بیان پروتئین پوششی نوترکیب ویروس X سیب زمینی در باکتری Escherichia coli

نوع مقاله : مقاله پژوهشی

نویسندگان

1 کرمان کرمان، دانشگاه شهید باهنر، دانشکده کشاورزی، بخش گیاهپزشکی

2 گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه شهید باهنر کرمان

3 استاد

چکیده

اهداف: ویروس X سیب زمینی (PVX) از خانواده Alphaflexiviridaeو جنس Potexvirus یکی از رایج­ترین و گسترده­ترین ویروس­های آلوده­کننده سیب زمینی در جهان می­باشد. به­دلیل گسترش و خسارت اقتصادی این ویروس در ایران، شناسایی و ردیابی آن با استفاده از روش­های با صرفه­تر ضروری می­باشد. تولید آنتی­بادی چندهمسانه­ای با استفاده از فناوری دی اِن اِی نوترکیب، یک راهکار مفید برای تسهیل شناسایی این ویروس در طبیعت می­باشد.
مواد و روش­ها: بر­این اساس، در بررسی حاضر، یک نمونه سیب زمینی آلوده به PVX از مزارع سیب‌زمینی در زرند، استان کرمان جمع آوری و آلودگی آن نسبت به ویروس مذکورتوسط آزمون DAS-ELISA تایید گردید. جدایه مذکور جهت تکثیر، بر روی توتون (Nicotiana glutinosa) مایه‌زنی گردید. پس از استخراج آر اِن اِی از گیاه آزمون، با استفاده از واکنش RT-PCR و آغازگرهای اختصاصی حاوی جایگاه برش برای آنزیم­های برشی، توالی پروتئین پوششی  ویروس تکثیر و در ناقل بیان پروکاریوتی (pQE60) همسانه­سازی گردید. پلاسمید نوترکیب، به باکتری Escherichia coli سویه M15، منتقل شد.
نتایج: پس از القای بیان، پروتئین مورد نظر استخراج و با تکنیک SDS-PAGE در ژل پلی آکریلامید 5/12 % مورد بررسی قرار گرفت. نتایج حاصل بیانگر آن است که وزن مولکولی باند حاصله در آزمون SDS-PAGE با وزن مولکولی پروتئین پوششی (kDa 24) مطابقت دارد. همچنین بیان پروتئین پوششی ویروس در تکنیک دات بلات نیز تأیید گردید.
نتیجه گیری نهایی: در این تحقیق، یک آنتی­ژن نوترکیب مناسب جهت تشخیص ویروس X سیب زمینی تهیه گردید که به­خوبی توسط آنتی­سرم تولید شده از طریق تزریق پیکره­های کامل ویروس به خرگوش در آزمون دات بلات شناسایی شد. تولید آنتی­بادی چندهمسانه­ای با استفاده از آنتی­ژن ویروسی نوترکیب، گامی موثر در زمینه تسریع و تسهیل فرآیند شناسایی سرولوژیکی نمونه­های آلوده به این ویروس مهم خواهد بود.

کلیدواژه‌ها

عنوان مقاله [English]

Expression of the recombinant coat protein of Potato virus X in Escherichia coli

نویسندگان [English]

  • Hossein Massumi 1
  • Parisa Hassan Chachi 2
  • Jahangir Heydarnejad 3
  • Akbar Hosseinipour 2
  • Mohammad Maddahian 2
1 Department of Plant Pathology, Shahid Bahonar University of Kerman
2 Department of Plant Pathology, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran
3 Professor, Department of Plant Pathology, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran.
چکیده [English]

Objective
Potato virus X (PVX) is a member of the genus Potexvirus in the family Alfaflexiviridae. This virus is one of the most common and widespread viruses infecting potato worldwide. Due to the wide distribution and economic damage of the virus in Iran, identification and detection of the virus is necessary using non-expensive methods. Production of polyclonal antibody through molecular approaches can be a useful method for detection of virus in the nature.
 
Materials and methods
 In this research, an infected potato sample was collected from Zarand (Kerman province) and its PVX infection was confirmed by ELISA test. This sample was inoculated on test plant and the coat protein (CP) gene of this isolate was amplified in RT-PCR test with specific primers comprising BglII and NcoI restriction enzymes. Amplified product was cloned into expression prokaryotic vector (pQE60 plasmid). followed by transformation of the E. coli strain M15 competent cells.
 
Results
Subsequent to induction of CP expression, total protein was extracted and run onto 12.5% SDS-PAGE. An approximate 24 kDa band was observed on the gel corresponded to the PVX coat protein. Furthermore, expression of the PVX CP was confirmed by dot blot technique.
 
Conclusions
In this study, a recombinant antigen suitable for the detection of potato X virus was prepared which was well identified by antiserum produced by injection of complete virus particles into rabbits in dot blot analysis. The production of multiclonal antibodies by recombinant viral antigen will be an important step in accelerating and facilitating the serological identification process of plants infected with this virus.

کلیدواژه‌ها [English]

  • Viral Coat Protein
  • E. coli
  • Recombinant Protein Expression
  • Potato virus X

مراجع

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مجله بیوتکنولوژی کشاورزی
دوره 11، شماره 4
دوره 11 شماره 4
اسفند 1398
صفحه 175-192

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