Open reading frame detection compositions and methods
OwnerIrene Teresa Rombel, Kathryn F. Sykes +1inventors
InventorsIrene Teresa Rombel, Kathryn F. Sykes, Stephen Albert Johnston
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Abstract
The present invention relates to a series of plasmid-based expression vectors and methods for systematically screening entire genomes for gene-coding fragments. The compositions and methods described herein facilitate the detection of open reading frames within a DNA sequence. In this manner, the ORF selection vectors of the invention may be utilized in the isolation of genetic vaccine candidates for expression library immunization. The invention allows for the rapid, efficient screening of large genomes of eukaryotic parasites, for example, for determining protective gene-coding DNA fragments
Claims90
An ORF selection vector comprising:
- (a) a promoter;
- (b) a start codon operably linked to the promoter;
- (c) a reporter gene that is positioned downstream from both the promoter and the start codon and is out of frame.
A method of producing an ORF selection vector comprising:
- (a) contacting genomic DNA with a restriction endonuclease;
- (b) obtaining an ORF selection vector comprising:
- (i) a promoter;
- (ii) a start codon, wherein the start codon operably linked to the promoter;
- (iii) a reporter gene that is positioned downstream from both the promoter and the start codon and is out of frame;
- (c) contacting the ORF selection vector with a restriction endonuclease;
- and
- (d) ligating a genomic restriction endonuclease DNA fragment generated from step (a) with the linearized ORF selection vector.
A method of identifying at least a portion of an ORF comprising:
- (a) contacting genomic DNA with a restriction endonuclease;
- (b) obtaining an ORF selection vector comprising:
- (i) a promoter;
- (ii) a start codon operably linked to the promoter;
- (iii) a reporter gene that is positioned downstream from both the promoter and the start codon and is out of frame;
- (c) contacting the ORF selection vector with a restriction endonuclease;
- (d) ligating a genomic restriction endonuclease DNA fragment generated from step (a) with the linearized ORF selection vector;
- (e) transfecting a host cell with the ligated selection vector;
- (f) determining whether the reporter gene is expressed.
A method of inducing an immune response in an animal comprising:
- (a) obtaining an ORF selection vector comprising:
- (i) a promoter;
- (ii) a start codon operably linked to the promoter;
- (iii) a reporter gene that is positioned downstream from both the promoter and the start codon;
- (iv) at least a part of a genomic ORF that is positioned between the start codon and the reporter gene;
- (b) identifying an ORF by determining whether the reporter gene is expressed;
- (c) if the reporter gene is expressed, subcloning the ORF into an expression construct lacking the reporter gene;
- (d) introducing the expression construct into an the animal in a manner effective to induce an immune response against one or more antigens that may be encoded by the construct.
A method of preparing an antigen comprising:
- (a) obtaining an ORF selection vector comprising:
- (i) a promoter;
- (ii) a start codon operably linked to the promoter;
- (iii) a reporter gene that is positioned downstream from both the promoter and the start codon;
- (iv) at least a part of a genomic ORF that is positioned between the start codon and the reporter gene;
- (b) identifying an ORF by determining whether the reporter gene is expressed;
- (c) if the reporter gene is expressed, subcloning the ORF into an expression construct lacking the reporter gene;
- (d) administering to an animal a pharmaceutical composition comprising one or more expression constructs;
- and
- (e) identifying the antigen or antigens so expressed.
A kit for identifying an antigen comprising:
- (a) an ORF selection vector comprising:
- (i) a promoter;
- (ii) a start codon operably linked to the promoter;
- (iii) a reporter gene that is positioned downstream from both the promoter and the start codon and is out of frame;
- and
- (iv) a restriction endonuclease site between the reporter gene and the start codon;
- (b) an expression construct lacking the reporter gene.
Dependencies are inferred from the claim text.