Plasmid maintenance system for antigen delivery
AssigneeUniversity of Maryland, Baltimore
InventorJames E. Galen
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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.
Claims27
A method of making a live attenuated bacterial vector vaccine, comprising transforming a bacterial strain with an expression vector, wherein said expression vector comprises a nucleotide sequence encoding:
- 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;
- at least one post-segregational killing cassette comprising (i) a nucleotide sequence encoding at least one post-segregational killing locus, (ii) a first unique restriction enzyme cleavage site located 5′ of the nucleotide sequence encoding the at least one post-segregational killing locus, and (iii) a second unique restriction enzyme cleavage site located 3′ of the nucleotide sequence encoding the at least one post-segregational killing locus, wherein said nucleotide sequence encoding at least one post-segregational killing locus encodes the ssb post-segregational killing locus;
- and
- at least one partitioning cassette comprising (i) a nucleotide sequence encoding at least one partitioning function, (ii) a first unique restriction enzyme cleavage site 5′ of the nucleotide sequence encoding the at least one partitioning function, and (iii) a second 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.