بیان پروتئین پوششی ویروس ایرانی پیچیدگی بوته چغندرقند در باکتری Escherichia coli

نوع مقاله: علمی-پژوهشی

نویسندگان

1 دانش‌آموخته دوره دکتری دانشگاه شیراز

2 استاد دانشگاه شیراز

3 استادیار، دانشگاه شهید چمران اهواز

10.22055/ppr.2019.14753

چکیده

بیماری پیچیدگی بوته چغندرقند در ایران با دو گونه ویروس ایرانی پیچیدگی بوته چغندرقند (Beet curly top Iran virus, BCTIV) و ویروس پیچیدگی بوته چغندرقند (BCTVBeet curly top virus,)ایجاد می­ شود. به منظور بیان پروتئین پوششی BCTIV، قطعه کامل چارچوب خوانش V1 اینویروس، با تعبیه محل­ های برشی دو آنزیم Bam HIو Hind III به ترتیب در انتهای ¢5 آغازگرهای اختصاصی رو به جلو و معکوس چارچوب خوانش V1، تکثیر شد. قطعه 751 جفت بازی محصول واکنش زنجیره­ای پلی­مراز، خالص شده و در ناقل همسانه­ سازی pTZ57R/T جای داده شد. توالی چارچوب خوانش V1 با واکنش هضم آنزیمی از ناقل همسانه­ سازی جدا و در ناقل بیان باکتریایی pQE30 همراه با یک دنباله هیستدینی در انتهای آمینی، جای داده شد. پلاسمیدهای نوترکیب حاوی توالی 6×His-V1، با استفاده از روش شوک الکتریکی به سلول­ های سویه M15 باکتری Escherichiacoli انتقال داده شدند. القای بیان توالی چارچوب خوانش V1 با استفاده از افزودن ایزوپروپیلتیو-دی-گالاکتوزیدبه محیط کشت LB مایع حاوی سلول­ های تراریخت باکتری صورت پذیرفت. الکتروفورز عمودی در ژل پلی­آکریل­آمید حاوی SDS، نشان­ دهنده وجود یک باند پروتئینی قوی به اندازه تقریبی 30 کیلودالتون بود که با وزن مولکولی مورد انتظار برای پروتئین پوششی BCTIV همخوانی داشت. آزمون لکه‌گذاری پروتئین با استفاده از آنتی­بادی آنتی­-هیستدین، صحت پروتئین بیان شده را به اثبات رساند. با توجه به میزان بالای خسارت حاصل از این ویروس­ ها در کشت چغندرقند، استفاده از پروتئین پوششی بیان شده BCTIVدر تولید آنتی­بادی­ اختصاصی می­ تواند روشی سریع، ارزان و ساده را برای شناسایی ویروس­ های پیچیدگی بوته چغندرقند در آزمون‌های سرولوژیکی از جمله الیزا فراهم آورد.

کلیدواژه‌ها


عنوان مقاله [English]

Bacterial expression of Beet curly top Iran virus coat protein in Escherichia coli

نویسندگان [English]

  • A. Anabestani 1
  • S. A. A. Bhejatnia 2
  • S. Tabein 3
  • K. Izadpanah 2
1 Ph.D. of Plant Pathology, Shiraz Univeristy
2 Professor, Shiraz University
3 Assistant Professor, Shahid Chamran University of Ahvaz
چکیده [English]

Background and Objectives
Beet curly top Iran virus (BCTIV) and Beet curly top virus (BCTV) are the causal agents of beet curly top disease in Iran. It has been reported that both viruses are distributed in different geographical regions and host plants in Iran. Development of a simple, fast and low cost serological method such as ELISA for detection of these viruses, is a necessary demand. To do serological tests such as ELISA, an antibody is needed. Production of polyclonal antibodies against geminiviruses is a hard candidate because of low concentration and localization of their particles in the phloem tissue of their hosts. Therefore, expression and production of geminiviral recombinant proteins in bacterial system is an alternative method to obtain polyclonal antibodies.
Material and Methods
Specific forward and reverse primers containing BamHI/HindIII digestion sites at the 5′ end of each primer, respectively, were designed to amplify the full-length fragment of BCTIV V1 open reading frame encoding the viral coat protein (CP). The amplified 751 bp fragment was cloned into the pTZ57R/T cloning vector. V1 gene was released from pTZ57R/T by enzymatic digestion and cloned into the expression vector pQE30. The recombinant pQE30-BCTIV V1 was transformed into Escherichia coli strain M15. The expression of protein was induced by adding 1 mM of isopropylthio-d-galactoside (IPTG) to the transformed bacterial cells and grown at 37 ºC in liquid LB medium. Expression of recombinant protein was analyzed by SDS-PAGE electrophoresis and Western blot hybridization.
Results
Analysis of the expressed proteins by SDS-PAGE electrophoresis, revealed a protein band with a position corresponding to molecular weight of approximately 30 kDa that was consistent with the predicted molecular weight of BCTIV CP.Time course of induction by IPTG influenced on level of the new recombinant protein expression, thus four hours induction had the highest level of expression. Western blot analysis confirmed the identity of expressed protein via using anti-His antibody to detect expressed recombinant His-BCTIV CP.
 
 
Discussion
One of the most popular bacterial protein expression system is IPTG inducible system wherein IPTG regulate level of new recombinant protein expression. Our results by SDS-PAGE electrophoresis and Western blot analysis revealed a high level of expression of the fusion protein in IPTG-induced bacterial cells. However, a negligible rate of expression of recombinant protein was also observed in non-induced bacterial cells. This suggests low appearance and activity of responsible promotor even without inducing protein expression with IPTG. Expression of conserved plant viral proteins in bacterial systems following purification of the resulted recombinant proteins would facilitate production of specific polyclonal antibodies.

کلیدواژه‌ها [English]

  • Beet curly top virus
  • Beet curly top Iran virus
  • Coat protein
  • Protein expression

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