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Biomedical subjects

D Mottershead

Publications and source records attributed to D Mottershead.

4 recordsLinked to original sources
PubMed1999

Mimicking rubella virus particles by using recombinant envelope glycoproteins and liposomes.

The envelope glycoproteins E1 and E2 of rubella virus (RV) were engineered to display the FLAG epitope tag and a polyhistidine tag, at their amino and carboxy termini, respectively. These modified envelope proteins were produced in Sf9 insect cells utilizing baculovirus expression vectors, the E1 and E2 vectors giving rise to protein products of about 58 and 42 kDa, respectively. The recombinant proteins were purified by immobilized metal-ion affinity chromatography and reconstituted into liposomes via their hydrophobic transmembrane anchors. The liposomes were prepared by detergent dialysis in the presence of europium-DTPA chelate, enabling the subsequent measurement of the binding of the resultant proteoliposomes to the antibodies by time resolved fluorescence. RV mimicking proteoliposomes were recognized by antibodies specific for the E1 and E2 proteins, as well as the FLAG epitope tag. This type of virosome may prove useful for studies on the basic biological events of an RV infection or as diagnostic reagents.

Amino Acid Sequence↗
PubMed1997

Baculoviral display of the green fluorescent protein and rubella virus envelope proteins.

The ability to display heterologous proteins and peptides on the surface of different types of bacteriophage has proven extremely useful in protein structure/function studies. To display such proteins in a eucaryotic environment, we have produced a vector allowing for fusion of proteins to the amino-terminus of the Autographa californica nuclear polyhedrosis virus (AcNPV) major envelope glycoprotein, gp64. Such fusion proteins incorporate into the baculoviral virion and display the FLAG epitope tag. We have further produced recombinant baculoviruses displaying the green fluorescent protein (GFP) and the rubella virus envelope proteins, E1 and E2. The incorporation of the GFPgp64, E1gp64, and E2gp64 fusion proteins into the baculovirus particle was demonstrated by western blot analysis of purified budded virus. This is the first report of the display of the GFP protein or the individual rubella virus spike proteins on the surface of an enveloped virus. Such a eucaryotic viral display system may be useful for the display of proteins dependent on glycosylation for activity and for targeting of recombinant baculoviruses to novel host cell types as a gene transfer vehicle.

Amino Acid Sequence↗
PubMed1993

Production and characterization of an Mls-1-specific monoclonal antibody.

Superantigens (SAGs) represent a new class of antigens, characterized as T cell receptor (TCR) V beta-reactive elements. Bacterial toxins constitute the major group of exogenous SAGs, while the mouse mammary tumor virus (MMTV)-encoded Mls molecules represent the endogenous SAGs. Mls-1 is the prototype of the latter SAGs, because it elicits a very potent T cell stimulatory response in vitro in unprimed T cells expressing the TCR V beta 6 or 8.1 chains. In vivo, Mls-1 causes deletion of immature T cells bearing the V beta 6, 7, 8.1, or 9 chains. Although Mls-1 was functionally discovered > 20 yr ago, it has not been possible to raise antibodies against this molecule. We have previously cloned and sequenced the Mtv-7 sag gene, which encodes Mls-1. Sequence comparisons with other MMTV sag genes suggested that the polymorphic 3' end encodes the TCR V beta specificity of these SAGs. We have, therefore, immunized hamsters with a 14-amino acid peptide from the deduced COOH-terminal sequence of the Mtv-7 sag gene. We describe here the production of a monoclonal antibody (mAb), 3B12, which is peptide specific and reacts with a recombinant baculovirus product of Mtv-7 sag. This mAb blocks Mls-1-specific T cell recognition and detects the Mls-1 protein on the surface of the B cell hybridoma LBB.A, but not on LBB.11, which is an Mtv-7 loss variant of LBB.A. Transfection of the Mtv-7 sag gene into LBB.11 renders this cell functionally Mls-1+ as well as positive for 3B12 binding, confirming the specificity of this mAb. It is well documented that B cells and CD8+ T cells express T cell stimulatory Mls-1 determinants, and we show here that this functional profile correlates with the expression of MMTV-specific mRNA. However, primary lymphocytes derived from Mls-1+ mice do not stain with 3B12, even after in vitro activation with mitogens or phorbol ester.

Amino Acid Sequence↗