IDENTIFICATION OF THE MEMBRANE-PROTEIN OF PORCINE EPIDEMIC DIARRHEA VIRUS
IDENTIFICATION OF THE MEMBRANE-PROTEIN OF PORCINE EPIDEMIC DIARRHEA VIRUS
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DOI：
10.1007/bf01702594
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发表时间：
1995-01-01
期刊：
影响因子：
1.6
通讯作者：
ACKERMANN, M
中科院分区：
文献类型：
--
作者：
UTIGER, A;TOBLER, K;ACKERMANN, M
猪流行性腹泻病毒（PEDV）基因组的序列信息最近才被确定。相比之下，人们对病毒蛋白知之甚少。在本报告中，我们通过使用兔抗肽血清和克隆的 M 基因在 Vero 细胞中的瞬时表达以及在杆状病毒系统中的表达，鉴定了 PEDV 的膜糖蛋白 (M)。 PEDV 的天然 M 蛋白被整合到病毒粒子中，进行 N-糖基化，并在聚丙烯酰胺凝胶中以 27 k 的相对迁移率 (Mr) 迁移。相比之下，重组杆状病毒合成的M蛋白以23k的Mr迁移，即与PEDV去糖基化产物具有相同的迁移率。因此，重组杆状病毒特异的M蛋白似乎糖基化程度很差（如果有的话）。使用对 M 蛋白 N 和 C 末端具有特异性的单克隆抗体和兔抗肽血清，我们能够证明在 PEDV 感染的细胞中检测到的 19 k 带（但在病毒颗粒中未检测到）代表 M 片段，其中 C 末端已被切除。最后，通过电子显微镜和免疫金标记，M在病毒体包膜内的相对方向被确定为NexoCcyt。总之，所有这些数据都强烈支持 PEDV 应归类为 I 类冠状病毒的假设。
Sequence information on the genome of porcine epidemic diarrhea virus (PEDV) has only recently been determined. In contrast, very little is known about the viral proteins. In the present report we have identified the membrane glycoprotein (M) of PEDV by use of rabbit anti-peptide sera and transient expression of the cloned M gene in Vero cells and by expression in the baculovirus system. The native M protein of PEDV is incorporated into virions, is N-glycosylated, and migrates with a relative mobility (Mr) of 27 k in polyacrylamide gels. In contrast, the M protein synthesized by recombinant baculoviruses migrates with a Mr of 23 k, that is, with identical mobility as the deglycosylated product of PEDV. Thus, it appears that M protein specified by the recombinant baculovirus is poorly, if at all, glycosylated. Using monoclonal antibodies and rabbit antipeptide sera specific for the N and C termini of the M protein, we were able to show that a 19 k band detected in PEDV-infected cells but not in virions represented a fragment of M from which the C terminus had been cleaved off. Finally, by electron microscopy and immunogold labelling, the relative orientation of M within the virion envelope was determined as NexoCcyt. In conclusion, all of these data strongly support the hypothesis that PEDV should be classified with the group I coronaviruses.