Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles.
Production of monoclonal antibodies against the ORF3 protein of rat hepatitis E virus (HEV) and demonstration of the incorporation of the ORF3 protein into enveloped rat HEV particles.
复制标题
生产抗大鼠戊型肝炎病毒 (HEV) ORF3 蛋白的单克隆抗体，并证明 ORF3 蛋白掺入有包膜的大鼠 HEV 颗粒中。
DOI：
10.1007/s00705-016-3047-9
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发表时间：
2016
影响因子：
2.7
通讯作者：
Okamoto H
中科院分区：
文献类型：
--
作者：
Takahashi M;Kobayashi T;Tanggis;Jirintai S;Mulyanto;Nagashima S;Nishizawa T;Kunita S;Okamoto H
针对大鼠戊型肝炎病毒（ratHEV）ORF 3蛋白C-末端15-氨基酸部分的合成肽，制备了8种鼠单克隆抗体（MAbs），并进行了表征。使用抗ratHEV ORF 3单克隆抗体的免疫荧光检测显示，ORF 3蛋白在用ORF 3表达质粒转染或用细胞培养物产生的ratHEV株接种的PLC/PRF/5细胞的细胞质中积累。抗ORF 3单克隆抗体能捕获0.5%脱氧胆酸盐处理后培养上清和血清中的ratHEV颗粒，但不能捕获未经去污剂处理的培养上清和血清中的ratHEV颗粒。经0.5%脱氧胆酸盐和0.5%胰蛋白酶处理后，在蔗糖梯度中，表面有ORF 3蛋白的培养上清液中的ratHEV颗粒的浮力密度从1.15 g/cm 3变为1.26 g/cm 3，所得颗粒可被抗ORF 2单克隆抗体捕获，但不能被抗ORF 3单克隆抗体捕获。这表明ORF 3蛋白（至少其C-末端部分）被掺入从感染细胞释放的有包膜的ratHEV病毒体中，但在粪便中的病毒体中未发现ORF 3蛋白，这支持了以下假设：ratHEV ORF 3蛋白与病毒体从感染细胞中流出有关，类似于人HEV，尽管事实上ratHEV ORF 3蛋白缺乏PSAP氨基酸基序。
Eight murine monoclonal antibodies (MAbs) against a synthetic peptide corresponding to the C-terminal 15-amino-acid portion of the ORF3 protein of rat hepatitis E virus (ratHEV) were produced and characterized. Immunofluorescence assays using the anti-ratHEV ORF3 MAbs revealed the accumulation of ORF3 protein in the cytoplasm of PLC/PRF/5 cells transfected with ORF3-expressing plasmids or inoculated with cell-culture-generated ratHEV strains. Anti-ORF3 MAbs could capture ratHEV particles in culture supernatant and serum following treatment with 0.5 % deoxycholate, but not those without prior detergent treatment or fecal ratHEV particles. Following treatment with 0.5 % deoxycholate and 0.5 % trypsin, the buoyant density of ratHEV particles in culture supernatant with ORF3 protein on the surface shifted from 1.15 g/cm3to 1.26 g/cm3in a sucrose gradient; the resulting particles were capturable by an anti-ORF2 MAb but not by an anti-ORF3 MAb. This indicates that the ORF3 protein (at least its C-terminal portion) is incorporated into the enveloped ratHEV virions released from infected cells but that it is not found in the virions in the feces, supporting the hypothesis that the ratHEV ORF3 protein is associated with the egress of virions from infected cells, similar to human HEV, despite the fact that the ratHEV ORF3 protein lacks a PSAP amino acid motif.