Otsuka H, Xuan X
Department of Animal Resource Science, Graduate School of Agricultural Sciences, University of Tokyo, Japan.
Arch Virol. 1996;141(1):57-71. doi: 10.1007/BF01718588.
We have improved the method for constructing recombinants of bovine herpesvirus type-1 (BHV-1). Using this method, we constructed three recombinants in which the pseudorabies virus (PRV) thymidine kinase (tk) gene was inserted at three different sites in the unique short region of BHV-1. These three sites are located in the open reading frame of gE, gG and gI genes. Previously, two sites (tk and gC) had been used to insert foreign DNA fragments to BHV-1 genome. Therefore we now have 5 sites in BHV-1 where DNA can be inserted. The gB, gC, gD, gE and gI genes of PRV were successfully inserted at the tk or the gC gene of BHV-1 genome and Western blot analyses confirmed that the recombinants express PRV gB, gC, gD and gE. Anti-PRV gB and gC antibodies as well as anti-PRV polyclonal serum neutralized BHV-1 recombinants which express PRV gB and gC. The latter was neutralized more strongly. However, anti-gD monoclonal antibody and anti-PRV polyclonal serum failed to neutralize gD-expressing recombinants. This suggests that PRV gC and some gB are integrated into the viral envelope of the recombinants, but very little gD is present in the viral envelope.
我们改进了构建牛疱疹病毒1型（BHV-1）重组体的方法。利用该方法，我们构建了三个重组体，其中伪狂犬病病毒（PRV）胸苷激酶（tk）基因插入到BHV-1独特短区域的三个不同位点。这三个位点分别位于gE、gG和gI基因的开放阅读框中。此前，已有两个位点（tk和gC）用于将外源DNA片段插入BHV-1基因组。因此，我们现在在BHV-1中有5个可插入DNA的位点。PRV的gB、gC、gD、gE和gI基因成功插入到BHV-1基因组的tk或gC基因处，蛋白质免疫印迹分析证实这些重组体表达PRV的gB、gC、gD和gE。抗PRV gB和gC抗体以及抗PRV多克隆血清可中和表达PRV gB和gC的BHV-1重组体。后者的中和作用更强。然而，抗gD单克隆抗体和抗PRV多克隆血清未能中和表达gD的重组体。这表明PRV的gC和部分gB整合到了重组体的病毒包膜中，但病毒包膜中gD的含量极少。
