مجله علوم پزشکی پارس

طراحی و ساخت سازه ژنی بیان کننده ایمنوتوکسین ضد EGFR VIII متصل به اگزوتوکسین A سودوموناس ائروجنوزا برای سرطان گلیوبلاستوما

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکترای میکروب شناسی ، دانشگاه آزاد اسلامی واحد قم، قم، ایران

2 دانشیار میکروبشناسی، دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد قم، قم، ایران

3 استاد بیوتکنولوژی، مرکز تحقیقات کاربردی دارویی دانشگاه علوم پزشکی تبریز، ، تبریز، ایران

4 استادیار میکروبشناسی، دانشکده علوم پایه، دانشگاه آزاد اسلامی واحد قم، قم، ایران

5 استادیار زیست فناوری پزشکی، دانشگاه علوم پزشکی تبریز، تبریز، ایران

چکیده

مقدمه: سرطان از رایجترین علل مرگ و میر انسانی در جوامع امروزی است که تلاش‌های بسیار گسترده‌ای برای مقابله با آن در حال انجام است. ایمونوتوکسین‌های مبتنی بر Anti-EGFRVIII می‌توانند با هدایت بخش‌های توکسینی به سلول‌های سرطانی که بیان بیش از حد گیرنده EGFRVIII دارند، منجر به مرگ سلولی آنها شوند. هدف مطالعه حاضر توسعه یک ایمونوتوکسین انسانی جدید ضد EGFRVIII (huscFv-PE38 EGFRVIII) با ادغام ژنتیکی آنتی بادی تک زنجیره ای ضد EGFRVIII انسانی با اگزوتوکسین A کوتاه شده سودوموناس آئروژینوزا (PE) (PE38KDEL) است.
روش کار: قطعه PE-38 اگزوتوکسین A با استفاده از PCR تکثیر و به PET22b-Anti-EGFRVIII huscFv متصل شد. واکنش با روش PCR وهضم آنزیمی تایید شد. ایمونوتوکسین حاصل در E. coli BL21 (plys S) بیان شد و سپس توسط ستون کروماتوگرافی Ni-NTA تخلیص شد. پس از آن، واکنش ایمونوتوکسین تخلیص شده با روش‌های الایزا و MTT مورد ارزیابی قرار گرفت.
یافته‌ها: آزمایش‌های PCRو هضم آنزیمی، صحت و یکپارچگی ساختار ایمونوتوکسین طراحی شده را تأیید کردند. تخلیص ایمونوتوکسین بیان شده توسط ستون کروماتوگرافی نیکل منجر به ساخت یک ایمونوتوکسین نوترکیب بسیار خالص با وزن مولکولی ۶۳ کیلو دالتون در ژل SDS-PAGE شد.
نتیجه‌گیری: نتایج  مطالعه حاضر نشان داد که ایمونوتوکسین طراحی شده می‌تواند به عنوان یک انتخاب امیدوارکننده برای مهار سولهای سرطانی EGFRVIII مثبت در نظر گرفته شود.

کلیدواژه‌ها

عنوان مقاله [English]

Design and construction of genetic construct expressing anti-EGFR VIII immunotoxin contains exotoxin A of Pseudomonas aeruginosa for glioblastoma cancer

نویسندگان [English]

  • maryam irani 1
  • MohammadReza Zolfaghari 2
  • Safar Farajnia 3
  • Razieh Nazari 4
  • Leila Rahbarnia 5

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 Department of Microbiology, Islamic Azad University, Qom Branch, Qom, Iran

5 Assistant Professor of Medical Biotechnology, Tabriz University of Medical Sciences, Tabriz, Iran

چکیده [English]

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.

کلیدواژه‌ها [English]

  • Targeted Cancer Therapy
  • EGFRVIII
  • huscFv-PE38
  • Immunotoxin
  • Pseudomonas Exotoxin A
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مجله علوم پزشکی پارس
دوره 21، شماره 1
فروردین 1402
صفحه 62-72