Date of Award

2008

Degree Type

Thesis

Department

Biological, Geological and Environmental Sciences

First Advisor

Sam-Yellowe, Tobili

Subject Headings

Plasmodium yoelii, Protein binding, Radioligand assay

Abstract

The Plasmodium Rhop-3 rhoptry protein is an erythrocyte binding protein that is secreted into the RBC membrane during merozoite invasion. Anti-Rhop-3 antibodies inhibit merozoite RBC invasion. The C-terminus of the Rhop-3 protein is highly conserved among Plasmodium species and antisera from endemic areas reacts with recombinant C-terminus of Rhop-3. The binding domain of the Rhop-3 protein is hypothesized to be within the C-terminal region of the protein. In the present study I investigated the conditions necessary for binding of the Rhop-3 protein to RBC by expressing recombinant proteins made from partial fragments of the Rhop-3 gene using the vector pDisplayTM. Recombinants were constructed, purified and used to transfect mammalian COS-7 cells. Surface expression of the proteins was detected by immunofluorescence assay (IFA) using Rhop-3 specific antibodies. A rosetting assay was used to determine whether uninfected mouse red blood cells would bind to COS-7 cells expressing Rhop-3 recombinant proteins on the surface. The pDisplayTM vector was used to express three Plasmodium falciparum Rhop-3 recombinants pDIS-PF17, pDIS-PF13, pDIS-PF7 and one P. yoelii Rhop-3 recombinant pDIS-PY1412 in COS-7 cells. Surface expression of recombinant Rhop-3 on COS-7 cells was identified using three mouse antibodies (MAb) F1, MAb FL1+FL2, MAb T1 and rabbit antibody # 686. Expression of the recombinant Rhop-3 recombinant pDIS-PY1412 remained consistent. Binding of the recombinant Rhop-3 recombinant pDIS-PY1412 to mouse RBC was obtained once but this binding was not consistent. The conditions used for the transfection and binding assays were modified to see if consistent binding with pDIS-PY1412 could be maintained. This is the first time the pDisplayTM vector has been used to study Plasmodium yoelii erythrocyte binding proteins. Expression of the PvDBPII control remained consistent and binding to human duffy positive RBC was also consistent. Optimizing the conditions for binding of pDIS-PY1412 to mouse RBC would be an essentia

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