Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

Abstract

We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli (3-galactosidase ("gal) Vaccinia virus, the most thoroughly studied member of the poxvirus family, was successfully used as a live vaccine to eradicate smallpox. Medical interest in vaccinia virus subsequently declined but was restimulated when live recombinants were shown to be capable of expressing foreign genes (8, 18) and of protectively immunizing animals against infections with influenza virus (26), herpes simplex virus types 1 (6, 19) and 2 (6), hepatitis B virus (14), rabies virus (31) and vesicular stomatitis virus (10). The ability to incorporate large amounts of foreign DNA without a loss of infectivity (25), the correct processing of expressed proteins (6,10,24, 26,31), and the wide host range of vaccinia virus also make this expression system of special value for research purposes. One novel application has been to identify target antigens for cell-mediated immune responses to viruses (1, 33).To facilitate the use of vaccinia virus as a cloning and expression vehicle, Mackett et al. (9) constructed plasmid vectors that contain defined vaccinia virus promoters, restriction sites for the insertion of foreign coding sequences, and flanking segments of the vaccinia virus thymidine kinase (TK) gene. Vaccinia virus promoters, which are found immediately upstream of early and late vaccinia virus genes (2, 5, 9, 30), are necessary because of the specificity of the viral transcription system (21) and the cytoplasmic site of replication. Flanking segments of vaccinia virus DNA are used for homologous recombination (16,23,28), the most practical way of inserting foreign sequences into the large 187,000-base-pair (187-kilobase) vaccinia virus genome (8,18). When an insertion is directed into the TK locus, recombinants have a TK-phenotype, providing a simple method of selection (8, 9). Although this selection method is extremely valuable, TK-recombinants and spontaneous TK-mutants still have to be distinguished either by DNA hybridization or by the expression of specific proteins. In our experience, usually between 10 and 80%o of TK-plaques are formed by recombinants, although at times the percentage is significantly lower. A rapid method of discriminating between TK-recombinants and spontaneous TK-mutants * Corresponding author.would enhance the vector system. In addition, a screening method that is not dependent on TK-cells or on the loss of the TK function would be useful.The formation of blue plaques by bacteriophages that express Escherichia coli 1-galactosidase (13-gal) has been used as the basis for rapid screening systems (12). Most commonly, insertion of DNA into the lacZ gene (which codes for ,B-gal) leads to the loss of 1-gal activity and the consequent production of white plaques. Recently, 13-gal fusion proteins were expressed in baculovirus (20)

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“…5,6 The wild-type (wt) virus used in the preparation of the recombinant was derived from the New York City Board of Health strain of vaccinia (Centers for Disease Control, Atlanta, Ga). The full-length cDNA for GM-CSF was obtained from the American Type Culture Collection (Manassas, Va) (pCSF-1, no.…”

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