected with these four viruses at different concentrations (1.52×1010 vp/mL (0.6 MOI),1.9×1010vp/mL(0.8MOI),2.5×1010vp/mL(1 MOI) and 3.8×1010 vp/mL(1.5 MOI)) for 48 h, respectively. The results from MTT test showed that the inhibition rates of Sf9 cells in the AcMNPV-BmK IT(P10)-vcath(PH) treatment group were 1.4 times of these in AcMNPV-BmK IT (P10) treatment group. And the inhibition rates of Sf9 cells in the AcMNPV-BmK IT(P10)-vcath(PH) treatment group were 2.1 times of these in AcMNPV- vcath(PH) treatment group. Sf9 cells were infected with these four viruses at the concentration of 0.56x1010vp/mL (0.2 MOI) for 48 h. Western blot analysis showed that compared with that in AcMNPV-BmK IT (P10) and AcMNPV-vcath(PH) treatment group, AcMNPV-BmK IT(P10)-vcath(PH) elevated the expression of c-Myc, cleaved-Caspase3 and Bax, but it decreased the stability of Bcl-2 in Sf9 cells.In this study, the effect and mechanism analysis of AcMNPV-mediated expression of BmK IT on Sf9 cells were performed. These results provide a theoretical basis for the development and mechanism analysis of recombinant virus biopesticides.