SEARCH

SEARCH BY CITATION

Keywords:

  • allergen;
  • Blattella germanica;
  • tropomyosin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Background: Cockroach infestation may sensitize and elicit allergic responses to genetically predisposed individuals. Invertebrate tropomyosins are a frequent cause of allergy and highly cross-reactive in nature. In this study, we aimed to produce recombinant German cockroach tropomyosin and investigate its allergenicity.

Methods: German cockroach tropomyosin (Bla g 7) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR). The cloned cDNA was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-nitrilotriacetic (NTA) acid resin. The allergenicity of the recombinant tropomyosin was examined by enzyme-linked immunosorbent assay (ELISA).

Results: The cloned Bla g 7 shared up to 91% amino acid sequence identity with other cockroach tropomyosins. ELISA showed a recombinant Bla g 7 sensitization rate of 16.2% to German cockroach allergic sera. Recombinant tropomyosin was able to inhibit 32.4% of the specific IgE binding to cockroach extract.

Conclusions: Tropomyosin represents a minor allergen in cockroach extracts. It is hoped that recombinant tropomyosin will be useful for further studies and clinical applications.

Cockroaches are known to produce several proteins that bind to IgE, and which are an important cause of asthma (1). Exposure to low levels of cockroach allergens is known to be associated with wheezing among infants in the first 3 months of life (2, 3). The principal domiciliary cockroaches in Korea are Blattella germanica and Periplaneta americana (4), and cross-reactivity between these and other animals, possibly caused by tropomyosin has been reported (5, 6). In particular, IgE antibodies to tropomyosin, which possibly induce food allergies during mite immunotherapy have been described (7). Several lines of evidence have described cockroach antigens, but no direct experiment was performed in a study about German cockroach tropomyosin as a highly cross-reactive antigen. To date, only two cDNA clones of cockroach tropomyosin were identified from American cockroach (8, 9).

Tropomyosin, a kind of muscle protein, is a highly cross-reactive allergen, which sensitizes predisposed individuals via food or house dust. It has been suggested that tropomyosin is an invertebrate pan-allergen due to its high cross-reactivity (10). More than 80% of patients allergic to house dust mites are reported to be sensitized to tropomyosin (11), and that is highly cross-reactive among invertebrate species, which is probably due to their conserved structure.

Molecular cloning and the characterization of the tropomyosins of domestic insects are inevitably required to investigate their allergenicity and how they sensitize individuals. This study was designed to clone German cockroach tropomyosin and to characterize its allergenic properties.

Subjects and serum samples

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Human allergic sera were obtained from patients attending the Allergy Clinic of the Severance Hospital, Yonsei University, Seoul, Korea. The diagnosis of allergy was based on case history and skin prick testing, and followed the revised nomenclature (12). Sera from patients were tested for the presence of IgE antibodies against Dermatophagoides farinae and B. germanica using the Uni-CAP system (Pharmacia, Uppsala, Sweden). Those with CAP results higher than 0.7 kU/l were used for the following study (n = 37, ages ranging from 9 to 75 years, average age 33 years).

Preparation of extract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Thirty grams of live or frozen cockroaches were pulverized in liquid nitrogen. The defatted sample in 200 ml of a 1 : 1 volume of ethyl ether : ethyl acetate was extracted with slow overhead stirring at 4°C overnight in phosphate buffered saline (PBS), pH 7.4 containing 6 mM of 2-mercaptoethanol, 1/1000 volume of protease inhibitor set III (Calbiochem, San Diego, CA) and 1 mg/ml of 1-phenyl-3-(2-thiazolyl)-2-thiourea (Sigma, St Louis, MO) to prevent melanization (13). The extract was then centrifuged at 10000 g for 30 min at 4°C and the supernatant was finally filtered through a 0.22 μm filter (Millipore, Bedford, MA).

