کلونینگ و بیان دومین III پروتئین پوششی ویروس دنگی نوع 2 در باکتری E. coli

نوع مقاله : مقاله های پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه ژنتیک، دانشکده‌ی علوم زیستی، دانشگاه تربیت مدرس، تهران، ایران

2 استاد، گروه ژنتیک، دانشکده‌ی علوم زیستی، دانشگاه تربیت مدرس، تهران، ایران

چکیده

مقدمه: هدف این مطالعه بیان آنتی‌ژن نوترکیبی از پروتئین پوششی ویروس دنگی، به منظور دست‌یابی به بیان بیشتر و به شکل محلول بود. بنابراین، قابلیت بیان دومین III این پروتئین در میزبان باکتریایی بررسی گردید.روش‌ها: هم‌ردیفی توالی‌های مختلف دومین III با استفاده از نرم‌افزار Megalign انجام شد. پیش‌بینی ساختار پروتئین با نرم‌افزار Modeller و بهینه‌سازی توالی ژن برای بیان در E. coli با نرم‌افزار Optimizer صورت گرفت. سنتز و کلونینگ ژن در حامل pET21a و بهینه‌سازی بیان پروتئین نوترکیب در E. coli انجام شد. برای تخلیص پروتئین از ستون‌های کروماتوگرافی Ni-NTA استفاده گردید. کارایی بیان پروتئین با روش‌های الکتروفورز و لکه‌گذاری وسترن بررسی شد.یافته‌ها: یک توالی مورد توافق برای دومین III پروتئین پوششی سروتیپ 2 ویروس دنگی، ایجاد شد. ویژگی‌های ساختاری پروتئین هدف با استفاده از روش‌های بیوانفورماتیکی ارزیابی شد. با هدف دستیابی به بیان بالا، توالی ژن از لحاظ الگوی به کارگیری کدون و درصد GC بهینه‌سازی گردید. توالی ژن هدف با استفاده از روش‌های توالی‌یابی و هضم آنزیمی مورد تأیید قرار گرفت. به منظور ایجاد پیوند دی‌سولفیدی در ساختار پروتئین، از میزبان بیانی Origami (DE3) استفاده شد. در نهایت بهینه‌سازی شرایط بیان پروتئین هدف انجام شد و غلظت مناسبی از پروتئین نوترکیب به صورت محلول به دست آمد.نتیجه‌گیری: نتایج این مطالعه نشان داد که بهینه‌سازی توالی دومین III منجر به تولید محصول پروتئینی با غلظت بالا (20 میلی‌گرم در لیتر محیط کشت) گردید. این سیستم بیانی می‌تواند جهت تولید دومین III پروتئین پوششی ویروس دنگی استفاده شود.

کلیدواژه‌ها


عنوان مقاله [English]

Cloning and Expression of Domain III of Dengue Virus Type 2 Envelope Protein in Escherichia Coli

نویسندگان [English]

  • Hossein Fahimi 1
  • Majid Sadeghizadeh 2
  • Mahshid Mohammadipoor 1
1 PhD Student, Department of Genetics, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran
2 Professor, Department of Genetics, School of Biological Sciences, Tarbiat Modares University, Tehran, Iran
چکیده [English]

Background: The aim of this study was production of a recombinant antigen from envelope protein of dengue virus, in order to achieve high level expression in soluble form. Therefore, the possibility of envelope protein domain III expression in bacterial host was studied.Methods: Multiple sequence alignment for domain III sequences was carried out using Megalign software. The Modeller and Optimizer software were used for protein structure prediction and sequence optimization. After gene synthesis and subcloning in the pET21a expression vector, the optimization of recombinant protein expression was carried out in Escherichia coli. Ni-NTA chromatography columns were used for protein purification. The efficiency of protein expression was analyzed using electrophoresis and western blotting methods.Findings: A consensus sequence for domain III of dengue virus type 2 envelope protein was provided. The structural properties of target protein were predicted using bioinformatics methods. For high expression level, the sequence of EDIII2 gene was optimized for appropriate codon usage and GC content. The coding gene sequence was approved by sequencing and enzymatic digestion. For disulfide bound formation in protein structure, the Origami (DE3) expression host was used. At the final step, expression of target protein was optimized, and a high concentration of recombinant protein was achieved in soluble form.Conclusion: The results of this study showed that the optimization of domain III sequence led to obtain high concentration (20 mg/l) of produced protein. This expression system can be used for production of dengue virus envelope protein domain III.

کلیدواژه‌ها [English]

  • Dengue virus
  • Recombinant
  • Envelope protein
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