ساخت وکتور دوسیسترونی مبتنی بر دومین فیوژن IntF2A برای بیان همزمان زنجیره‌های پلی‌پپتیدی آنتی‌بادی بواسیزوماب و بررسی تظاهر موقت آن در گیاه توتون

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ارومیه، ارومیه، ایران

2 دانشیار، گروه مهندسی تولید و ژنتیک گیاهی، دانشکده کشاورزی، دانشگاه ارومیه، ارومیه، ایران

10.22103/jab.2023.21398.1477

چکیده

هدف: بواسیزوماب (با نام تجاری آوستین) یک آنتی‌بادی مونوکلونال (mAb) انسانی شده است که به‌طور گسترده‌ای در درمان سرطان‌های مختلفی کاربرد دارد. این دارو جزء گرانقیمت‌ترین و پرفروش‌ترین داروهای دنیا است. بواسیزوماب از طریق تکنولوژی DNA نوترکیب در سیستم بیان پستانداری، تخمدان همستر چینی تولید می‌شود. در سال‌های اخیر، سیستم‌های بیان گیاهی به دلیل مزایای مختلف برای تولید پروتئین‌های نوترکیب دارویی، توجه جهانی را به خود جلب کرده‌اند. تولید موفقیت‌آمیز mAbها به تاشدگی و گردایش مناسب زنجیره سبک (HC) و زنجیره سنگین (LC) بستگی دارد. بیان همزمان کارآمد و کنترل شده این زنجیره‌های پپتیدی یک ملاحظه مهم در طراحی وکتورهای بیان آنتی‌بادی است. هدف این تحقیق ساخت وکتور دو‌سیسترونی برای بیان همزمان زنجیره‌های پپتیدی HC و LC آنتی‌بادی بواسیزوماب با استفاده از دومین فیوژنIntF2A و ارزیابی آن از طریق بیان موقت در گیاه توتون است.

مواد و روش‌ها: به منظور ساخت وکتور نوترکیب، توالی ژن‌های رمزکننده بواسیزوماب و IntF2A از نظر کدونی بهینه‌سازی شد و برای بیان در گیاه توتون به‌طور مصنوعی سنتز شدند. وکتور نوترکیب دوسیسترونی بواسطه IntF2A با روش معمول هضم آنزیمی-اتصال ساخته شد. در نهایت، سازه نوترکیب برای بیان گذرا بواسیزوماب در گیاهان توتون از طریق روش آگروفیلتراسیون استفاده گردید. برای تعیین حضور رونوشت‌ تراژن‌ها از آنالیز RT-PCR استفاده شد. تولید و میزان تجمع آنتی‌بادی نوترکیب از طریق آنالیز وسترن بلات مورد ارزیابی قرار گرفت.
نتایج: ساختار مولکولی وکتور نوترکیب ساخته شده از طریق آنالیز‌های PCR، هضم آنزیمی و توالی‌یابی تایید شد. آنالیز RT-PCR نشان داد تراژن‌ها به‌صورت یک رونوشت واحد بیان می‌شوند. آنالیز وسترن بلات تولید آنتی‌بادی هتروتترامر کامل در برگ‌های آگرواینفیلتر شده را تایید نمود. سطح تجمع آنتی‌بادی به طور متوسط ۲۰/۶۷ میلی‌‌گرم بواسیزوماب به ازای هر کیلوگرم وزن تر (معادل ۳۶/۳ درصد پروتئین محلول کل) برآورد شد.
نتیجه‌گیری: نتایج نشان داد وکتور دو‌سیسترونی مبتنی بر IntF2A بیان همزمان کارآمد ژن‌های رمزکنندة زنجیره‌های پپتیدی آنتی‌بادی و تجمع بالای mAb با فرم کامل را در سلول‌های گیاهی فراهم می‌کند. از وکتور ساخته شده می‌توان برای امکان توسعه و ساخت آنتی‌بادی بواسیزوماب در سیستم بیان گیاهی استفاده کرد.

کلیدواژه‌ها


عنوان مقاله [English]

Construction of the IntF2A fusion domain-based bicistronic vector for simultaneous expression of the polypeptide chains of bevacizumab antibody and investigating its transient expression in tobacco plants

نویسندگان [English]

  • Pari Andaz 1
  • Morad Jafari 2
1 Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran
2 Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran.
چکیده [English]

Objective
Bevacizumab (trade name Avastin®) is a humanized monoclonal antibody (mAb) that is widely used in the treatment of various cancers. It is one of the most expensive and best-selling cancer drugs in the world. Bevacizumab is produced by recombinant DNA technology in a mammalian expression system, the Chinese hamster ovary. In recent years, plant expression systems have attracted global attention due to their various benefits for producing recombinant pharmaceutical proteins. The successful expression of the mAbs is dependent on the proper folding and assembly of the light chains (LC) and heavy chains (HC). The efficient and controlled co-expression of the peptide chains is an essential consideration in designing antibody expression vectors. This study aimed to construct a plant expression vector for the simultaneous expression of HC and LC peptide chains of bevacizumab mAb using the IntF2A fusion domain.
Materials and methods
In order to construct the recombinant vector, the sequences of genes encoding bevacizumab and IntF2A were codon-optimized and artificially synthesized for expression in tobacco plants. The bicistronic recombinant vector was constructed by the routine restriction digestion-ligation method. Finally, the recombinant construct was used for the transient expression of bevacizumab in tobacco plants through agroinfiltration approach. RT PCR analysis was used to determine the presence of transgene transcripts. The production and accumulation level of the recombinant antibody was evaluated through western blotting analysis.
Results
Recombinant plasmid construction is verified by colony PCR, restriction digestion, and sequencing analyses. Expression of the transgenes as a single-transcript unit was confirmed by RT-PCR analysis. Western blot analysis showed the production of the full-length heterotetrameric antibody in agroinfiltrated leaves. The level of antibody accumulation was estimated at an average of 67.20 mg of bevacizumab per kg of fresh weight (3.36% TSP).
Conclusions
The results showed that the IntF2A-based bicistronic vector provides an efficient simultaneous expression of genes encoding the polypeptide chains of antibody and high accumulation of the full-length mAb in plant cells. The constructed vector can be used for the possibility of development and manufacturing of bevacizumab antibody in plant expression system.

کلیدواژه‌ها [English]

  • Agroinfiltration
  • Bevacizumab
  • Bicistronic vector construction
  • IntF2A
  • Tobacco
جعفری مراد، دلیرژ نوروز، حبیبی مهدی (۱۴۰۱) تولید آزمایشگاهی ریتوکسی‌ماب، آنتی‌بادی ضد CD20، در گیاه توتون (Nicotiana tabacum L.). گزارش نهایی طرح تولید محور،‌ دانشگاه ارومیه، ۱۴۲-۱.
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