The goal of this research is to understand the functional relationships of the secretory and systemic immune systems in fish. It is now clear that fish, like higher vertebrates, have distinct H and L chain isotypes. Secondly, fish have a secretory immune system which can function independently of the systemic system hence may provide the first line of immune defense. Also, information on H chain cDNA structure has opened new horizons for understanding the phylogenetic basis of Ig gene diversity.
The specific aims are four-fold. 1). Quantitative gene titration experiments will be used to confirm initial Southern blots which show that catfish do not exhibit extensive C region gene heterogeneity. Genomic libraries will then be screened with H chain cDNA probes and CH genes sequenced. Additional approaches to screen cDNA libraries will determine if different CH genes are represented. 2). The structure of V region genes will be characterized. Strong sequence similarities of FR4 regions in H chain cDNA with mammalian JH genes indicates that FR4 cDNA probes can be used to isolate JH clones from genomic libraries. Using approaches to link CH to JH genes, overlapping clones will be identified. Sequence information coupled with genomic Southern blot analysis should define the JH locus. As detailed within, fish are likely the first vertebrates to have evolved different VH gene families. Because additional VH families are represented in defined cDNA clones, these clones will be sequenced. The cDNA library will then be rescreened to define other VH families. Genomic clones representing each VH family will be sequenced. This analysis will define the recombination signal sequences and allow important comparisons between different VH gene families. 3). cDNA clones encoding the different classes of L chains will be defined in cDNA expression libraries. Sequence analysis of cDNA clones should provide formal proof that divergence of L chain genes occurred in fish and allow initial investigations into their genomic organization. 4). Enteric vaccination methods to elicit secretory immunity will be explored. The Ab populations in the serum, bile, and cutaneous mucus will be analyzed with mAbs to H and L isotypes and compared with samples from systemically immunized fish. These studies will provide basic information on the functional relationships of the secretory and systemic immune systems in fish, provide a phylogenetic basis for understanding Ig gene diversity, and provide economically important information to the aquaculture industry.
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