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تولید فاژهای پلیکلونال حاوی قطعات ژنی آنتیبادی اختصاصی باکتری Xanthomonas citri subsp. citri با استفاده از فنآوری نمایش فاژی | ||
| آفات و بیماری های گیاهی | ||
| مقاله 11، دوره 85، شماره 2 - شماره پیاپی 105، اسفند 1396، صفحه 265-276 اصل مقاله (236.33 K) | ||
| نوع مقاله: بیماریشناسی گیاهی | ||
| شناسه دیجیتال (DOI): 10.22092/jaep.2017.115980.1194 | ||
| نویسندگان | ||
| حمیده رئیسی1؛ محمد رضا صفرنژاد* 2؛ سید مهدی علوی3؛ سیدعلی الهی نیا4؛ ناصر فرخی5 | ||
| 1دانش آموخته دکتریt ﺑﺨﺶ گیاهپزشکی، دانشکده علوم کشاورزی، ﺩﺍﻧﺸﮕﺎﻩ گیلان، رشت | ||
| 2دانشیار پژوهش، ﺑﺨﺶ ﻭﻳﺮﻭﺱ ﺷﻨﺎﺳﻲ ﮔﻴﺎﻫﻲ، ﻣﻮﺳﺴﻪ ﺗﺤﻘﻴﻘﺎﺕ ﮔﻴﺎﻩ ﭘﺰﺷﻜﻲ ﻛﺸﻮﺭ، سازمان تحقیقات آموزش و ترویج کشاورزی، ﺗﻬﺮﺍﻥ | ||
| 3استادیار پژوهش، ﺑﺨﺶ ﺑﻴﻮﺗﻜﻨﻮﻟﻮژﻱ ﮔﻴﺎﻫﻲ، ﭘﮋﻭﻫﺸﮕﺎﻩ ﻣﻠﻲ ﻣﻬﻨﺪﺳﻲ ژﻧﺘﻴﻚ ﻭ ﺯﻳﺴﺖ ﻓﻦ ﺁﻭﺭﻱ، تهران | ||
| 4استاد، ﺑﺨﺶ گیاهپزشکی، دانشکده علوم کشاورزی، ﺩﺍﻧﺸﮕﺎﻩ گیلان، رشت | ||
| 5دانشیار، گروه بیولوژی سلولی و مولکولی، دانشکده علوم زیستی و بیوتکنولوژی، دانشگاه شهید بهشتی، تهران | ||
| چکیده | ||
| شانکر باکتریایی مرکبات از بیماریهای باغات لیمو در جنوب کشور با عامل Xanthomonas citri subsp. citriمیباشد. تکنیک فاژنمایی از راهکارهای تولید آنتیبادیهای اختصاصی به منظور شناسایی گیاهان آلوده و همچنین تولید گیاهان مقاوم به بیماری است. در این تحقیق قابلیت استفاده از این سیستم برای تولید فاژهای نوترکیب حاوی قطعات آنتیبادی اختصاصی برعلیه باکتری عامل بیماری شانکر مرکبات بررسی شد. برای این منظور، پروتئین تاثیرکننده (pthA) و پیلوس (HrpE) که از اجزای اساسی سیستم ترشحی نوع سه باکتری هستند و نقش ضروری در بیماریزایی پاتوژن دارند، به عنوان آنتیژن انتخاب شدند. ابتدا پروتئینهای pthA و HrpE به صورت نوترکیب در میزبان باکتریایی تولید شدند و با روش کروموتوگرافی خالص گردیدند. از کتابخانه های فاژی حاوی قطعات ژنی نواحی متغیر آنتیبادی برای جداسازی فاژهای اختصاصی استفاده شد. غنیسازی جمعیت فاژهای اختصاصی با انجام سه مرحله غربالگری بر علیه پروتئین های خالص شده pthA و HrpEانجام شد و اختصاصیت فاژهای حاصله بر علیه آنتیژن توسط آزمون الیزا بررسی شد. فاژهای نوترکیب قابلیت اتصال به پروتئینهای pthA و HrpE را داشته و قادر به ردیابی گیاهان آلوده به بیماری شانکر باکتریایی مرکبات نیز بودند. از فاژهای جداسازی شده میتوان به منظور تولید آنتیبادی اختصاصی مونوکلونال استفاده نمود. | ||
| کلیدواژهها | ||
| افکتور پروتئین pthA؛ پروتئین HrpE؛ شانکر باکتریایی مرکبات؛ غربالگری؛ نمایش فاژی | ||
| عنوان مقاله [English] | ||
| Production of polyclonal phages harbouring antibody fragment genes against Xanthomonas citri subsp. citri using phage display technology | ||
| نویسندگان [English] | ||
| H. RAEISI1؛ M. R. SAFARNEJAD2؛ S. M. ALAVI3؛ S. A. ELAHINIA4؛ N. FARROKHI5 | ||
| 1Plant protection,agriculture,guilan university | ||
| 2Department of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural research education and extension organization of Iran. Tehran | ||
| 3Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran | ||
| 4Department of Plant Protection, College of Agricultural Sciences, Guilan University, Rasht | ||
| 5Departement of Plant Sciences & Biotechnology, Faculty of Life Sciences & Biotechnology, Shahid Beheshti University G.C, Tehran | ||
| چکیده [English] | ||
| Citrus bacterial canker, caused by Xanthomonas citri subsp. citri (Xcc),is amongst the important diseases of lime orchards in southern parts of Iran. Phage display has been used to produce specific antibodies for detection of pathogen-infected plants as well as development of resistant varieties. An effector, namely pthA and a pilus protein, HrpE, the major critical components of type III secretion (T3S) system with roles in pathogenesis, were chosen as antigens. Recombinant forms of the proteins (pthA and HrpE) were expressed in a bacterial host and purified via affinity chromatography. Tomlinson phage display libraries including single chain variable fragments were used for isolation of the specific antibodies. Biopanning, 3 rounds against pthA and HrpE proteins, allowed enriching antigen-specific phages. The specificity of phages was tested using ELISA. Moreover, the phages were able to detect the plants infected with citrus bacterial canker. | ||
| کلیدواژهها [English] | ||
| Biopanning, citrus bacterial canker, effector protein pthA, HrpE protein, phage display | ||
| مراجع | ||
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