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Title

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

AuthorsAlcolea, Pedro J. ; Alonso, Ana ; Larraga, Vicente
KeywordsTriclosan
LACK/p36
Third generation vaccine
Plasmid
Selection marker
Enoyl-ACP reductase
CpG islands
Leishmaniasis
Th1 response
Issue DateJan-2019
PublisherElsevier
CitationPlasmid 101:35-42 (2019)
AbstractDNA 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, the selection of positive clones depends on the enzyme:inhibitor molar ratio. According to western blot analysis, the pPAL vector is functional for expression of the Leishmania infantum (Kinetoplastid: Trypanosomatidae) gene encoding for the protein kinase C receptor analog (LACK/p36) in the HEK293T human 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 contains the essential elements for manipulation and expression of any cloned DNA sequence in prokaryotic and mammalian cells using an E. coli endogenous gene as a selectable marker, which also provides a long CpG island. This element enhances Th1 immune response against L. infantum infection in dogs using the gene encoding for the LACK antigen. Therefore, this antibiotic resistance-free plasmid is a vaccine vector actively participating in protection against canine leishmaniasis and may be potentially tested as a vaccine vector with other antigens against different pathogens.
Description26 p.-6 fig.-1 tab.
Publisher version (URL)https://doi.org/10.1016/j.plasmid.2018.12.002
URIhttp://hdl.handle.net/10261/175610
DOI10.1016/j.plasmid.2018.12.002
ISSN0147-619X
E-ISSN1095-9890
Appears in Collections:(CIB) Artículos
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