B., J. tropomyosin should be used for the development of diagnostic and restorative reagents. The tropomyosins are a family of closely related proteins with multiple functions, including the rules of the actin-myosin connection, transport of mRNA (8), and mechanical support of the cytoplasmic membrane (19). Tropomyosin Rabbit Polyclonal to CCR5 (phospho-Ser349) has been recognized as probably one of the most important allergens in crustacean foods (7, 20, 27). It is highly conserved, to the degree that tropomyosin may serve as a candidate marker for phylogenetic studies of mollusks by parsimony analysis (4). Allergic reactions to shellfish and mollusks are often cross-reactive, which may be explained by the highly conserved amino acid sequences of tropomyosins, but vertebrate tropomyosin is Aconine not known to be allergenic (2). Comparisons of the immunoglobulin E (IgE) epitope areas among tropomyosins from different molluscs by Ishikawa et al. (11) showed the presence of polymorphic sites, indicating that the oyster epitope is definitely species specific (18). The presence of unique as well as shared epitopes in Blo t 10 and Der p 10 Aconine have also been described (34). At least 18 different isoforms are known to be generated by option RNA splicing in mammalian cells. The synthesis of isoforms is definitely developmentally regulated, and cells from different embryonic lineages communicate different isoforms (26). The alternate exon splicing patterns of were reported to involve 27 Aconine amino acids in the C terminus (3), which regularly contain IgE-binding areas (24). Specifically, eight different IgE-binding epitopes were identified in the American cockroach tropomyosin (Per a 7) by using a set of overlapping synthetic peptides (1). The amino acid sequence diversity of individual allergens has been described in wild or cultured house dust mites (5, 29, 30, 32, 35) or storage mites (16). Small changes in the amino acid sequences of given allergens can influence their allergenicities (10). For example, certain natural isoforms of Bet v 1, the major birch pollen allergen, were found to have high T-cell reactivities and low or no IgE-binding activities (21). Analysis of these isoforms may lead us to a better understanding of the different allergenicities of many invertebrate tropomyosins and the development of immunotherapeutic strategies and products, such as hypoallergenic (low IgE-binding activity) products. We have previously isolated the cDNA encoding German cockroach tropomyosin (15) and named it Bla g 7, according to the guidelines of the International Union of Immunological Aconine Societies Allergen Nomenclature Subcommittee (17). Recombinant tropomyosin expressed in showed low levels of IgE-binding reactivity. Recombinant tropomyosin was also expressed as a nonfusion protein Aconine in BL21(DE3) and purified by Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen, Valencia, Calif.), according to the instructions of the manufacturer (15), in 100 l of phosphate-buffered saline emulsified with an equal volume of alum adjuvant. Booster injections were given twice at 3-week intervals. The production of specific antibodies was monitored by enzyme-linked immunosorbent assay (ELISA), and the mice were killed 3 days after the second booster injection. The polyclonal antitropomyosin antiserum (1:1,000) was used to probe and identify the German cockroach tropomyosin. Two-dimensional gel electrophoresis and immunoblotting. Two-dimensional gel electrophoresis was performed with precast gels (Invitrogen, Carsbad, Calif.), according to the instructions of the manufacturer. Cockroach extract was prepared as described previously (15). Fifty micrograms of whole-body extracts mixed with an equal volume of the sample buffer was loaded into the first-dimension gel. After isoelectric focusing (pH 3 to 10), second-dimension gel electrophoresis was carried out in a 4 to 20% gradient polyacrylamide gel made up of sodium dodecyl sulfate. The proteins were then electrophoretically transferred onto a nitrocellulose membrane (pore size, 0.45 m; Osmonics, Westborough, Mass.). After the membrane was blocked overnight with 3% skim milk, it was incubated for 1 h with mouse anti-recombinant Bla g 7 serum. The blots were then incubated with goat anti-mouse IgG conjugated with alkaline phosphatase (Sigma, St. Louis, Mo.) for 1 h at room temperature and developed in a substrate solution of Nitro Blue Tetrazolium and 3-bromo-4-chloro-5-indolyl-phosphate (Promega, Madison, Wis.). RT-PCR. cDNA encoding tropomyosin was amplified by using high-fidelity DNA polymerase (Stratagene, La Jolla, Calif.). A total of.