Skate Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
Virol Sin. 2013 Dec;28(6):360-7. doi: 10.1007/s12250-013-3393-7. Epub 2013 Dec 2.
P74 is a per os infectivity factor of baculovirus. Here, we report the production of three monoclonal antibodies (mAbs), denoted as 20D9, 20F9 and 21E1, raised against P74 of Helicoverpa armigera nucleopolyhedrovirus (HearNPV), and the identification of their recognition epitopes. The full-length P74, without the transmembrane domains at the C-terminus, was first divided into three segments (N, M and C, respectively), based on the proposed cleavage model for the protein, which were then expressed individually. Western blot analyses revealed specific cross-reactions with the N fragment, for both 20D9 and 21E1. Extensive truncation, followed by prokaryotic expression, of the P74 N fragment was then performed in order to screen for linear epitopes of P74. The recognition regions of 20D9 and 21E1 were revealed to be localized at R144-T153 and T199-C219, respectively. In addition, immunofluorescence microscopy indicated that 20D9 and 20F9 could recognize native P74 in HearNPV-infected cells. These findings will facilitate further investigations of the proteolytic processing of HearNPV P74, and of its involvement in virus-host interactions.
P74 是杆状病毒的一种口服感染因子。在这里，我们报告了针对棉铃虫核多角体病毒（HearNPV）P74 产生的三种单克隆抗体（mAbs），分别命名为 20D9、20F9 和 21E1，并鉴定了它们的识别表位。根据该蛋白的拟议切割模型，首先将全长 P74（不包括 C 末端的跨膜结构域）分为三个片段（N、M 和 C），然后分别进行表达。Western blot 分析显示，20D9 和 21E1 均与 N 片段发生特异性交叉反应。然后，为了筛选 P74 的线性表位，对 P74 N 片段进行了广泛的截断和原核表达。20D9 和 21E1 的识别区域分别定位于 R144-T153 和 T199-C219。此外，免疫荧光显微镜表明，20D9 和 20F9 可以识别感染细胞中的天然 P74。这些发现将有助于进一步研究 HearNPV P74 的蛋白水解加工及其在病毒-宿主相互作用中的参与。