Molecular cloning of tropomyosin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Total RNA was prepared from female German cockroaches with TRIzol reagent (GibcoBRL, Rockville, MD) according to the manufacturer's instructions. First strand cDNA was synthesized from 5 μg of total RNA, and PCR was performed using a pair of degenerate primers designed on the basis of known allergenic tropomyosins: TrpmF1 (forward primer): GGABGCBATCAAGAADAARAYT, (where B stands for C, G, or T; R for A or G; D for A, T, or G and Y denotes C or T), TrpmR1 (reverse primer): CAACCATDGCYAAYTTACGT. After 5 min of initial denaturation at 95°C, 35 cycles of PCR (each of 1 min at 94°C, 1 min at 55°C, and 2 min at 72°C) were performed and this was followed by 8 min at 72°C for a final extension. The PCR amplified DNA fragment was gel-purified and subcloned into pGEM-T Easy vector (Promega, Madison, WI) before sequence determination. Nested PCR was performed with two specific primers corresponding to the subcloned cDNA sequence (TrpmF2: GGCCTGGCAGATGAAGAGCG, TrpmF3: CGCACGTAAATTAGCAATGG).

DNA sequence analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Nucleotide sequences were determined using a ThermoSequenase kit (Amersham Life Science, Cleveland, OH) and run on a Long ReadIR 4200 DNA sequencer (LI-COR, Inc., Lincoln, NE). Sequence alignments were performed using the CLUSTAL X program (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX) (14).

Expression of tropomyosin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

The open reading frame of tropomyosin was obtained by RT-PCR using two primers (TrpmF4: ATGGATGCCATCAAGAAGAAG, TrpmR2: GTTTAGTTGCCAATAAGTTCGG) using the same conditions, cloned into pGEM-T-easy vector, and subsequently transferred into the EcoR I site of pET-28b vector (Novagen, Madison, WI). The orientation of the insert was confirmed by PCR using T7 primer annealing to the vector and TrpmR2 primer annealing to the tropomyosin. The resultant sequence had additional 37 amino acids (MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRDPNWI) at the N-terminus.

The recombinant tropomyosin was over-expressed in E. coli BL21 (DE3). These were grown at 37°C in Luria Bertani broth to 0.5 OD600, to induce the expression of recombinant protein. Isopropyl-1-thio-β-galactopyranoside (1 mM) was then added to the bacteria culture. The culture was harvested 4 h after induction. The cellular pellet was resuspended in lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH2PO4, pH 8.0), and lysed using a French presser. Recombinant tropomyosin was purified by using Ni NTA-agarose (Qiagen, Valencia, CA) according to the manufacturer's instructions under native condition. Purified recombinant tropomyosin was analyzed by 10% polyacrylamide gel containing sodium dodecyl sulfate (15).

Specific IgE-binding to recombinant tropomyosin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

The prevalence of specific IgE antibodies against tropomyosin was determined by ELISA. Briefly, each well of the microtiter plates was coated with 200 ng of recombinant tropomyosin. Alkaline phosphatase-conjugated mouse monoclonal anti-human IgE (Sigma) was diluted 1 : 1000 in PBS–0.05% Tween 20 containing 1% bovine serum albumin (BSA) (Sigma). For color development, 100 μl of para-nitrophenyl phosphate (mg/ml) (Sigma) was added and the absorbance was measured with an automatic microplate reader (TECAN, Salzburg, Austria).

The assays were done in triplicate. The mean absorbance level plus two standard deviations (SD) of the sera from 13 healthy controls was used as the cut-off value.

ELISA inhibition test

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Polystyrene microtiter plates were coated with 100 μl of 20 μg/ml of B. germanica extract 0.05 mM of carbonate buffer (pH 9.6) overnight at 4°C. The 1 : 4 diluted serum was preincubated with various quantities of the crude extract or recombinant protein for 2 h at room temperature and overnight at 4°C. After washing, each well of the microtiter plate was blocked with 200 μl of 1% BSA in PBS for 1 h at room temperature. Fifty microliters of the preincubated serum were then incubated for 1 h at room temperature. Specific IgE was detected using 50 μl of 1 : 1000 diluted biotinylated goat anti-human IgE (Vector, Burlingame, CA) followed by streptavidin-peroxidase (Sigma) diluted 1 : 1000 and incubated for 1 h and 30 min, respectively. The signal was developed by adding 3,3′,5,5′-tetramethyl-benzidine (TMB, KPL, Gaithersburg, MD) (16) at a concentration of 0.4 g/l.

