Plasmid maintenance system for antigen delivery
AssigneeUniversity of Maryland, Baltimore
InventorsJames E. Galen, Christofer Vindurampulle
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Abstract
The present invention relates generally to a Plasmid Maintenance System for the stabilization of expression plasmids encoding foreign antigens, and methods for making and using the Plasmid Maintenance System. The invention optimizes the maintenance of expression plasmids at two independent levels by: (1) removing sole dependence on balanced lethal maintenance functions; and (2) incorporating at least one plasmid partition function to prevent random segregation of expression plasmids, thereby enhancing their inheritance and stability. The Plasmid Maintenance System may be employed within a plasmid which has been recombinantly engineered to express a variety of expression products.
Claims23
An expression vector comprising a nucleotide sequence encoding:
- (a) a restricted-copy-number origin of replication cassette comprising
- (i) a nucleotide sequence encoding an origin of replication that limits the expression vector to an average plasmid copy number of about 2 to 75 copies per cell,
- (ii) a first unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the origin of replication, and
- (iii) a second unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the origin of replication;
- (b) at least one post-segregational killing cassette comprising
- (i) a nucleotide sequence encoding a ssb post-segregational killing locus, wherein the nucleotide sequence encoding the ssb post-segregational killing locus is the nucleotide sequence of SEQ ID NO:41 or a nucleotide sequence having at least 95% sequence identity over its entire length to the nucleotide sequence of SEQ ID NO:41, wherein the nucleotide sequence of SEQ ID NO:41 comprises the ssb inducible promoter, the ssb constitutive promoter and the ssb coding region of Shigella flexneri 2a strain 2457t, and wherein both the inducible and constitutive promoters of said nucleotide sequence having at least 95% sequence identity have promoter activity, and wherein the SSB polypeptide encoded by said nucleotide sequence having at least 95% sequence identity has DNA binding and DNA replication activity,
- (ii) a third unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the ssb post-segregational killing locus, and
- (iii) a fourth unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the ssb post-segregational killing locus;
- and
- (c) at least one partitioning cassette comprising
- (i) a nucleotide sequence encoding at least one partitioning function,
- (ii) a fifth unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the at least one partitioning function, and
- (iii) a sixth unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the at least one partitioning function.
Dependencies are inferred from the claim text.