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


OBJECTIVE
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.


METHODS
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 proIL1 beta.


CONCLUSIONS
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.
Clarke
N J
NJ
Molecular Pathology Research Unit, Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, UK.
Hissey
P
P
Buchan
K
K
Harris
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001483:Base Sequence; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000276:immunology; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D010714:Phosphatidylethanolamines; D010957:Plasmids / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification; D012270:Ribosomes / Q000235:genetics*; D019444:Vaccines, DNA / Q000737:chemistry* / Q000276:immunology
D007375:Interleukin-1; D008081:Liposomes; D010714:Phosphatidylethanolamines; D019444:Vaccines, DNA; C020888:1,2-dielaidoylphosphatidylethanolamine