Sauer B, Whealy M, Robbins A, Enquist L
Central Research and Development Department, E. I. du Pont de Nemours and Company, Wilmington, DE 19898.
Proc Natl Acad Sci U S A. 1987 Dec;84(24):9108-12. doi: 10.1073/pnas.84.24.9108.
A simple, efficient method for introducing recombinant DNA into a herpesvirus vector and retrieving it at a later time has been developed. By using the Cre-lox site-specific recombination system of coliphage P1, DNA can be readily inserted in vitro into a pseudorabies virus (PRV) vector containing the lox recombination site. The vector PRV42 contains a lox site within the nonessential gIII gene, which encodes a virion envelope glycoprotein. Incubation in vitro of PRV42 DNA with Cre protein and a circular plasmid containing a lox site generates approximately 5% recombinant molecules having the plasmid integrated into the PRV genome at the lox site. Transfection of the reaction mixture into cultured cells allows recovery of the infectious recombinant virus, which is readily identified by a nondestructive "black-plaque assay" using a gIII-specific monoclonal antibody. PRV42 plaques stain black when treated with the gIII monoclonal antibody and a peroxidase-linked second anti-antibody because the lox site placed within the gIII gene of PRV42 does not destroy the gIII epitope. However, Cre-mediated integration of heterologous DNA at the lox site disrupts the gIII epitope so that the resulting recombinant virus produces white plaques. The recombinant virus is infectious, stable, and grows as well as the parental PRV42 vector. The inserted plasmid can be efficiently excised (greater than 50%) from viral DNA by Cre and recovered by transformation of Escherichia coli.
一种简单、高效的将重组DNA导入疱疹病毒载体并在之后回收的方法已经开发出来。通过使用大肠杆菌噬菌体P1的Cre-lox位点特异性重组系统，DNA可以很容易地在体外插入到含有lox重组位点的伪狂犬病病毒（PRV）载体中。载体PRV42在非必需的gIII基因内含有一个lox位点，该基因编码一种病毒粒子包膜糖蛋白。将PRV42 DNA与Cre蛋白以及一个含有lox位点的环状质粒在体外孵育，可产生约5%的重组分子，这些分子将质粒整合到PRV基因组的lox位点处。将反应混合物转染到培养细胞中可回收感染性重组病毒，使用gIII特异性单克隆抗体通过非破坏性的“黑斑试验”可很容易地鉴定出该病毒。当用gIII单克隆抗体和过氧化物酶连接的二抗处理时，PRV42斑块会染成黑色，因为位于PRV42的gIII基因内的lox位点不会破坏gIII表位。然而，Cre介导的异源DNA在lox位点的整合会破坏gIII表位，从而使产生的重组病毒产生白色斑块。该重组病毒具有感染性、稳定性，其生长情况与亲本PRV42载体相同。插入的质粒可以通过Cre从病毒DNA中高效切除（大于50%），并通过转化大肠杆菌进行回收。
