New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins.

(1990) New vaccinia virus recombination plasmids incorporating a synthetic late promoter for high level expression of foreign proteins. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge The content must not be changed in any way or reproduced in any format or medium without the formal permission of the copyright holder(s) When referring to this work, full bibliographic details must be given Vaccinia virus has found considerable utility as a vector for expressing foreign genes in eukaryotic cells. Foreign genes may be regulated by early or late vaccinia promoters, but much higher levels of expression may be achieved with the latter (1,2). Based on a detailed analysis of vaccinia late promoters, we designed a synthetic promoter that is at least half as strong again as the most active poxvirus natural late promoters presently used in expression systems (3). We describe here a pair of recombination plasmids incorporating this powerful late promoter. The plasmids, pMJ601 and pMJ602, are 7.2 kbp in size, and comprise a cassette inserted into the EcoRI site of the vaccinia virus thymidine kinase (TK) gene, which is carried on a portion of pBR328 that includes the betalactamase gene. They were made by insertion of synthetic oligonucleotide duplexes into a parental plasmid, pMJ7 (4). The cassette consists of the powerful late promoter immediately upstream from a multiple cloning site (MCS) containing 11 unique restriction endonuclease sites and lacking ATG codons, followed by lacZ under the control of the vaccinia 7.5-kD early promoter. The two plasmids differ only in the orientation of the MCS. Features of pMJ601 and the sequence upstream oflacZ are shown in Fig. 1. The mRNA starts within the indicated AAA sequence, and contains a poly(A) leader. A DNA fragment containing a continuous ATG-initiated open reading frame may be inserted into the MCS, and the resulting cassette recombined into the vaccinia virus genome at the TK locus (4). Recombinant virus may be isolated by selecting for lack of TK expression and by screening for expression of betagalactosidase from lacZ (4). We have used pMJ601 and pMJ602 to construct several recombinant viruses that express herpesvirus proteins. As an example, Fig. 2 shows the proteins expressed by two recombinant viruses. The virus made using the parental plasmid pMJ601 expressed betagalactosidase at a high level (lane 1) because, in the absence of an insert, lacZ is regulated by both the powerful late …