Monoclonal antibodies were prepared against herpes simplex virus type 1 (strain 14012) by two immunization procedures. Procedure A utilized infectious virus propagated in mouse cells, and procedure B utilized mouse cells infected with herpes simplex virus in the presence of cycloheximide and harvested 1 h after removal of the inhibitor. A total of 52 monoclonal antibodies were obtained against 10 herpes simplex virus proteins, including four glycosylated proteins (a 110,000-molecular-weight protein, gB, gC, and gD) and six nonglycosylated proteins (a 68,000-molecular-weight protein, ICP 9, ICP 8, ICP 6, ICP 5, and the immediate-early ICP 4). The antibodies were assayed against herpes simplex virus types 1 and 2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioimmunoprecipitates, immunofluorescence, and neutralization. Using the reagents prepared, we concluded that the 110,000-molecular-weight protein, gD, ICP 9, ICP 9, ICP 6, and the 68,000-molecular-weight protein express both type-specific and cross-reactive antigenic determinants. In contrast, nine antibodies against gB all cross-reacted with herpes simplex virus type 2, whereas eight antibodies to gC all reacted type specifically.

We examined the properties of 17 monoclonal antibodies to glycoprotein gD of herpes simplex type 1 (HSV-1) (gD-1) and HSV-2 (gD-2). The antibodies recognized eight separate determinants of gD, based on differences in radioimmuno-precipitation and neutralization assays. The determinants were distributed as follows: three were gD-1 specific, one was gD-2 specific, and four were type common. Several type-specific and type-common determinants appeared to be involved in neutralization. We developed a procedure for examining the effect that binding of monoclonal antibody has on proteolysis of native gD-1 by Staphylococcus aureus protease V8. We showed that several different patterns of protease V8 cleavage were obtained, depending on the monoclonal antibody used. The proteolysis patterns were generally consistent with the immunological groupings. With four groups of antibodies, we found that fragments of gD-1 remained bound to antibody after V8 treatment. A 38,000-dalton fragment remained bound to antibodies in three different groups of monoclonal antibodies. This fragment appeared to contain one type-common and two type-specific determinants. A 12,000-dalton fragment remained bound to antibodies belonging to one typecommon group of monoclonal antibodies. Tryptic peptide analysis revealed that the 12,000-dalton fragment represented a portion of the 38,000-dalton fragment and was enriched in a type-common arginine tryptic peptide.

The Raji line of human lymphoblastoid cells, which does not show expression of Epstein-Barr virus, was made resistant to 5-bromodeoxyuridine. Within several weeks after removal of the drug, Epstein-Barr virus particles were detected in the cells. The

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of 3-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ-because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ' gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and J3-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.One of the goals of genetic engineering is the expression of all or part of a gene to produce an antigenic protein segment that can be used as a vaccine or to raise or detect antibodies for research and diagnosis. Expressing A(argF-lac)U169 rpsL150 (strR) relAl flbB5301 deoCl ptsF25 malPQ: :Tn5 ompBcsl. The ompR101 mutation (2) drastically decreases ompF expression, whereas the ompBcsl mutation decreases ompF expression at temperatures near 30°C and shows increased ompF expression above 37°C (unpublished data). MH3000 was used to construct LacZ+ clones because it is highly transformable and prevents the overproduction lethality of ompF-lacZ fusions (see Results and Discussion). We also observed that ompR+ cells containing the pORF vectors formed light blue colonies on 5-bromo-4-chloro-3-indolyl-f3-D-galactoside (XG) plates, even though ompF and lacZ were out of frame, whereas with MH3000 the colonies were white, allowing clones to be more clearly identified. TK1046 transformed poorly and was not suitable for the initial construction of clones; its use was therefore limited to testing for overproduction lethality and producing high levels of tribrid proteins. The cs mutation in TK1046 is either in ompR or envZ and allows more expression of ompF at 30°C than does ompR101. Vero The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.