Construction and evaluation of a new triple-gene expression cassette vaccinia virus shuttle vector

A vaccinia virus shuttle vector pSTKE with a triple-gene expression cassette was designed, and the derived recombinant virus could express at least three different target genes. A vaccinia virus and its mutant as the original viruses and EGFP as the reporter gene were used to verify the three expression cassettes. Two recombinant viruses containing EGFP were obtained by homologous recombination and plaque screening. The expression and genetic stability of the recombinant virus and foreign genes were analyzed using PCR, real-time PCR, and Western blot. And then EGFP, RFP and BFP were inserted into MCS1, MCS2 and MCS3 of pSTKE respectively, resulting in the generation of recombinant expressing three fluorescent proteins mentioned above, and the recombinant was continuously passaged 20 times. The results showed that EGFP, RFP and BFP were highly expressed in vaccinia virus, and no interaction between the three expression cassettes was observed. Recombinant viruses were stable genetically. The shuttle vector pSTKE can be used for efficient and stable gene expression to address problems in recombinant vaccinia viruses, such as low expression efficiency, limited number of inserted genes. In addition, this study provides a solid foundation for the development of a new genetically engineered vaccine.