This study represents a multidisciplinary experimental approach to obtaining genetic and protein structural information on shark complement proteins, specifically C3, C4 and putative """"""""factor B"""""""", utilizing a combination of molecular biological, immunological and functional methods.
The specific aim of the study is (1) gene cloning, and (2) protein isolation and purification. Gene cloning will involve (a) cross hybridization with heterologous (human and other animal) cDNA probes by genomic Southern hybridization, Northern blotting, and colony and/or plaque hybridization; and (b) reverse transcriptase-polymerase chain reaction (RT-PCR). For RT-PCR cDNA template will be synthesized from mRNA prepared from liver or, alternatively, nucleated peripheral blood cells (a more accessible source). PCR amplified sequences will be digested, gel purified and cloned by ligation into a vector (a variety of commercial kits are available). Transformants will be selected using the appropriate selective medium. Following miniplasmid preparations, successful cloning will be validated by nucleotide sequence analysis and the inserts will serve as homologous probes to later screen appropriate libraries for cDNA or genomic clones. Supplemental to the molecular biological approach, isolation and purification of the complement proteins selected for the study, will be undertaken in order to obtain additional information on protein structure, and to study function and component interaction. Chromatographic separation of the proteins from shark serum will employ ion exchange and size exclusion chromatography of serum pseudoglobulin fraction obtained by low ionic strength precipitation of serum. Protein purification will be monitored by 14/C-methylamine incorporation, functional assays and immunoscreening. Sufficient quantities of pure protein will permit further amino acid sequence analysis, and the data will facilitate the design of appropriate synthetic oligonucleotide primers for cloning experiments. With purified protein available, specific antisera will be raised in rabbits and used for the development of specific immunoassays, for preparation of affinity column for subsequent protein purification, and later for screening expression libraries.
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