Slack Jeffrey M, Lawrence Susan D
USDA/ARS, Insect Biocontrol Laboratory, BARC-West, Bldg 011A, Rm 214, Beltsville, MD 20852-2350, USA.
J Gen Virol. 2005 Jun;86(Pt 6):1637-1643. doi: 10.1099/vir.0.80832-0.
Baculovirus occlusion-derived virions (ODVs) are released from occlusion bodies by the alkaline environment of the insect midgut. The ODV envelope protein P74 is required for oral infectivity. A soluble form of the Autographa californica multiple nucleopolyhedrovirus P74 protein, P74sol, was engineered as part of a chimeric protein with jellyfish green fluorescent protein (GFP). P74sol-GFP was overproduced by the baculovirus expression system and purified away from the wild-type P74. Brush border membrane vesicles (BBMVs) were prepared from the midguts of third-instar Helicoverpa zea larvae. When P74sol-GFP was incubated under alkaline conditions with BBMVs, a P74sol-GFP product with a smaller molecular mass was produced. Immunoblots indicated that the smaller product was generated by N-terminal cleavage of P74. This cleavage was prevented by soybean trypsin inhibitor. Analysis of the peptide sequences of P74 homologues identified a conserved trypsin cleavage site that could generate the observed P74sol-GFP BBMV-specific cleavage product.
杆状病毒包涵体衍生病毒粒子（ODV）通过昆虫中肠的碱性环境从包涵体中释放出来。ODV包膜蛋白P74是口服感染性所必需的。苜蓿银纹夜蛾多核多角体病毒P74蛋白的可溶性形式P74sol，被设计成与水母绿色荧光蛋白（GFP）组成嵌合蛋白的一部分。P74sol-GFP由杆状病毒表达系统过量表达，并从野生型P74中纯化出来。从三龄玉米螟幼虫的中肠制备刷状缘膜囊泡（BBMV）。当P74sol-GFP在碱性条件下与BBMV一起孵育时，产生了分子量较小的P74sol-GFP产物。免疫印迹表明，较小的产物是由P74的N端裂解产生的。大豆胰蛋白酶抑制剂可阻止这种裂解。对P74同源物的肽序列分析确定了一个保守的胰蛋白酶裂解位点，该位点可产生观察到的P74sol-GFP BBMV特异性裂解产物。
