بهینه‏سازی کدونی و مقایسه‏ی بیان فاکتور رشد عصبی بتا انسانی (β-NGF) بصورت نوترکیب در میزبان‏های باکتریایی( BL21(DE3 و BL21(DE3)pLysS

نوع مقاله: مقاله پژوهشی

نویسندگان

1 کارشناس ارشد، دانشکده علوم و فنون نوین دانشگاه تهران، گروه مهندسی علوم زیستی، تهران، ایران

2 استادیار، دانشکده علوم و فنون نوین دانشگاه تهران، گروه مهندسی علوم زیستی، تهران، ایران

3 دانشیار، دانشگاه علوم پزشکی شهید بهشتی، گروه بیوشیمی بالینی، تهران، ایران

چکیده

فاکتور رشد عصبی بتا (β-NGF) برای اولین بار بدلیل نقش حیاتی‌اش در رشد و بقای سلول‏های عصبی مورد مطالعه قرار گرفت. این پروتئین که به خانواده‌ی نوروتروفین‌ها تعلق دارد در درمان بیماری‌های تخریب عصبی نظیر آلزایمر دارای نقش قابل ملاحظه‏ای می‏باشد. بدین منظور، β-NGF انسانی نوترکیب برای اولین‏بار در ایران در دو سویه‏ی متفاوت باکتری E.coli بیان شد. از آنجاییکه β-NGF در ساختار فعال و عملکردی خود دارای سه پیوند دی‏سولفیدی است، محیط احیا کننده‏ی سیتوپلاسم E.coli برای بیان آن مناسب نمی‌باشد. بنابراین، محیط اکسید کننده‌ی پری‏پلاسم جهت تولید β-NGF همراه با تاخوردگی صحیح مورد توجه قرار گرفت. در این تحقیق، cDNA β-NGFانسانی بدست آمده از بانک اطلاعاتی NCBI پس از بهینه‌سازی کدونی و ساب کلونینگ در وکتور pET39b(+)، حاوی ژن دی سولفید ایزومراز A، به روش ترنسفورماسیون (شوک حرارتی) به سویه‌های BL21(DE3)pLysS و BL21(DE3) انتقال یافت. بیان همزمان با استفاده از القای پروموتر با غلظت‌ 1 میلی مولار IPTG صورت گرفت. سپس، محتوای پروتئینی استخراج شده از دو سویه‏ی مذکور با استفاده از تکنیک‌های SDS-PAGE و دات بلات با یکدیگر مقایسه گردید. نتایج نشان دهنده بیان، بویژه در سویه باکتریایی BL21(DE3) می باشد.

کلیدواژه‌ها

عنوان مقاله [English]

Codon Optimization and Comparison of Recombinant Human β-NGF Expression in BL21(DE3)pLysS and BL21(DE3) Bacterial Hosts

نویسندگان [English]

  • Masoumeh Rajabi bazl 1
  • zahra Hajihassan 2
  • Mona Sohrabi 3

چکیده [English]

Beta Nerve growth factor (β-NGF) was first known for its vital role in development and survival of neurons. This protein, which belongs to the neurotrophin family, plays a considerable role in the treatment of neurodegenerative diseases such as Alzheimer’s disease (AD). As a result of its characteristic, recombinant human β-NGF (rhβNGF) was expressed in two different strains of E.coli for the first time in Iran. β-NGF has three disulfide bonds in its native and functional structure, so the cytoplasmic reducing environment of E.coli is not appropriate for its expression. Therefore, the oxidative space of periplasm for production of correctly folded β-NGF was considered. In this study, hβNGF cDNA obtained from NCBI data bank after codon optimization and subcloning in pET39b(+) vector containing DsbA gene was transformed (using heat shock) to BL21(DE3)pLysS and BL21(DE3) strains of E.coli. Co-expression occurred via induction of promoter with 1 mM of IPTG. Consequently, extracted proteins from these two strains were compared with each other with SDS-PAGE and Dot blot techniques. The data shows βNGF expression especially in BL21(DE3) strain of E.coli.

کلیدواژه‌ها [English]

  • Nerve Growth Factor
  • Periplasmic Expression
  • Recombinant Protein
  • Escherichia coli

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