AcMNPV介导BmKIT和组织蛋白酶协同表达对Sf9细胞凋亡的分析

摘要

In order to investigate the mechanism underlying the BmK IT mediated insecticidal activity of AcM-NPV,an excitatory insect toxin ,BmK IT,gene was inserted into the geneome of AcMNPV to construct a recombinant baculovirus.The scorpion insect neurotoxin BmK IT and vcath gene were constructed into the genome of AcMNPV using Bac-to-Bac system.Effects of BmK IT under these promoters of AcMNPV on Sf 9 cells were analyzed and it re-vealed that the expression level of BmK IT was high under the regulation of PH promoter at 36 ,48 h after the virus infected.Effects of AcMNPV mediated BmK IT and vcath synergism expression on the proliferation of Sf 9 cells was also analyzed.Compared to AcMNPV-BmK IT (P10),the inhibition rate of AcMNPV-BmK IT (P10)-vcath(PH) increased 14.5%averagely,the expression level of c-Myc,cleaved-Caspase3 and Bax was raised and the expression of Bcl-2 was reduced .%为了研究BmK IT提高AcMNPV抗虫活性的作用机制，将BmK IT基因插入到AcMNPV中形成重组病毒。采用重组单价病毒AcMNPV-BmK IT（IE1）、AcMNPV-BmK IT（P10）、AcMNPV-BmK IT（PH）和一种重组双价病毒AcM-NPV-BmK IT（P10）-vcath （PH），通过四唑盐比色法（MTT）和蛋白质印迹法（Western Blot）分析了BmK IT在AcMNPV的3个启动子调控下对Sf9细胞增殖和细胞凋亡的影响，结果显示，感染病毒36，48 h BmK IT在不同启动子调控下表达量从高到低依次为PH、P10、IE1。同时分析了AcMNPV介导的BmK IT与组织蛋白酶的协同表达对昆虫Sf9细胞增殖和调亡的机制，结果表明，AcMNPV-BmK IT（P10）-vcath（PH）处理组对Sf9细胞抑制率比AcMNPV-BmK IT（P10）处理组平均提高了14．5％，凋亡相关蛋白c-Myc、cleaved-Caspase3、Bax表达量增加，Bcl-2表达量减少。