fa بیان ژن leoA هلیکوباکترپیلوری در سلول‌های تخمدان همستر چینی به روش RT-PCR ژنتیک Genetic تحقيقي Original Articles <h1 dir="RTL" style="margin-top:0cm;margin-right:14.15pt;margin-bottom:0cm;margin-left:14.2pt;margin-bottom:.0001pt;page-break-after:auto;"><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;">زمینه و هدف : عفونت هلیکوباکترپیلوری یکی از شایع‌ترین عفونت‌های مزمن باکتریایی در جهان به‌ویژه در کشورهای در حال توسعه است. ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> نقش مهمی در بیماری‌زایی ایفا می‌کند و نقش اصلی این ژن افزایش ترشح سم توسط باکتری است. این مطالعه به منظور جداسازی و کلون‌سازی ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> در وکتور بیانی </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pEGFP-C2</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> و بررسی بیان آن به روش </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">RT-PCR</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> در سیستم یوکاریوتی انجام شد.</span></span></span></span></h1> <h1 dir="RTL" style="margin-top:0cm;margin-right:14.15pt;margin-bottom:0cm;margin-left:14.2pt;margin-bottom:.0001pt;page-break-after:auto;"><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;">روش بررسی : در این مطالعه توصیفی آزمایشگاهی ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> از ژنوم هلیکوباکترپیلوری سویه استاندارد (</span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">ATCC 43504</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;">) به روش </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">PCR</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> تکثیر و جداسازی شد. سپس با روش کلون‌سازی </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">T/A</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> در ناقل </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pTZ</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> درج گردید. ساب کلونینگ این ژن در وکتور بیانی </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pEGFP-C2</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> با آنزیم لیگاز انجام شد. سپس سازواره نهایی </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pEGFP-C2-leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> به روش الکتروپوریشن در سلول‌های تخمدان همستر چینی</span></span></span></span><br> <span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">(Chinese hamster ovary: CHO)</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> ترانسفرم گردید و بیان ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> با روش </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">SDS-PAGE</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> و </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">RT-PCR</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> ارزیابی شد.</span></span></span></span></h1> <h1 dir="RTL" style="margin-top:0cm;margin-right:14.15pt;margin-bottom:0cm;margin-left:14.2pt;margin-bottom:.0001pt;page-break-after:auto;"><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;">یافته‌ها : نتایج </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">PCR</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> نشان‌دهنده تکثیر قطعه 1758 جفت بازی مربوط به ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> بود. کلون‌سازی این ژن به ترتیب در وکتورهای </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pTZ</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> و </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pEGFP-C2</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> با موفقیت انجام شد. هضم آنزیمی با دو آنزیم </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">KpnI</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> و</span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">SacII</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> و همچنین تعیین توالی، صحت کلون سازی ژن را تایید کرد. مشاهده محصول پروتئینی ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> در سلول‌های </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">CHO</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> نشان‌دهنده بیان موفقیت‌آمیز ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> هلیکوباکترپیلوری در سیستم یوکاریوتی بود.</span></span></span></span></h1> <h1 dir="RTL" style="margin-top:0cm;margin-right:14.15pt;margin-bottom:0cm;margin-left:14.2pt;margin-bottom:.0001pt;page-break-after:auto;"><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;">نتیجه‌گیری : سازواره نهایی </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">pEGFP-C2-leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> ، قدرت بیان موفق ژن </span></span></span></span><span dir="LTR" style="layout-grid-mode:line;"><span style="color:windowtext;"><span style="font-family:times new roman,serif;"><span style="font-size:8.0pt;">leoA</span></span></span></span><span style="layout-grid-mode:line;font-weight:normal;"><span style="color:windowtext;"><span style="font-family:tahoma,sans-serif;"><span style="font-size:10.0pt;"> در سلول‌های جانوری را داشت.</span></span></span></span></h1> <strong>Background and Objective: </strong><em>Helicobacter pylori</em> infection is one of the most common chronic bacterial infections all over the world, particularly in the developing countries. LeoA gene plays an important role in pathogenesis, and the main role of this gene is to increase the bacterial toxin secretion. This study was conducted to isolate and clone the leoA gene in a pEGFP-C2 expression vector and evaluate its expression in eukaryotic system.<br> <strong>Methods: </strong>In this laboratory study, the leoA gene was amplified from the standard strain of <em>Helicobacter pylori</em> genome (ATCC 43504) by PCR method. It was then inserted into the pTZ vector by cloning T/A. Sub cloning of this gene was performed in a pEGFP-C2 expression vector with a ligase enzyme. The final structure of pEGFP-C2-leoA was transformed by electroporation in CHO (Chinese hamster ovary) cells and the expression of the leoA gene was evaluated by SDS-PAGE and RT-PCR.<br> <strong>Results: </strong>The results of PCR indicated that the 1758 bp fragment was amplified from the leoA gene. Cloning of this gene was performed successfully in pTZ and pEGFP-C2 vectors, respectively. The enzyme digestion with two KpnI and SacII enzymes, as well as sequencing, confirmed the accuracy of gene cloning. The observation of the protein product of the leoA gene in CHO cells indicated the successful expression of the LeoA gene in the eukaryotic system of <em>Helicobacter pylori</em>.<br> <strong>Conclusion: </strong>The final construct of pEGFP-C2-leoA had a successful expression of the leoA gene in animal cells. هلیکوباکترپیلوری , ژن leoA , الکتروپوریشن , واکسن نوترکیب Helicobacter pylori, LeoA gene, Electroporation, Recombinant vaccine 92 99 http://goums.ac.ir/journal/browse.php?a_code=A-10-1-1006&slc_lang=fa&sid=1 Naghmeh Mirabolghasemi نغمه میرابوالقاسمی 5900319475328460049353 5900319475328460049353 No M.Sc in Genetics, Department of Biology, Faculty of Basic Science, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran کارشناس ارشد ژنتیک، گروه زیست شناسی، دانشکده علوم پایه، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران Abbas Doosti عباس دوستی abbasdoosti@yahoo.com 5900319475328460049354 5900319475328460049354 Yes Associate Professor, Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran دانشیار، مرکز تحقیقات بیوتکنولوژی، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران