بیان هترولوگ ژن پروتئین پوششی ویروس موزائیک گل‌کلم در E. coli و تشکیل ذرات شبه ویروسی

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان

2 گروه گیاهپزشکی دانشکده کشاورزی، دانشگاه شهید مدنی آذربایجان

3 نانوفناوری پزشکی. گروه نانوتکنولوژی پزشکی، دانشکده علوم نوین پزشکی. مرکز تحقیقات علوم کاربردی دارویی، دانشگاه علوم پزشکی تبریز. ایران

چکیده

ویروس موزائیک گل‌کلم  (Cauliflower Mosaic Virus= CaMV)نخستین ویروس گیاهی شناسایی شده دارای ژنوم DNA است که خسارت اقتصادی زیادی به گیاهان خانواده Brassicaceae وارد می­کند. تولید موفق کپسید­های این ویروس هم در تشخیص سرولوژیکی آن و هم در تشکیل ذرات شبه ویروسی Virus-Like Particles = VLPs مفید است. در این تحقیق، DNA نمونه­های گل کلم دارای علایم مشکوک به ویروس، استخراج و ژن پروتئین پوششی (Coat protein= CP) ویروس توسط آغازگرهای اختصاصی در PCR تکثیر شد. سپس توسط آنزیم­های برشی BamHI و SmaI در وکتور بیانی  pGEX2TKدر پایین دست GST وارد و بعد از تایید وکتور نوترکیب، توالی­یابی انجام شد و توالی بدست آمده در پایگاه داده NCBI با برنامه BLASTn با توالی­های موجود در بانک ژن مقایسه شد و مشخص گردید که این توالی مربوط به ویروس CaMV با شماره دسترسی KF357590.1 در پایگاه NCBI است. بیان پروتئین CP در باکتری E. coli سویه Rosetta القاء و بهینه­سازی شد. بیان بسیار بالای این پروتئین در دمای 37 درجه سانتی­گراد و شش ساعت پس از القا به کمک IPTG با غلظت نهایی 1mM بدست آمد. سپس این پروتئین با نانوذرات سفاروز که توانایی اتصال به GST را دارند خالص­سازی و در SDS-PAGE مشاهده شد. پروتئین­های خالص شده CP با میکروسکوپ الکترونی بررسی شد و ذرات شبه ویروسی (VLPs) مشاهده گردید. تصاویر مربوط به میکروسکوپ الکترونی تولید VLPs این ویروس را بدون ایجاد اینکلوژن بادی و با مونتاژ صحیح تائید می­کند که می­تواند در حمل دارو در پزشکی مورد آزمایش قرار گیرد.
 

کلیدواژه‌ها


عنوان مقاله [English]

Heterologous expression of Cauliflower Mosaic Virus coat protein gene in E. coli and formation of virus-like particles

نویسندگان [English]

  • Mahin Pouresmaeil 1
  • Maghsoud Pazhouhandeh 1
  • Akbar Shirzad 2
  • Ahmad Yari Khosroushahi 3
1 Department, of Biotechnology Agriculture Faculty, Azarbaijan Shahid Madani University, Km 35 Tabriz-Azarshahr Road, Tabriz, Iran.
2 Plant Protection Department, Agriculture Faculty, Azarbaijan Shahid Madani University, Tabriz, Iran.
3 Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
چکیده [English]

Cauliflower Mosaic Virus (CaMV) is the first identified plant virus with a DNA genome and causes great economic damage to Brassicaceae plants. The successful production of capsids of this virus is useful both in its serological diagnosis and in the formation of virus-like particles (VLPs). In this research, DNA from cauliflower samples with suspected virus symptoms was extracted and the coat protein (CP) gene of the virus was amplified using specific primers in PCR. Then it was inserted into the expression vector pGEX2TK downstream of GST by digestion endonuclease enzymes BamHI and SmaI and after confirmation of the recombinant vector, its sequencing was carried out. Sequencing was performed and the sequence obtained in the NCBI database was compared with the sequences in the gene bank with the BLASTn program. It was found that this sequence is related to the CaMV virus with accession number KF357590.1 in the NCBI database. The expression of the CP was induced and optimized in E. coli (Rosetta strain). A very high expression of this protein was obtained at 37°C and 6 hours after induction using IPTG with a final concentration of 1 mM. Then, this protein was purified with sepharose nanoparticles which have an affinity to GST, and observed in SDS-PAGE. The related electron microscope images confirm the production of VLPs of this virus with correct assembly without creating inclusion bodies that can be tested in drug delivery in medicine.
 

کلیدواژه‌ها [English]

  • Cauliflower Mosaic Virus
  • Cloning
  • Coat protein gene
  • protein expression
  • Virus like particles (VLPs)
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