Tropomyosin gene was cloned out using heterologous primers based on existing bivalvia and actinopterygii tropomyosin sequences obtained from Genbank, after which subcloning into E. coli vector through use of pGEM­T was carried out, followed by DNA sequencing and alignment creation. Cloning into pETm vector for protein expression was then done, and the recombinant proteins purified using affinity chromatography. Dot blotting was carried out with 45 sera samples, and the results subjected to correlation analyses. Results DNA and amino acid sequences A total of eight new tropomyosin DNA sequences from Anadara granosa, Meretrix meretrix, Meretrix lyrata, Pholas orientalis, Glaucomone rugosa, Paphia undulata, Mugil cephalus and Oreochromis hybrid were obtained, all of which were 855 base pairs long. Amino acid sequences were deduced using the Translate tool on the ExPASy Proteomics Server, which gave 284 amino acids in each sequence. DNA sequence identity expectedly decreases as phylogenic distance increases. Protein expression and purification Based on newly obtained sequences, four different recombinant proteins were obtained from Andara granosa, Meretrix meretrix, Meretrix lyrata and Mugil cephalus. The recombinant proteins from two in­house tropomyosin clones Argopecten irradians and Perna viridis were also expressed and purified using affinity chromatography. All proteins were soluble and had similar pI and mW as deduced by ProtParam on ExPASy. Dot blotting Dot blotting was carried out using the six recombinant tropomyosin proteins of which five were subjected to analyses, as well as two cockroach (Periplaneta americana, Blattella germanica) and eight dust mite (Blomia tropicalis, Aleuroglyphus ovatus, Dermatophagoides pteronyssinus, Tyrophagus putrescentiae, Suidaisa medanensis, Glycyphagus domesticus, Acarus siro, Leptidoglyphus destructor) tropomyosin proteins available from previous studies, with 45 atopic paediatric patient sera samples. Strong correlation was observed within and between cockroach and dust mite tropomyosin reactivity, while correlation was not as strong within the bivalves, nor between cockroach/dust mite tropomyosin reactivity. Discussion DNA and amino acid sequences There was no 100% identity and similarity observed between any currently available DNA and protein sequences, although protein identities within Class Bivalvia were between 66.1­98.9%. Highly conserved regions in protein sequences were noted in positions 140­199 and 213­284, which may be areas of concern in future epitope mapping studies. Cross­reactivity and epitope mapping
Correlation studies suggests that atopic individuals reacting to dust mite/cockroach tropomyosins are not likely to react as strongly to the bivalve topomyosins in this study, and vice versa, although they are likely to react to another tropomyosin from the same class of organisms if they react to a particular invertebrate tropomyosin. However, at this stage, it is not possible to state with certainty that differences in reactivity is due to any particular region of the invertebrate tropomyosin gene and/or its structure, or if cross­reactivity is present for any two isoforms. Inhibition assays should be carried out following this study to determine the cross­reactivity potential of the new recombinant tropomyosin, as well epitope mapping and positional differences causing different reactions, if possible. References Burger, J., J. Fleischer, et al. (2003). "Fish, shellfish, and meat meals of the public in Singapore." Environmental Research 92: 254­261. Gell, P. G. H. and R. R. A. Coombs (1963). The classification of allergic reactions underlying disease, Blackwell Science. Marti, P., R. Truffer, et al. (2007). "Allergen motifs and the prediction of allergenicity." Immunology Letters 109: 47­55. Mikita, C. P. and E. A. Padlan (2007). "Why is there a greater incidence of allergy to the tropomyosin of certain animals than to that of others?" Medical Hypotheses 69: 1070­1073. Motoyama, K., Y. Suma, et al. (2007). "Molecular Cloning of Tropomyosins Identified as Allergens in Six Species of Crustaceans." Journal of Agricultural and Food Chemistry 55: 985­991. Nieuwenhuizen, N., A. L. Lopata, et al. (2006). "Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis." Journal of Allergy and Clinical Immunology 117(5): 1098­1105. Reese, G., R. Ayuso, et al. (1999). "Tropomyosin: an invertebrate pan­allergen." International Archives of Allergy and Immunology 119(4): 247­258. Thong, H., Y. K. Cheng, et al. (2005). "Anaphylaxis in adults referred to a clinical immunology/allergy centre in Singapore." Singapore Medical Journal 46(10): 529­534.