طراحی و بررسی کارایی کیت الایزا مبتنی بر آنتی بادی تولید شده علیه پروتئین پوششی ویروس ساقه شیاری سیب در Escherichia coli

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانش آموخته کارشناسی ارشد بیماری شناسی گیاهی، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه زنجان، زنجان،

2 دانشیار، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه زنجان، زنجان، ایران

3 دانشیار، گروه گیاهپزشکی، دانشکده کشاورزی، دانشگاه کردستان، سنندج، ایران

چکیده

هدف: تولید آنتی‌بادی و کاربرد آن در آزمون‌های متنوع و طراحی بیوسنسورها یکی از راهبردهای مهم در تشخیص ویروس‌های گیاهی است. ویروس ساقه شیاری سیب (Apple stem grooving virus (ASGV)) یکی از ویروس‌های مهم درختان دانه‌دار و سیب ‌می-باشد. هدف از این تحقیق، بیان ژن پروتئین پوششی این ویروس در E. coli و تولید آنتی بادی علیه پروتئین بیان شده و ارزیابی آن در آزمون‌های تشخیصی است.
مواد و روش: در واکنش زنجیره‌ای پلی‌مراز، قطعه‌ای به طول 714 جفت باز مربوط به ژن کامل پروتئین پوششی ASGV تکثیر و در پلاسمید pTG19 همسانه‌سازی شد. سپس، قطعه مورد نظر با آنزیم‌های برشی BamHI و EcoRI خارج و در حامل بیانی pET28a(+) همسانه‌سازی شد و سازه مورد نظر به‌منظور بیان ژن هدف، به باکتری E. coli سویه BL21(DE3) منتقل شد. بهینه سازی بیان در غلظت یک میلی مولار IPTG و زمان‌های سه، چهار، شش و 16 ساعت پس از القا صورت گرفت و تایید بیان پس از الکتروفورز عمودی و آزمون وسترن بلات انجام شد. پروتئین بیان شده تخلیص و به‌عنوان آنتی‌ژن به خرگوش تزریق شد. پس از ایمن سازی سرم، ایمنوگلوبولین‌ها از سرم خون تخلیص و بخشی از آنها برای تولید کانجوگیت استفاده شدند. در نهایت، کارایی آنها در آزمون‌های سرولوژیکی ارزیابی شد.
یافته‌ها: نتایج تعیین توالی یابی نشان داد قطعات تکثیر یافته در پی‌سی‌آر مربوط به ژن پروتئین پوششی ویروس ساقه شیاری سیب است. نتایج پی‌سی‌آر کلونی و هضم آنزیمی تایید کننده ساخت سازه pET28-ASGV-CP بود. تعیین توالی سازه ساخته شده در دو جهت حاکی از قرار گرفتن ژن پروتئین پوششی در قاب صحیح درون پلاسمید بیان و فقدان جهش نوکلئوتیدی بود. پس از بهینه‌سازی، بیان و استخراج پروتئین پوششی، نوار پروتئینی با اندازه تقریبی 27 کیلودالتون چهار ساعت بعد از القاء در الکتروفورز عمودی و وسترن بلات تایید شد. در الایزی مستقیم، غیر مستقیم و آزمون دیبا، نتایج نشان داد غلظت حدود 1:1000 آنتی‌بادی تخلیص شده در غلظت مناسب آنتی‌ ژن توانایی واکنش مناسب با آنتی‌ژن را دارد.
نتیجه گیری: آنتی بادی تولید شده و کانژوگه شده در رقت مناسب در تناسب با غلظت آنتی ژن توانایی اختصاصیت و کارایی لازم برای ردیابی و شناسایی ویروس ساقه شیاری سیب را بخوبی دارد. یکی از کاربردهای این آنتی بادی در طراحی انواع کیت های تشخیصی سرولوژیکی مانند الایزا است.

کلیدواژه‌ها

عنوان مقاله [English]

Designing and evaluation of ELISA kit based on preparing antibodies against Apple stem grooving virus coat protein gene expressed in Escherichia coli

نویسندگان [English]

  • Mahsa Mohammadloo 1
  • Davoud Koolivand 2
  • Mohammad Hajizadeh 3
  • Omid Eini 2
1 Master Graduated in Plant Pathology, Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran
2 Associate Professor, Department of Plant Protection, Faculty of Agriculture, University of Zanjan, Zanjan, Iran,
3 Associate Professor, Department of Plant Protection, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran
چکیده [English]

Goal: production of antibodies against the coat protein of Apple stem grooving virus and their application in diagnostic assays and biosensor design represents a crucial strategy for effective plant virus detection. ASGV is a significant pathogen affecting pome fruit trees. The objective of this study was to express the coat protein gene of ASGV in E. coli, generate antibodies against the expressed protein, and assess their effectiveness in diagnostic assays.
Methods: In the PCR, we successfully amplified a 714 bp fragment corresponding to the complete coding region of the ASGV coat protein gene. The amplified fragment was then cloned into the pTG19 vector. Subsequently, the desired fragment was subcloned into the pET28a (+) expression vector using BamHI and EcoRI restriction enzymes. The resulting construct was then introduced into E. coli BL21(DE3) cells for gene expression. To determine the optimal conditions for protein expression, the transformed cells were induced with one mM IPTG for various durations (3, 4, 6, and 16 hours). The expression of the protein was confirmed through SDS-PAGE electrophoresis and western blot analysis. Following the purification of the expressed protein, it was utilized as an antigen for immunizing rabbits. Immunoglobulins (IgGs) were subsequently purified from the rabbit serum, and some of them were employed for conjugate production. The efficacy of the conjugates was evaluated in serological assays.
Results: Sequencing analysis of the amplified fragments conclusively verified their correspondence to the ASGV coat protein gene. Moreover, PCR and enzymatic digestion of the resulting clone provided further evidence of the successful construction of pET28-ASGV-CP. Comprehensive sequence analysis of the constructed plasmid in both directions confirmed the accurate insertion of the coat protein gene into the expression vector, while also confirming the absence of any nucleotide mutations. Following optimization, the expression of the ASGV coat protein was successfully achieved, resulting in an approximate size of 27 kDa. This confirmation was attained through SDS-PAGE electrophoresis and subsequent western blotting, conducted four hours after induction. The direct, indirect, and dot-blot assays were performed to assess the efficiency of the conjugate. The results indicated that the optimal concentration of the conjugate was approximately 1:1000.
Conclusion: The generated and conjugated antibodies exhibit suitable titers relative to the antigen concentration and demonstrate specificity and effectiveness in the detection and identification of Apple stem grooving virus (ASGV). These antibodies can be utilized for various serological diagnostic kits, including ELISA, offering valuable applications in virus detection and identification.

کلیدواژه‌ها [English]

  • Antibody
  • Gene Expression
  • Capillovirus
  • Western Blot
  • Apple stem grooving virus

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مجله بیوتکنولوژی کشاورزی
دوره 15، شماره 3
مهر 1402
صفحه 1-24

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