Fuchs W, Bauer B, Mettenleiter T C, Rziha H J
Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany.
J Virol Methods. 1994 Jan;46(1):95-105. doi: 10.1016/0166-0934(94)90019-1.
Restriction enzyme analysis of DNA of a number of Pseudorabies virus (PRV) single plaque isolates revealed in several cases the existence of a unique EcoRI cleavage site, which has not been observed in PRV DNA before. This EcoRI site was mapped to the right end of the unique long region of the PRV genome, in BamHI-fragment 6. Sequence analysis of this region demonstrated the presence of an 11 bp tandem repeat in variable copy numbers in different PRV strains, suggesting the creation of the EcoRI recognition site by a recombinational event. The occurrence of variable reiterations and Northern blot analysis indicated an intergenic region. We therefore, used this site for integration and expression of heterologous DNA (the multiple cloning site of phage M13 and the E. coli lacZ gene). Viable PRV recombinants could be obtained which showed no detectable differences in virus growth in vitro compared to wild-type PRV. The novel insertion site can be used for the construction of PRV recombinants expressing foreign genes without apparent impairment of PRV genes.
对多个伪狂犬病病毒（PRV）单斑分离株的DNA进行限制性内切酶分析，结果在几例中发现存在一个独特的EcoRI切割位点，这在以前的PRV DNA中未曾观察到。该EcoRI位点定位于PRV基因组独特长区域的右端，在BamHI片段6中。对该区域的序列分析表明，不同PRV毒株中存在可变拷贝数的11 bp串联重复序列，提示该EcoRI识别位点是由重组事件产生的。可变重复序列的出现和Northern印迹分析表明这是一个基因间区域。因此，我们利用该位点进行异源DNA（噬菌体M13的多克隆位点和大肠杆菌lacZ基因）的整合和表达。可以获得有活力的PRV重组体，与野生型PRV相比，其在体外病毒生长方面没有可检测到的差异。这个新的插入位点可用于构建表达外源基因的PRV重组体，而不会明显损害PRV基因。