Molecular cloning of tropomyosin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

The cloned sequence encoded 284 amino acids with an estimated molecular mass of 32.836 kDa and an estimated isoelectrical point of 4.57. There were no potential N-linked glycosylation sites (NXT/S) and no proline or tryptophan residues in the amino acid composition. This sequence, Genbank accession number AF260897, was designated as Bla g 7 according to the guidelines of the International Union of Immunological Societies Allergen Nomenclature Subcommittee (17) (Fig. 1). The deduced amino acid sequence of the cloned cDNA showed 79–97% identity with previously reported allergenic tropomyosins (Fig. 2).

image

Figure 1. Nucleotide and deduced amino acid sequences of Bla g 7. This sequence was submitted to Genbank under accession number AF260897.

Download figure to PowerPoint

image

Figure 2. Sequence alignment of Bla g 7 with other tropomyosins: Bla g 7 (Blattella germanica, AF260897), Per a 7 (Periplaneta Americana, AF106961), Der p 10 (Dermatophagoides pteronyssinus, Y14906), Der f 10 (Dermatophagoides farinae, D17682), Lep d 10 (Lepidoglyphus destructor, AT250096), Met e 1 (Metapenaeus ensis, U08008), Hom a 1 fast (Homarus americanus fast muscle, AF034954), Hom a 1 slow (Homarus americanus slow muscle, AF034953), Cha f 1 (Charybdis feriatus, AF061783), Pan s 1 (Panulirus stimpsoni, AF030063). The percentage of each sequence identity is given in parentheses.

Download figure to PowerPoint

Expression and purification of recombinant tropomyosin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

The open cDNA reading frame was subcloned into pET-28b expression vector. The molecular weight of the expressed protein was 42 kDa by SDS-PAGE. The yield of the purified protein was 5.495 mg/l as measured by the Bradford assay (Bio-rad, Hercules, CA) (Fig. 3).

image

Figure 3. Expression and purification of recombinant tropomyosin. Proteins were run on 10% acrylamide gel and stained with Coomassie brilliant blue. Lanes: M, MW markers in kDa; L, total cell lysate of E. coli BL21 cells bearing rBla g 7 after IPTG induction; P, fraction passed through the column; W, fraction washed the column; E, fraction eluted; B, bovine serum albumin.

Download figure to PowerPoint

IgE reactivity of the recombinant tropomyosin

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Specific IgE against recombinant tropomyosin was determined by ELISA. The results showed that six of the 37 sera tested (16.2%) could be considered positive (Fig. 4).

image

Figure 4. IgE reactivity of human sera against recombinant Bla g 7. The horizontal line indicates the cut-off value. bsl00046, Healthy control sera; •, German cockroach sensitized sera.

Download figure to PowerPoint

Inhibition of specific IgE binding to crude extract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

The crude extract of German cockroach reached a maximum of 92.2% at an inhibitor concentration of 10 μg/ml and recombinant tropomyosin inhibited IgE by 32.4% at an inhibitor concentration of 1 μg/ml (Fig. 5).

image

Figure 5. ELISA inhibition of B. germanica specific IgE with whole body extract and recombinant tropomyosin.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References

Tropomyosin is suggested to be a cross-reacting allergen between foods and inhalant allergens originating from animals. Sensitization to a particular food allergen may ultimately lead to sensitization to certain inhalant allergens (18) and vice versa (7, 19).

