재조합 Der f 4를 포함한 여러 재조합 진드기 알레르겐의 면역글로불린E 반응성과 전염증성 싸이토카인 유발능
IgE-reactivity and proinflammatory cytokine -inducibility of various recombinant house dust mite allergens including novel rDer f 4
- 주제(키워드) 집먼지진드기 , 저장진드기 , 알레르기 , 면역글로불린E 반응성 , 교차반응성 , 성분 항원 기반 진단 , 재조합 알레르겐 , 큰다리먼지진드기 , 긴털가루진드기
- 발행기관 아주대학교
- 지도교수 신호준
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021552
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
House dust mite (HDM) is the most important inducer of allergy and asthma in the world. More than thirty three group of mite allergens have been reported from HDMs and storage mites (SMs). The accurate diagnosis of mite allergy is a very important for effective treatment. For diagnosis and immunotherapy of mite allergy, the crude extract of mites have been used, which may cause side effects. Recently, specific allergens selectively developed using molecular biological technology came into the spotlight. This study intended to examine diagnostic and immunotherapeutic potentials of the recombinant allergens from house dust mite Dermatophagoides farinae. Thirteen D. farinae-derived recombinant allergens were produced using Escherichia coli or Pichia pastoris expression system. Native Der f 4 was also purified for comparison. rDer f 1 (89.2%) and rDer f 2 (96.4%) showed high reactivity to IgE in Korean HDM-sensitized patient’s sera. rDer f 3 (61.9%), nDer f 4 (75.8%), rDer f 5 (64.9%), rDer f 7 (43.8%), rDer f 21 (66.0%), and rDer f 23 (43.3%) exhibited moderate IgE reactivity them. IgE reactivity of rDer f 10 (24.7%), rDer f 13 (3.6%), rDer f 15 (4.1%), rDer f 18 (32.0%) and rDer f 20 (38.7%) displayed low IgE reactivity. IgE reactivities against Der f 2 were shown in all patients with asthma, rhinitis or atopic dermatitis. Each serum recognized with 2 to 12 D. farinae proteins, and 190 of 194 (97.9%) recognized with three or more proteins. A cross-reactivity of recombinant group 5 and 21 allergens from three mite species (D. farinae, Tyrophagus putrescentiae, and Blomia tropicalis) was evaluated. Cross-reactivity between rDer f 23 and rDer p 23 were also examined by using inhibition-ELISA. IgE reactivity to rDer f 5 was highly correlated with dose of rDer f 21 (r = 0.733). rTyr p 5 exhibited the highest level of correlation with rTyr p 21 (r = 0.950). rDer f 21 exhibited a high inhibitory effect against rTyr p 5 (93.0%), rTyr p 21 (92.1%), rBlo t 5 (87.0%), and rBlo t 21 (70.3%), implying cross-reactivity across mite species. rDer p 23 effectively inhibited a IgE binding to rDer f 23. Not only ability of IgE responses but also capability to induce inflammatory cytokine production is important properties of allergens. In this study, IL-6 and IL-8 production in BEAS-2B cells (bronchial epithelial cell-line) were examined by challenging of the recombinant mite allergens. rDer f 4, rDer f 5, rDer f 10 and rDer f 21 induced IL-6 or IL-8 secretions in BEAS-2B. In particular, rDer f 21 could trigger IL-6 and IL-8 production in BEAS-2B through TLR4-dependent signaling. In summary, Der f 1 and Der f 2 was shown to be the most potent allergens. Der f 3, Der f 4, Der 5, Der f 7, Der f 21 and Der f 23 were found to be the major allergens sensitized more than 50% of HDM allergy patients. Der f 21 showed cross-reactivity to T. putrescentiae and B. tropicalis group 5 and 21 allergens. In addition, Der f 21 was found to trigger of IL-6 and IL-8 production in a bronchial epithelial cell line through a TLR4. The information on allergenicity and IgE reactivities of various mite allergens shown in this study will be useful for development of a component-resolved diagnosis and effective treatment to HDM allergy.
more목차
I. INTRODUCTION 1
1. Overview of house dust mite allergens 1
2. Component-resolved diagnosis 2
3. Cross-reactivity of mite allergens 3
4. Recombinant mite allergens in diagnosis and treatment 4
5. Immune response on airway epithelial cell by mite allergens 6
6. Aims of this study 7
II. MATERIALS AND METHODS 11
1. Subjects and serum samples 11
2. Preparation of native Der f 4 and other recombinant mite allergens 11
1) Purification of native Der f 4 (nDer f 4) 11
2) Cloning of Der f 4 full cDNA sequence 13
3) Expression of recombinant allergens in E. coli using the pET expression vector 14
4) Expression of recombinant allergens in P. pastoris using the pPIC9 expression vector 15
5) Removal of endotoxin from the recombinant allergens (proteins) 17
3. IgE reactivity of recombinant mite allergens 17
4. Inhibition ELISA and 3D protein structure modeling 18
5. Stimulation of BEAS-2B cells with recombinant mite allergens 19
1) Cell culture 19
2) Determination of cytokine production 19
III. RESULTS 23
1. IgE reactivity profile of Korean mite-sensitized allergic patients to recombinant D. farinae allergens 23
1) IgE reactivity to D. farinae, T. putrescentiae and B. tropicalis extracts 23
2) Preparation of native and recombinant Der f 4 26
3) IgE reactivities and correlations of thirteen D. farinae allergens 38
2. Evaluation of cross-reactivity among mite allergens 50
1) Cross-reactivities of six recombinant group 5 and 21 mite allergens 50
2) Cross-reactivity between rDer f 23 and rDer p 23 63
3. Production of proinflammatory cytokines, IL-6 and IL-8, in BEAS-2B by stimulation of recombinant allergens derived from HDM, D. farinae. 68
IV. DISCUSSION 76
V. CONCLUSION 82
REFERENCE 83
국문요약 94
