The antibiotic resistance-free mammalian expression plasmid vector pPAL for development of third generation vaccines

Publication date: Available online 6 December 2018Source: PlasmidAuthor(s): Pedro J. Alcolea, Ana Alonso, Vicente LarragaAbstractDNA vaccines require a vector to replicate genes and express encoding antigens. Antibiotic resistance genes are often used as selection markers, which must not be released to the environment upon final product commercialization. For this reason, generation of antibiotic resistance-free vectors is imperative. The pPAL vector contains the cytomegalovirus enhancer and promoter for expression in mammalian cells, and the E. coli fabI chromosomal gene as a selectable marker. The fabI gene encodes the enoyl-ACP reductase (FabI). The bacteriostatic compound triclosan is an inhibitor of this enzyme. Therefore, selection of positive clones depends of the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for mammalian expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding the protein kinase C receptor analogue (LACK/p36) in the HEK293T cell line transfected with pPAL-LACK. The fabI gene sequence contains a 210 bp CpG island, suggesting a potential role as an adjuvant of the antibiotic resistance-free pPAL vector. In fact, Th1 response induction levels against canine leishmaniasis only using pPAL-LACK was shown to be as strong as in previous strategies using a recombinant vaccinia virus in combination with standard mammalian expression plasmid vectors. In summary, the pPAL plasmid cont...
Source: Plasmid - Category: Biotechnology Source Type: research