Various IgE reactivities to tropomyosin has been reported. There could be three possible reasons for various IgE reactivities to invertebrate tropomyosins. First, IgE reactivity could be affected by the structural differences between native tropomyosin and its recombinant counterpart. The studies using purified native tropomyosin found that the frequencies of IgE reactivity to D. farinae and P. americana were 80.6% (25/31) (11) and 41.4% (12/29) (8), respectively. In a previous study of recombinant Der f 10, it is reported that the IgE-binding response of recombinant fusion protein was at least 25 times weaker than that of the purified native tropomyosin (11), and that recombinant Der p 10 reacted with only 5.6% (4/71) of house dust mite allergic sera even though it contains nonfusion protein (20). Recombinant Blomia tropicalis tropomyosin showed a IgE reactivity of 29% (27/93) (21). In our study, 16.2% (6/37) of the allergic sera showed positive IgE reactivity to recombinant Bla g 7. Posttranslational modifications may reflect the reduced allergenicity of bacteria-expressed tropomyosin. The N-terminus of vertebrate tropomyosin is known to be acetylated and that this influences its biological activity (22, 23). Moreover, phosphorylation is thought to influence head to tail interaction and substantially increases viscosity at low ionic strength (24). Recombinant Bla g 7 has additional 37 amino acids at the N-terminus instead of acetylation. Second, cross-reactivity with other invertebrates in polysensitized populations, and genetic variants affecting the specific sensitizations could contribute to the different allergenicities. Lastly, the little amino acid change in the epitope region of tropomyosin could be the other explanation, because isoforms in different tissues and at different developmental stages have been observed in another insect, Locusta migratoria (25) and these isoforms could have different allergenicities.

