PPV: a novel IRES-containing vector to facilitate plasmid immunization and antibody response characterization

Abstract

Background: The ability to derive immunological reagents for basic and applied research in a timely fashion is a basic requirement of many research projects and is becoming increasingly important as the number of novel gene products of potential interest continues to evolve rapidly. DNA immunization provides a means of facilitating the production of antibody reagents by circumventing the need to derive either purified protein or define peptides before initiating an in vivo immunization protocol. Objectives: The DNA construct pPV, for plasmid vaccination, has been designed to facilitate the generation and characterization of antibody reagents against either random or defined molecular targets. Study design: pPV incorporates mammalian regulatory and structural features that promote expression of a bifunctional messenger RNA (mRNA) from a single promoter within mammalian cells both in vitro and in vivo. The bifunctional mRNA encodes a control epitope (human IL5), and the `test' epitope expressed as a tagged recombinant polypeptide in either a random `shot-gun' mode or a predetermined fashion. In addition, to aid subsequent characterization of antibody responses elicited in vivo, a T7 promoter is included to enable in vitro expression of tagged recombinant polypeptides. Results: The utility and functionality of pPV for the in vitro expression of recombinant protein and the in vivo elicitation of antibody responses is illustrated using a defined `test' epitope, human proIL1β. Conclusion: It is anticipated pPV will find particular utility in the future rapid generation and characterization of antibody reagents against the plethora of novel genes emerging from ongoing genomics activity in a directed or genome wide fashion.