A composite DNA sequence of regions of hepatitis B virus, determined from a series of recombinant plasmids, reveals the genes for the surface antigen and the core antigen of the virus. The sequence of the core antigen shows it to be a DNA binding protein. The core antigen gene is expressed in Escherichia coli and when injected into rabbits the bacterial product induces antibodies which react with core antigen isolated from human sources.
A cDNA for endothelial leukocyte adhesion molecule 1 (ELAM-1) was isolated by transient expression in COS-7 cells of a subtracted cDNA library from cytokinetreated human umbilical vein endothelial cells (HUVECs), with selection of ELAM-1-expressing clones by adhesion of transfected cells to the human promyelocytic cell line HL-60. This cloning method requires neither antibody nor purified ligand. ELAM-1-expressing COS cells bind the promyelocytic cell line HL-60 by a Ca2+-dependent but temperature-independent mechanism. Although ELAM-1 is homologous to mammalian lectins, its interaction with HL-60 cells is not inhibited by simple carbohydrate structures. ELAM-1-expressing COS cells also bind human neutrophils and the human colon carcinoma cell line HT-29, but not the B-cell line Ramos. However, Ramos cells adhere to cytokine-treated HUVECs but not control HUVECs, confirming the existence of other inducible adhesion molecules. In addition, the binding of HL-60 cells or neutrophils to ELAM-1-expressing COS cells is not inhibited by a monoclonal antibody (60.3) directed to an inhibitory epitope on CD18, indicating that the ELAM-1 ligand, although uncharacterized, is not a member of the CD11/CD18 family.
Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value ofan in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.Hepatitis B virus (HBV) is widespread, but detailed studies of its molecular structure and of its pathobiology have been frustrated by the inability to propagate the virus in cultured cells (1). DNA from HBV has now been cloned and propagated in Escherichia colA, both as a series offragments and as entire linear molecules afterjoining to plasmid or lambdoid phage vectors (2-4), to provide HBV DNA for nucleotide sequence determination from which the general organization of the viral genome was deduced (5-7).Several (10), pKT234 and pKT279 (11), and pEXlac2O5 (12) in the E. coli strain HB101. The AT4 lig phage NM989 (13) was used as a vector into which recombinant plasmids were inserted after digestion with EcoRI. The transfection host strain and strain used for lysogenization were as described (13). Details ofrestriction and ligation reactions, polynucleotide terminal transferase reactions, and transformation and transfection operations have been reported (2).Recombinant plasmids were characterized by their drug-resistance markers and their structures were confirmed by the fragments released on digestion with restriction enzymes and by nucleotide sequence analysis (14, 15). Recombinant phage were identified by plaque hybridization (16) against a 2P-labeled probe made by nick-translation (17) of the plasmid pHBV114 (7). Most enzymes were prepared in the various laboratories or were purchased from New England BioLabs, Bethesda Research Laboratories (Rockville, MD), or Boehringer Mannheim AG.Radioimmunoassays. Reagents used for the solid-phase radioimmunoassay adapted for direct screening of bacterial colonies or phage plaques (8) were as described (2). In double-antibody radioimmunoprecipitation assays (18), rabbit serum (50 Al, diluted appropriately) was incubated with 1"I-labeled HBsAg (125I-HBsAg; 50 1.l, containing 3000 cpm) at 40C for at least 18 hr and complexes precipitated by addition of donkey anti-rabbit serum (DARS; 100 Al, at the appropriate dilution) and pelleted by centrifugation after at least 18 hr at 40C; supernatants were removed and the radioactivity in the pellets was counted. In competitive inhibition assays the appropriately diluted rabbit serum (50 Al) was incubated w...
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