The long-terms goal of this work is to understand the novel phenomenon of immunoglobulin transport in the midgut of disease-vector ticks. To achieve this goal we seek to determine immunoglobulin uptake pathways in the midgut cells, to identify participating molecules in the transport mechanism, and to investigate the specificity and location of the molecule interactions with immunoglobulins in the cells.
Our first aim i s to identify and characterize midgut epithelium cell proteins that specifically interact IgG. Studies for this aim are the following: (a) Identification of candidate proteins involved in IgG transport across tick epithelium cells; (b) Cloning, analysis, and recombinant expression of the genes encoding the proteins; (c) Production of monospecific Fab fragments that will be used as reagents for specific aim II. Identification of the IBP proteins will employ different but complementary strategies. These include the following: (i) binding of labeled IgG and Fc fragments to midgut components separated by one- and two-dimensional electrophoresis blotted to membranes, (ii) affinity chromatography using columns with IgG and Fc fragments as ligands, (iii) protein nearest-neighbor detection using cross-linking reagents, and (iv) screening of cDNA expression libraries produced from RNA from unfed and partially fed ticks.
Our second aim i s to localize IBP in the tick midgut. The general approach is to determine the distribution of IBP in the midgut cells that support IgG transport. First we will use the monospecific Fab antibodies to recombinant IBP to detect these proteins on Western blots of different midgut fractions, such as membranes and cytoplasmic contents, and to assess by Western blots or other immunoassay the expression of the proteins before, during, and after feeding by the ticks on rabbits. The location and temporal expression of the IBPs will be further characterized by tissue immunofluorescence.
