Purification, identification, and partial characterization of proteins associated with the adaptive immune response to soluble protein antigen in the American cockroach (Periplaneta americana)

The study of insect immunity has traditionally focused on innate immune responses, under the longstanding assumption that insects are incapable of generating an anticipatory immune response. Research in our lab has focused on a specific, adaptive response in Periplaneta americana, the American cockroach. This response was shown to be specific and exhibit immunological memory in survivorship studies. Protein factors in cell-free immune hemolymph demonstrated the ability to precipitate antigen and confer immunity on naive roaches. One reduced protein (102 kDa) was implicated in the immune response due to its enhancement in immune hemolymph and association with antigen binding. The goals of the research presented in this dissertation were to characterize the 102 kDa protein, identify and characterize other proteins involved in antigen binding, and to illuminate the relationship between reduced antigen binding proteins and native bands capable of antigen binding.; In order to identify the 102 kDa protein, a combination of preparative electrophoresis and MALDI mass spectrometry was employed. A positive match was achieved between the 102 kDa protein and a 40--45 kDa region of vitellogenin from the American cockroach. Affinity chromatography using immunizing antigen was used to determine other proteins involved in antigen binding, including reduced proteins at 115 kDa and 95 kDa. Isolation and MALDI mass spectrometry analysis of these proteins yielded a positive match for the 115 kDa protein with vitellogenin-2 from the American cockroach, but no significant match for the 95 kDa protein. Schiff's staining and enzymatic deglycosylation of the antigen binding proteins, 115, 102, and 95 kDa, indicated that all three were glycosylated. Analysis of native protein bands indicated the presence of the immune associated proteins in native bands 2 and 4 (the native bands capable of binding antigen). The above research strongly indicates a role for a vitellogenin-like protein in the adaptive immune response to soluble protein antigen in the American cockroach, and links the native antigen-binding bands with reduced proteins at 115, 102, and 95 kDa.