Molrcular Immunology, Vol. 33. No. 3, pp. 31 l-319, 1996 Pergamon 0161~5890(95)00127-l Copyright 1’; 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0161-5890~96515.00+0.00 CLONING AND CHARACTERIZATION OF A NEW ALLERGEN, Mag 3, FROM THE HOUSE DUST MITE, DERMA TOPHAGOIDES FARINAE: CROSS-REACTIVITY WITH HIGH-MOLECULAR-WEIGHT ALLERGEN AKIHIRO FUJIKAWA,” NORIYUKI ISHIMARU,* ATSUKO SETO,* HIROYUKI YAMADA,* TSUNEHIRO AKI,* SEIKO SHIGETA,* TAKESHI WADA,? TOSHIHIKO JYO,; YOSHIKATU MUROOKA,” SATORU OKA* and KAZUHISA ONO*$ *Department of Fermentation Technology. Faculty of Engineering, Hiroshima University, Higashi- Hiroshima, 724, Japan; TDevelopment and Administration section, Fumakilla Ltd, Hiroshima 739- 04, Japan; fDepartment of Internal Medicine, Hiroshima Prefectural Hospital, Hiroshima 734, Japan (First received 28 Mu,v 1995; utcepted in rrvisedfhrm 20 August 1995) Abstract-A new immunoreactive clone whose sequence is not homologous to that of any of the previously identified mite allergens was isolated by successive immunoscreening of a Derm- atophugoides,furinue cDNA library with rabbit antisera to an extract of the house dust mite and IgE in pooled sera from patients allergic to mites. Rabbit antibodies specific for the recombinant protein recognized a 177 kD protein in a mite body extract. This immunoreactive protein was located in the circumferential tissues of esophagus, gut and the other internal organs in mites. The reaction of human IgE to the purified natural antigen was inhibited competitively to 30% by the recombinant antigen. In terms of the frequency and the intensity of response to specific IgE in sera from asthmatic patients, the natural protein was similar to Der .f’2, while the recombinant protein was slightly less allergenic by these criteria. We conclude that the natural protein from the house dust mite, D.,fkrinue, is an important allergen. Copyright ‘(; 1996 Elsevier Science Ltd. Kej, nerds: Dermutophugoides firrinae, recombinant allergen, cross-reactivity, high-molecular-weight allergen. INTRODUCTION Since house dust mites (Dermatophagoides sp.) were reported as one of the major agents causing allergic dis- eases such as bronchial asthma, perennial rhinitis and atopic dermatitis (Voohorst et al., 1967), many efforts have been made to identify allergens for humans from two closely related mite species, D. farinae and D. pteron- yssinus (Arlian, 1991; Platts-Mills et al., 1992). A large number of allergenic components has been identified in extracts of mite culture, although the exact numbers of reported components varies from 4 to 26 and from 5 to 19 in mite bodies and feces, respectively (Arlian et al., 1987a, 1987b). Among them, antigens in groups of Der 1 (25 kD), Der 2 (14 kD), Der 3 (28/30 kD), Der 4 (60 kD), Der 5 (14 kD), Drr 6 (25 kD) and Der 7 (22% 28 kD) have been characterized as major or important allergens (King et al., 1994). We have found and identified - §Author to whom correspondence should be addressed. Abhrtwiations: Dfb, mite body extract from Dermatophugoides furinue; GST, glutathione S-transferase; IPTG, isopropyl-fi- D-thio-galactopyranoside; mug, cDNA coding for recom- binant mite antigen; PBS, phosphate-buffered saline. new D.,farinae antigens, such as Mag 1 (39 kD), Mag 29 (68 kD) and Mag 44 (37 kD) as the corresponding allergens (Aki et al., 1994a, 1994b, 1994c, 1995) and additional low-molecular-weight antigens which appear to be asthma-inducible (Y amaguchi et al., 1994a, 1994b). Mag 29 is nearly identical in sequence to heat shock protein 70, while Mag 44 has been identified as tropo- myosin. Recently, another antigen which is highly hom- ologous to glutathione S-transferase was reported to be a major mite allergen (O’Neil et al., 1994). Most of the major or important allergens in mite body and feces extracts are found in a molecular weight range of lo-70 kD. In some cases, larger allergens (> 110 kD, > 158 kD, 174 kD and 175 kD) could be detected by immunoelectrophoresis and/or immunoblotting, but they are not fully characterized (Stewart and Turner, 1980a; Baldo et al., 1989; Wahl et al., 1989; Colloff et al., 1992). We have found that high-molecular-weight antigens iso- lated in our laboratory are mitogenic allergens (Shigeta et al., 1995). However, much work remains to be done in characterizing al1 potentially allergenic antigens. In this paper, we report the molecular cloning of a cDNA encoding a new allergenic component named Mag 3 from a house dust mite D. farinae cDNA library. We 311