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رکورد قبلیرکورد بعدی
نوع مدرک : TF
زبان مدرک : فارسی
شماره رکورد : 66823
شماره مدرک : ‭پ۵۳۳۵۵‬
شماره راهنما : ‭آ۲۷۷‬
سر شناسه : فلک، رضا
عنوان اصلي : شناسایی، تعیین مشخصات مولکولی و تولید پروتئین نوترکیب آلرژن های انگور و بررسی تاثیر سموم کشاورزی بر بروز یا آلرژی زایی آنها
نام عام مواد : [پایان‌نامه]
نام نخستين پديدآور : /رضا فلک
نام ساير پديدآوران : ؛استاد راهنما: عبدالرضا وارسته، مجتبی سنکیان
نام ساير پديدآوران : ؛استاد مشاور: فرحزاد جباری آزاد
عنوان ديگر : عنوان به انگلیسی‭Identification, molecular charavterization and production of recombinant form of grape allergens as well as evaluation of pasticide impact on expression and allergenicity of grape allergenic proteins :‬
وضعيت نشر : دانشگاه علوم پزشکی مشهد، ‭۱۳۹۱‬، دانشکده پزشکی
صفحه شمار : ‮‭[۱۴۰]‬ ص.‬: مصور
يادداشت : خلاصه فارسی
يادداشت : چاپی
خلاصه يا چکيده : ‭PAGE analysis and assessment of protein concentration of the extract revealed that a suitable allergenic extract was prepared from local grape berries containing 250 -time PCR. Results: SDS-PAGE. Moreover, a set of monoclonal antibodies were produced against grape class IV chitinase using hybridoma technology. Finally, the impact of Niconazol was studied on expression level of grape allergens using real-glycosylation structure in plants. The effect of gastric fluid on grape proteins was evaluated using SDS-allergic source of MUXF3; a common N-CCD antibodies was checked using immunoreactivity of patients sera with bromlein as a non-Linked Immunosorbent Assay (ELISA) and immunoblotting procedures, via total extract or fractionated proteins. Grape proteins were separated by anion exchange chromatography and the immunoreactive components were characterized by mass spectrometry. Some of the characterized proteins were produced as recombinant forms using PCR cloning method. For this purpose, total RNA was extracted from grape berries and the specific cDNA of each allergen was amplified and directionally inserted into bacterial plasmid or baculoviral donor system. The recombinant proteins were produced in bacterial hosts or insect cell line and their immunoreactivity were checked using grape allergic patients sera. Finally, the glycosylation state of grape proteins was studied via specific blotting methods. Moreover, the presence of anti-reactivity of grape proteins was determined by Enzyme-allergic controls. The pattern of IgE-CCDs) in grape allergic patients sera was examined. We also determined to produce monoclonal antibodies against grape allergens and apply them in diagnostic procedures. We supposed that these complementary studies will help us in better understanding of the immunochemical characteristics of grape allergens as well as better interpretation and elucidation of the immunodiagnostic findings in this commonly observed fruit allergy. Finally, we determined to check the impact of pesticides as common environmental pollutants on expression level of grape allergens. Methods: A crude extract was prepared from ripen berries of a local grape cultivar (i.e. Vitis vinifera vitis). Participants with a clinical history of grape allergy underwent standard grape skin prick test (SPT) with the prepared extract. Serum was obtained from selected grape sensitive patients as well as non-reactive carbohydrate determinants (anti-277) Background and Rationale: Initial questionnaire based study of food allergy revealed that grape is a common allergenic fruit in Iran. The current study was performed to characterize the main allergic proteins of a local grape cultivar and subsequently produce the recombinant forms of its allergens. Moreover, in this study the glycosylation state of grape proteins as well as the presence of antibodies to cross-Title: Identification, molecular characterization and production of recombinant form of grape allergens as well as evaluation of pesticide impact on expression and allergenicity of grape allergenic proteins (Thesis number A‬إ‭reactive carbohydrate determinants [CCD], Pesticide, Niconazol. -Like Protein, Glucanase, Glycosylation, cross-time PCR results showed that spraying of the grapevines with fungicidal agents slightly increase the plants resistance against pathogenic fungi and consequently reduce their needs for high level expression of pathogenesis related proteins. However, this reduction was less than two folds and statistically could not be considered as significant. Key words: Grape, Allergy, Allergen, Recombinant Protein, Skin Prick Test, Mass Spectrometry, ELISA, Western Blotting, Lipid Transfer Protein, Chitinase, Thaumatin-allergen named lipid transfer protein (LTP) could be categorized as grape major allergen; while the other IgE binding proteins could be categorized as grape minor allergens. Gastric fluid had minimal effect on grape proteins revealing that these proteins may play role as true food allergens. The real-vitro reactions. Meanwhile, the results of the component resolved ELISA in combination with clinical history and SPT results indicates that a 10 kDa pan-CCDs may play role in some positive in-like protein and class IV chitinase play role in allergy to grapes. The results confirmed that cross reactive antibodies such as anti-glucanase, thaumatin-1,3-regulate the expression level of chitinase and thaumatin like protein. Conclusion: In conclusion, Vitis vinifera vitis showed a rather similar pattern of IgE reactivity with other reported grape cultivars. We found that a mixture of grape proteins including beta-fungal pesticide slightly down-A blotting showed that 24 and 60 kD bands were extremely mannosylated. Treatment of grape proteins with gastric fluid showed their resistance to pepsin and low pH conditions. Niconazol spraying of the grapevines revealed that this newly applied anti-CCD antibodies in development of some positive in vitro reactions. Moreover, IgE reactivity of some patients sera with bromlein strongly supported this possibility. Con-like protein was produced in bacterial hosts as inclusion body. The refolding procedures could not help for achieving immunoreactive form of those aggregated proteins in E. coli; however, application of the baculovirus system resulted to production of soluble intracellular protein in insect cells host which showed significant IgE reactivity with grape allergic patients sera. Partial chemical oxidation of carbohydrate moieties of grape proteins by sodium metaperiodate reduced IgE reactivity of patients sera with total extract proteins, showing possible role of anti-glucanase, respectively. The recombinant form of the chitinase and thaumatin-1,3-like protein, class IV chitinase and beta-g/ml protein. IgE Immunoblotting showed that sera from grape allergic patients' mainly were reactive with 10, 28 and 60 kDa proteins; meanwhile 16, 24, 30, 34, 38 and 45 kDa proteins could be categorized as its minor IgE binding proteins. Several grape proteins were fractionated by chromatographic methods. Among those proteins, a 10 kDa allergen showed the highest immunoreactivity with patients sera and also strong association with grape total extract SPT results. Mass spectrometry analysis of the 24, 28 and 30 kDa allergens revealed their identity as thaumatin‬
 
 
 
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