An alternative pathway of complement activation (ACP) has not been described for the nurse shark, though the classical pathway of complement activation has been observed and an activation cascade involving 6 functionally distinct components has been described. The goal of this investigation is to study the alternative pathway in the nurse shark; its proteins, their structure, interactions, genetic association and role in immune function. Information on complement proteins of this primitive animal would provide a clearer picture of the evolution of the complement system, specifically the alternative pathway. Shark serum will be examined for hemolytic activity in the presence of chelating buffers which inhibit the classical pathway. The effect of activators of mammalian ACP, i.e., CVF, LPS, insulin and zymosan, on the hemolytic activity of shark serum will be examined. The ability of a shark factor to replace a missing component in human serum will be explored, using factor-depleted serum. By Southern and Northern blot analysis shark genomic DNA and mRNA respectively will be examined using a cDNA human factor B probe, pFB3b, to determine the extent of sequence homology. This method of analysis will also be employed to seek sequence homologies in nurse shark using a cDNA clone of the C3 gene. Proteins, analogous to mammalian ACP proteins will be detected by immunoblotting of shark serum after SDS-PAGE using monospecific polyclonal antisera to mammalian complement components; provided the antisera recognized shared antigenic determinants. On identifying a shark ACP protein, chromatographic procedures will be developed for its rapid isolation and purification. As structural analysis is of fundamental importance in studying the evolutionary origins of complement proteins, primary structure of shark proteins will be determined from aminoacid sequence analysis, which will be achieved through collaboration.
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