Design and construction of genetic construct expressing anti-EGFR VIII immunotoxin contains exotoxin A of Pseudomonas aeruginosa for glioblastoma cancer
Document Type : Original Article
Authors
1 PhD student in microbiology, Islamic Azad University, Qom Branch, Qom, Iran
2 Department of Microbiology, Islamic Azad University, Qom Branch, Qom, Iran
3 Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4 Assistant Professor of Medical Biotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
Abstract
Introduction:
Cancer is one of the most common causes of human mortality in today's societies, and extensive efforts are being made to combat it. EGFRVIII is a tumor-specific antigen that is not observed in normal tissues, making it possible to target cancerous tissues without damaging normal tissues. ANTI-EGFRVIII-based immunotoxins can lead to the death of cancer cells that express an excessive amount of the EGFRVIII receptor by directing toxic components to these cells.
Objectives: The objective of the present study is to develop a new human immunotoxin against EGFRVIII (huscFv-PE38) by genetically fusing a single-chain human anti-EGFRVIII antibody with a truncated form of the Pseudomonas aeruginosa exotoxin A (PE) (PE38KDEL).
Materials and Methods:
The PE-38 exotoxin a fragment was amplified using PCR and attached to
pET22b-antiEGFRVIII huscFv. The reaction was confirmed by PCR and enzymatic digestion. The
immunotoxin was expressed in E. coli BL21 (plysS) and then purified using Ni-NTA chromatography.
After that, the purified immunotoxin reaction was evaluated using ELISA methods and MTT test.
Results:
The PCR and restriction digestion experiments confirmed the accuracy and integrity of the designed
immunotoxin structure. Purification of the expressed immunotoxin using Ni-NTA chromatography
column resulted in a highly pure, non-recombined immunotoxin with a molecular weight of 63 kDa
shown on SDS-PAGE gel.
Conclusion:
The results of this study demonstrate that the designed immunotoxin was successfully cloned, expressed, and purified, and can be considered as a promising candidate for the inhibition of EGFRVIII-positive cancer cells.
Keywords
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