Huang Xiaozhi, Shi Tingting, Mo Kaikun, Wang Dandan, Peng Xing, Liao Min, Zhou Jiyong
Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University , Hangzhou, China .
Monoclon Antib Immunodiagn Immunother. 2017 Apr;36(2):57-61. doi: 10.1089/mab.2016.0044. Epub 2017 Apr 14.
Premembrane (prM) is a viral protein of flavivirus, which is important for the generation of infectious virion and for virus infection to the host. However, the biological properties and function of the prM of Avian Tembusu virus (ATMUV) have scarcely been studied to date. Monoclonal antibodies (mAbs) are a powerful tool for functional analysis of viral protein. To produce a mAb against prM protein of ATMUV, the prM gene sequence was amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into the prokaryotic expression vector pET-28a (+). The recombinant prM protein was successfully expressed in BL21 (DE3). Using the purified prM as immunogen in mice, three hybridoma cells secreting mAbs against prM protein were obtained. These mAbs showed a strong reaction with ATMUV-infected DF-1 cells and pEGFP-C3-prM transfected 293-T cells in both Western blotting analysis and immunofluorescence assay. The mAbs developed in this study will be useful tools for analysis of the prM protein functions on ATMUV infection and the interaction between prM and its host molecules.
前膜蛋白（prM）是黄病毒属的一种病毒蛋白，对传染性病毒粒子的产生以及病毒感染宿主都很重要。然而，迄今为止，禽坦布苏病毒（ATMUV）的prM的生物学特性和功能鲜有研究。单克隆抗体（mAb）是病毒蛋白功能分析的有力工具。为制备抗ATMUV的prM蛋白的单克隆抗体，通过逆转录聚合酶链反应（RT-PCR）扩增prM基因序列，并将其克隆到原核表达载体pET-28a（+）中。重组prM蛋白在BL21（DE3）中成功表达。以纯化的prM作为免疫原免疫小鼠，获得了三株分泌抗prM蛋白单克隆抗体的杂交瘤细胞。在蛋白质印迹分析和免疫荧光试验中，这些单克隆抗体与感染ATMUV的DF-1细胞以及转染了pEGFP-C3-prM的293-T细胞均表现出强烈反应。本研究中制备的单克隆抗体将成为分析prM蛋白在ATMUV感染中的功能以及prM与其宿主分子之间相互作用的有用工具。
