با همکاری انجمن‏‌ بیماری شناسی گیاهی ایران

نوع مقاله : بیماری‌شناسی گیاهی

نویسندگان

1 دانش آموخته دکتریt ﺑﺨﺶ گیاهپزشکی، دانشکده علوم کشاورزی، ﺩﺍﻧﺸﮕﺎﻩ گیلان، رشت

2 دانشیار پژوهش، ﺑﺨﺶ ﻭﻳﺮﻭﺱ ﺷﻨﺎﺳﻲ ﮔﻴﺎﻫﻲ، ﻣﻮﺳﺴﻪ ﺗﺤﻘﻴﻘﺎﺕ ﮔﻴﺎﻩ ﭘﺰﺷﻜﻲ ﻛﺸﻮﺭ، سازمان تحقیقات آموزش و ترویج کشاورزی، ﺗﻬﺮﺍﻥ

3 استادیار پژوهش، ﺑﺨﺶ ﺑﻴﻮﺗﻜﻨﻮﻟﻮژﻱ ﮔﻴﺎﻫﻲ، ﭘﮋﻭﻫﺸﮕﺎﻩ ﻣﻠﻲ ﻣﻬﻨﺪﺳﻲ ژﻧﺘﻴﻚ ﻭ ﺯﻳﺴﺖ ﻓﻦ ﺁﻭﺭﻱ، تهران

4 استاد، ﺑﺨﺶ گیاهپزشکی، دانشکده علوم کشاورزی، ﺩﺍﻧﺸﮕﺎﻩ گیلان، رشت

5 دانشیار، 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

موضوعات

عنوان مقاله [English]

Production of polyclonal phages harbouring antibody fragment genes against Xanthomonas citri subsp. citri using phage display technology

نویسندگان [English]

  • H. RAEISI 1
  • M. R. SAFARNEJAD 2
  • S. M. ALAVI 3
  • S. A. ELAHINIA 4
  • N. FARROKHI 5

1 Plant protection,agriculture,guilan university

2 Department of Plant Viruses, Iranian Research Institute of Plant Protection, Agricultural research education and extension organization of Iran. Tehran

3 Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, Tehran

4 Department of Plant Protection, College of Agricultural Sciences, Guilan University, Rasht

5 Departement 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
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