Vaccinia virus has been developed into a eukaryotic expression vector. A chimeric gene is formed by ligating vaccinia virus transcriptional regulatory signals to a foreign protein coding sequence. Homologous recombination is used to insert the chimeric gene into a non-essential region of the vaccinia virus genome. Several different methods have been developed to select or screen the recombinant vaccinia viruses. The recombinant viruses can then be used to express proteins in a variety of tissue culture cells or animals. A novel modification of the recombinant vaccinia virus expression system which utilizes the bacteriophage T7 RNA polymerase and T7 transcriptional regulatory signals was developed. The prokaryotic RNA made in mammalian cells by this system, however, was not efficiently capped. Since the 5' cap structure is important for stability and translatability, this presented a severe problem. Stability was achieved by using DNA encoding the 5' end of a T7 transcript with potential to form a stem-loop structure. Translatability was obtained by employing the untranslated leader sequence of encephalomyocarditis virus, which confers cap-independent ribosome binding. With the newly modified system, after 24 hours of infection the recombinant protein comprises approximately 10% of the total cell protein. Several new plasmid victors that facilitate and increase the expression of genes were developed. In addition, a new method of selecting recombinant vaccinia viruses that allows the insertion of several genes was introduced.
