A plasmid vector for cloning and expression of gene segments: expression of an HTLV-I envelope gene segment.
We describe a cloning-expression vector system for selecting DNA fragments containing open reading frames (ORFs) and expressing them as beta-galactosidase (beta Gal) hybrid fusion proteins. The plasmid vector, pWS50, utilizes the very strong and easily regulated bacteriophage lambda promoter pL, and the efficient translation initiation signals of the N-terminal segment of the lambda cII gene. Fused distally to and out of translational phase with cII is the E. coli lacZ gene, lacking its own transcriptional and translational initiating signals. A unique restriction enzyme site (NruI) is located between the upstream regulatory sequences and the lacZ gene, which provides a cloning site for the insertion of blunt ended DNA fragments. In addition, there are two other unique restriction sites (NheI and BamHI) located in this region which can also be used as closing sites. If a DNA fragment does not contain any translation termination codons (i.e., an ORF), and is inserted correctly into the vector, the translational reading frame between cII and lacZ can be restored. Colonies containing these recombinants can be easily screened as LacZ+ on lactose indicator media. The beta-galactosidase fusion proteins produced from the LacZ+ recombinants are identified on sodium dodecyl sulfate polyacrylamide gels by their large size and high level of production. To test the ORF cloning-expression system, a segment of the human T-cell lymphotrophic virus type I envelope gene was cloned and expressed at high levels. The envelope-beta Gal fusion protein was recognized by antibodies in serum from a patient with adult T-cell leukemia.
D000912:Antibodies, Neoplasm / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000235:genetics; D017977:Deltaretrovirus / Q000235:genetics* / Q000276:immunology; D015480:Deltaretrovirus Antibodies; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007938:Leukemia / Q000276:immunology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology
D000912:Antibodies, Neoplasm; D000914:Antibodies, Viral; D004279:DNA, Viral; D015480:Deltaretrovirus Antibodies; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D004262:DNA Restriction Enzymes