In summary, we isolated and sequenced the cDNA encoding tropomyosin to elucidate its role in cockroach allergy. Tropomyosin showed 79–97% amino acid sequence identity to the other invertebrate tropomyosins. Six (19.4%) of the 31 subjects’ sera showed positive IgE reactivity to recombinant Bla g 7, which is not thought to be a major allergen of the German cockroach, but the importance of invertebrate tropomyosin should not be underestimated because of its strong cross-reactivity. However, much remains to be clarified about the cross-reactivity of invertebrate tropomyosins. Attempts to produce the recombinant protein in the eukaryotic system and to purify native tropomyosin are in progress to further understand their allergenicities.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Subjects and serum samples
  5. Preparation of extract
  6. Molecular cloning of tropomyosin
  7. DNA sequence analysis
  8. Expression of tropomyosin
  9. Specific IgE-binding to recombinant tropomyosin
  10. ELISA inhibition test
  11. Results
  12. Molecular cloning of tropomyosin
  13. Expression and purification of recombinant tropomyosin
  14. IgE reactivity of the recombinant tropomyosin
  15. Inhibition of specific IgE binding to crude extract
  16. Discussion
  17. Acknowledgments
  18. References
  • 1
    Arruda LK, Chapman MD. The role of cockroach allergens in asthma. Curr Opin Pulm Med 2001;7: 1419.
  • 2
    Finn PW, Boudreau JO, He H, Wang Y, Chapman MD, Vincet C, Burge HA, Weiss ST, Perkins DL, Gold DR. Children at risk for asthma: home allergen levels, lymphocyte proliferation, and wheeze. J Allergy Clin Immunol 2000;105: 933942.
  • 3
    Huss K, Adikinson NF, Eggleston PA, Dawson C, Van Natta ML, Hamilton RG. House dust mite and cockroach exposure are strong risk factors for positive allergy skin test responses in the childhood asthma management program. J Allergy Clin Immunol 2001;107: 4854.
  • 4
    Jeong KY, Lee IY, Lee J, Ree HI, Hong CS, Yong TS. Effectiveness of health education for the control of dust mites and cockroaches in Seoul, Korea. Med Vet Entomol 2003, in press.
  • 5
    Crespo JF, Pascual C, Helm R, Sanchez-Pastor S, Ojeda I, Romualdo L, Martin-Esteban M, Ojeda JA. Cross-reactivity of IgE-binding components between boiled Atlantic shrimp and German cockroach. Allergy 1995;50: 918924.
  • 6
    Pascual CY, Crespo JF, San Martin S, Ornia N, Ortega N, Caballero T, Munoz-Pereira M, Martin-Estaban M. Cross-reactivity between IgE-binding proteins from Anisakis, German cockroach, and chironomids. Allergy 1997;52: 514520.
  • 7
    van Ree R, Antonicelli L, Akkerdaas JH, Garrtani MS, Aalberse RC, Bonifazi F. Possible induction of food allergy during mite immunotherapy. Allergy 1996;51: 108113.
  • 8
    Asturias JA, Gomez-Bayon N, Arilla MC, Martinez A, Palacios R, Sanchez-Gascon F, Martinez J. Molecular characterization of American cockroach tropomyosin (Periplaneta americana Allergen 7), a cross-reactive allergen. J Immunol 1999;162: 43424348.
  • 9
    Santos ABR, Chapman MD, Aalberse RC, Vailes LD, Ferriani VPL, Oliver C, Naspitz MC, Naspitz CK, Arruda LK. Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross reactivity with mite and shrimp allergens. J Allergy Clin Immunol 1999;104: 329337.
  • 10
    Reese G, Ayuso R, Lehrer SB. Tropomyosin: an invertebrate pan-allergen. Int Arch Allergy Immunol 1999;119: 247258.
  • 11
    Aki T, Kodama T, Fujikawa A, Miura K, Shigeta S, Wada T, Jyo T, Murooka Y, Oka S, Ono K. Immunochemical characterization of recombinant and native tropomyosin as a new allergen from the house dust mite, Dermatophagoides farinae. J Allergy Clin Immunol 1995;96: 7483.
  • 12
    Johansson SGO, Hourihane JO'B, Bousquet J, Bruijnzeel-Koomen C, Dreborg S, Haahtela T, Kowalski ML, Mygind N, Ring J, van Vauwenberge P, van Hage-Hamsten M, Wuthrich B. A revised nonmenclature for allergy. An EAACI position statement from EAACI nomenclature task force. Allergy 2001;56: 813824.
  • 13
    Johnson GA, Boukma SJ, Kim EG. In vivo inhibition of dopamine β-hydroxylase by 1-phenyl-3-(2-thiozolyl)-2-thiourea. J Pharmacol Exp Ther 1970;171: 8087.
  • 14
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL-X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;25: 48764882.
  • 15
    Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227: 680685.
  • 16
    Adams JC. Stabilizing and rapid thionin staining of TMB-based HRP reaction product. Neurosci Lett 1980;17: 79.
  • 17
    King TP, Hoffman D, Lowenstein H, Marsh DG, Platts-Mills TAE, Thomas W. Allergen nomenclature. Int Arch Allergy Immunol 1994;105: 224233.
  • 18
    Witteman AM, Akkerdaas JH, van Leeuen J, van der Zee JS, Aslberse RC. Identification of a cross-reactive allergen (presumably tropomyosin) in shrimp, mite and insects. Int Arch Allergy Immunol 1994;105: 5661.
  • 19
    De Maat-Bleeker F, Akkerdaas JH, van Ree R, Aslberse RC. Vineyard snail allergy possibly induced by sensitization to house dust mite (Dermatophagoides pteronyssinus). Allergy 1995;50: 428430.
  • 20
    Asturias JA, Arilla MC, Gomez-Bayon N, Martinez A, Martinez J, Palacios R. Sequencing and high level of expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus. Biochem Biophys Acta 1998;1397: 2730.
  • 21
    Yi FC, Cheong N, Shek PCL, Wang DY, Chua KY, Lee BW. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus. Clin Exp Allergy 2002;32: 12031210.
  • 22
    Cho YJ, Liu J, Hitchcock-DeGregori SE. The amino terminus of muscle tropomyosin is a major determinant for function. J Biol Chem 1990;265: 538545.
  • 23
    Urbancikova M, Hitchcock-DeGregori SE. Requirement of amino-terminal modification for striated muscle α-tropomyosin function. J Biol Chem 1994;269: 2431024315.
  • 24
    Heeley DH, Watson MH, Mak AS, Dubord P, Smillie LB. Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle α-tropomyosin. J Biol Chem 1989;264: 24242430.
  • 25
    Krieger J, Raming K, Knipper M, Grau M, Mertens S, Breer H. Cloning, sequencing and expression of locust tropomyosin. Insect Biochem 1990;20: 173184.