Abstract

A simple yet powerful selection system was developed for the insertion of foreign genes in vaccinia virus. The selection system utilizes the vaccinia virus KIL (29K) host range gene which is located in HindIII M. This gene is necessary for growth in RK-13 cells but not in BSC40 or CV-1 cells. A vaccinia mutant (vAbT33) unable to grow on RK-13 cells was constructed having sequences at the 3′ end of the KIL gene and the adjacent M2L gene deleted and replaced with the β-galactosidase gene regulated by the BamHI F (F7L) promoter. A recombination plasmid containing the hepatitis B surface (HBs) antigen gene regulated by the M2L promoter and the complete sequence of the KIL gene was used to insert the HBs gene into vAbT33. The M2L negative KIL positive recombinant was easily isolated in two rounds of plaque purification by plating the virus on RK-13 cell monolayers. The KIL gene selection system allows the isolation of recombinants arising at frequencies as low as 1 100 000 . It was noted that recombinants containing vaccinia sequence duplications (promoters) resulted in intragenomic recombinations that eliminated all sequences between the duplications. A second recombination plasmid was constructed that allowed insertion into the vaccinia genome without the loss of vaccinia coding sequences. This was achieved by insertion of the pseudorabies virus GIII gene regulated by the vaccinia H5R (40K) promoter between the translation and transcription stop signals at the 3′ end of the KIL gene. The KIL gene transcription stop signal thus became the stop signal for the inserted GIII gene and an upstream transcription stop signal present in the H5R promoter fragment provided the stop signal for the KIL gene. This manipulation of the vaccinia genome had no effect on the accumulation or 5′ end of the M2L gene transcripts. Although the insertion lengthened the 3′ end and lowered the accumulation of KIL transcripts it altered neither the virulence nor the immunogenicity of the recombinant.

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