Parris D S, Cross A, Haarr L, Orr A, Frame M C, Murphy M, McGeoch D J, Marsden H S
Department of Medical Microbiology and Immunology, Ohio State University, Columbus 43210.
J Virol. 1988 Mar;62(3):818-25. doi: 10.1128/JVI.62.3.818-825.1988.
Hybrid arrest of in vitro translation was used to localize the region of the herpes simplex virus type 1 genome encoding the 65-kilodalton DNA-binding protein (65KDBP) to between genome coordinates 0.592 and 0.649. Knowledge of the DNA sequence of this region allowed us to identify three open reading frames as likely candidates for the gene encoding 65KDBP. Two independent approaches were used to determine which of these three open reading frames encoded the protein. For the first approach a monoclonal antibody, MAb 6898, which reacted specifically with 65KDBP, was isolated. This antibody was used, with the techniques of hybrid arrest of in vitro translation and in vitro translation of selected mRNA, to identify the gene encoding 65KDBP. The second approach involved preparation of antisera directed against oligopeptides corresponding to regions of the predicted amino acid sequence of this gene. These antisera reacted specifically with 65KDBP, thus confirming the gene assignment.
利用体外翻译杂交阻止法将编码65千道尔顿DNA结合蛋白（65KDBP）的1型单纯疱疹病毒基因组区域定位到基因组坐标0.592至0.649之间。该区域DNA序列的信息使我们能够确定三个开放阅读框作为编码65KDBP基因的可能候选者。采用了两种独立的方法来确定这三个开放阅读框中哪一个编码该蛋白。第一种方法是分离出一种与65KDBP特异性反应的单克隆抗体MAb 6898。该抗体与体外翻译杂交阻止技术和所选mRNA的体外翻译技术一起用于鉴定编码65KDBP的基因。第二种方法是制备针对与该基因预测氨基酸序列区域相对应的寡肽的抗血清。这些抗血清与65KDBP特异性反应，从而证实了基因的归属。
