ارزیابی گیاه تراریخت کلزای بیان‌کننده پروتئین نوترکیب از زیر واحد B سم‌های STX2 و CTX باکتری‌های اشریشیا کلای و ویبریوکلرا

نوع مقاله : علمی پژوهشی

نویسندگان

1 کارشناس ارشد، پژوهشکده بیوتکنولوژی کشاورزی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران

2 دانشجوی دکتری، پژوهشکده بیوتکنولوژی کشاورزی، پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری، تهران، ایران

3 دانشیار، مرکز تحقیقات میکروبیولوژی کاربردی، دانشگاه علوم پزشکی بقیه‌الله (عج)، تهران، ایران

4 استاد، پژوهشکده بیوتکنولوژی کشاورزی، پژوهشگاه ملی مهندسی ژنتیک و زیست‌فناوری، تهران، ایران

چکیده

بیماری‌های اسهالی یکی از مهمترین معضلات بهداشت جهانی، در تمامی رده‎های سنی مطرح می‌باشند. از میان عوامل ایجاد کننده این بیماری‌ها می-توان به Enterohemorrhagic Escherichia coli (EHEC) وVibrio cholerae اشاره کرد. در گروه EHEC سروتایپ O157:H7، شایع‌ترین عامل ایجاد اسهال، از طریق سم STX2 ایجاد بیماری می‌کند. در ویبریوکلرا نیز توکسین CTX منجر به بروز بیماری می‌گردد. هر دوی این سموم دارای ساختار AB5 هستند. با توجه به غیر سمی بودن زیرواحد B و ایمنی‌زایی ذاتی این زیرواحد، می‌توان آن را کاندیدای مناسبی برای ایجاد ایمنی علیه این سموم معرفی کرد. بذور گیاهان رآکتور زیستی مناسبی برای تولید ایمونوژن‌های نوترکیب محسوب شده و پروتئین‌های بیگانه‌ای که در گیاه بیان می‌شوند می توانند ساختار اصلی خود را حفظ می‌کنند. در این تحقیق بیان پروتئین نوترکیب متشکل از زیرواحد اتصالی دو سم کلرا و STX2 با ترجیح کدونی گیاه به‌عنوان کاندیدای یک ایمونوژن خوراکی در بذر بررسی شد. بدین منظور قطعه ژنی sc (stx2B و ctxB) به ناقل بیانی گیاهی تحت کنترل پیش‌برنده اختصاصی بذر کلزا FAE (Fatty Acid Elongase) منتقل و پس از تائید حضور سازه ژنی با روش‌های مولکولی، به اگروباکتریوم تومی فاشینس جهت تراریختی گیاه انتقال و جهت بیان پروتئین به گیاه کلزا منتقل شد. پس از تأیید تراریختی، میزان بیان گیاهی پروتئین نوترکیب در بذر به روش الایزا ارزیابی و میزان پروتئین حدود 40 میلی‌گرم در یک گرم بذر تخمین زده شد. نتایج این تحقیق بیانگر کارایی بالای استفاده از بذر برای تولید پروتئین نوترکیب می‌باشد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Molecular analysis of transgenic canola plant containing chimeric B subunit of bacterial toxin STX2 and CTX from Escherichia coli and Vibrio cholerae

نویسندگان [English]

  • Atena Mozafari 1
  • Rouhollah Kazemi 2
  • Jafar Amani 3
  • Mahyat Jafari 1
  • Ali Hatef Salmanian 4
1 M.Sc., Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
2 Ph.D student, Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
3 Associate Professor, Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
4 Professor, Department of Agricultural Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.
چکیده [English]

Diarrheal diseases have been considered as one of the most major world health problems in all age categories. Among all the pathogens caused to diarrhea, entrohemorrhagic Escherichia coli (EHEC) and Vibrio cholerae are the most important agents. E. coli O157:H7 as the most important serotype of EHEC bacteria caused diarrhea by producing STX2 toxin. In vibrio cholerae the cholera toxin (CTX) also is the main virulence factor that leads to diarrhea. Both of these toxins are belong to AB5 family and due to non-toxicity and natural immunogenic characteristic of B subunit, it could be introduced as an appropriate candidate for immunogenicity against these toxins. Plant seeds are an effective biological bioreactor for production of recombinant immunogenic antigens and also foreign proteins can be expressed in plants with the native structure. In this research, the production of recombinant protein composed of binding subunits of STX2 and cholera toxin was evaluated in canola seed as an oral vaccine candidate. The SC construct composed of (stx2B and ctxB) was subcloned to plant vector under the control of canola seed specific promoter (fae) and transformed to Agrobacterium tumefaciens. Then the recombinant vector was transformed to plant host via Agrobacterium mediated transformation. Transgenic plants was evaluated and the amount of chimeric protein expressed in transgenic canola seed was determined by semi quantitative ELISA and subsequently the amount of chimeric proteins was estimated 40 milligram per 1 gram seed. The results showed that the canola seed can efficiently produce recombinant protein.

کلیدواژه‌ها [English]

  • Transgenic canola plant
  • EHEC
  • Vibrio cholerae
  • Edible immunogen

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