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

Document Type : Research Paper

Authors

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.

Abstract

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.

Keywords

References

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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Agricultural Biotechnology Journal
Volume 16, Issue 1
February 2024
Pages 45-68

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