fa همسانه سازی، بیان و تخلیص بخش خارج غشایی پروتئین Tir از باکتری E.coli O157:H7 و ارزیابی ساختاری آن ژنتیک Genetics مقاله پژوهشی Research Article <span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">سابقه و هدف</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">باکتری اشرشیاکلی </span></span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">انتروهموراژیک </span></span><span b="" style="font-family:" zar="">(</span><span dir="LTR" new="" roman="" style="font-family:" times="">Enterohemorrhagic <i>E</i>.&nbsp;<i>coli</i></span><span b="" style="font-family:" zar="">)</span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> سویه&shy;ی</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">O157:H7</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">یک پاتوژن مهم انسان و حیوان است که در انسان موجب بروز اسهال خونریزی دهنده و سندروم اورمی همولیتیک می شود. اتصال به سلول&shy;های میزبان اولین گام در تثبیت این باکتری است که به واسطه&shy;ی سیستم ترشحی نوع</span></span> &nbsp;<span dir="LTR" new="" roman="" style="font-family:" times="">(T3SS)</span> <span dir="LTR" new="" roman="" style="font-family:" times="">III</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">و از طریق میان&shy;کنش بین پروتئین&shy;های ترشحی صورت می&shy;گیرد.</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">پروتئینی است که پس از بیان در باکتری و از طریق</span></span>&nbsp; <span dir="LTR" new="" roman="" style="font-family:" times="">T3SS</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">به سلول میزبان منتقل و در غشای آن مستقر می شود و نقش مهمی در اتصال باکتری به میزبان دارد. هدف از این تحقیق ساخت سازه ژنی است که دارای فاکتورهای ویرولانس</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">می</span></span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">&shy;</span></span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">باشد. </span></span><span lang="FA" style="font-size:12.0pt"><span style="font-family:"B Zar""></span></span></span></span></span><br> <span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">مواد و روش&shy; ها:</span></span></b> <b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">:</span></span></b><b> </b>&nbsp;<span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">طراحی پرایمر اختصاصی برای ژن</span></span> <i><span dir="LTR" new="" roman="" style="font-family:" times="">tir</span></i> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">با استفاده از نرم افراز</span></span><span dir="LTR" new="" roman="" style="font-family:" times="">Oligo</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">&nbsp;انجام شد و تکثیر ژن به وسیله واکنش</span></span>&nbsp; &nbsp;&nbsp;<span dir="LTR" new="" roman="" style="font-family:" times="">PCR</span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">از روی</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">DNA</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">الگو صورت گرفت. پس از واکنش&shy;های هضم آنزیمی و الحاق ژن درون ناقل بیانی</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">pET28a</span>&nbsp; <span b="" style="font-family:" zar="">، </span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">انتقال ناقل به درون میزبان بیانی </span></span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">انجام شد. پس از تایید همسان&shy;سازی ژن، بیان نوترکیب پروتئین </span></span>&nbsp;&nbsp;<span dir="LTR" new="" roman="" style="font-family:" times="">Tir</span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">با استفاده از القاگر</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">IPTG</span> &nbsp;<span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">صورت گرفت. برای تایید پروتئین</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir </span>&nbsp;<span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">آنالیز وسترن بلات انجام شد.</span></span></span></span></span><br> <span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">&nbsp;یافته‌ها:</span></span></b> <b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">:</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">همسان سازی ژن</span></span> <i><span dir="LTR" new="" roman="" style="font-family:" times="">tir</span></i> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">درون وکتور</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">pET28a</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">به شکل مناسب بین جایگاه&shy;های مطلوب انجام شد و پروتئین</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir </span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">پس از القا، پروتئین نوترکیب بیان &nbsp;مناسب و قابل توجهی نشان داد. آنتی بادی مورد استفاده در وسترن بلات نیز توانست به شکل مناسبی</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir </span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">را شناسایی کند.</span></span></span></span></span><br> <span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">نتیجه‌گیری:</span></span></b> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">در این مطالعه، سازه پایدار محتوی ژن</span></span> <i><span dir="LTR" new="" roman="" style="font-family:" times="">tir</span></i> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">ساخته شد که بیان نوترکیب قابل توجهی از پروتئین</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir </span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">را از خود نشان داد. بنابراین این سازه را می توان برای تولید پروتئین</span></span> <span dir="LTR" new="" roman="" style="font-family:" times="">Tir </span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">جهت مطالعات ایمنی زایی علیه</span></span> <i><span dir="LTR" new="" roman="" style="font-family:" times="">E</span></i><span dir="LTR" new="" roman="" style="font-family:" times="">.<i>coli</i> O157:H7</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">&nbsp;به کاربرد</span></span><span dir="LTR" style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">.</span></span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">.</span></span></span></span></span><br> &nbsp; <span style="line-height:normal"><b><span new="" roman="" style="font-family:" times="">Aim and Background :</span></b> <span new="" roman="" style="font-family:" times="">Enterohemorrhagic <i>E. coli </i>O157: H7 is an important human and animal pathogen that causes hemolytic uremic hemorrhagic diarrhea in humans. The binding to host cells is the first step in stabilizing this bacterium through the secretion system of the type (T3SS) III and through the interactions between secretory proteins. Tir is a protein that is transferred to the host cells after expression in the bacterium and via T3SS and is located in the membrane and plays an important role in the bacterial binding to the host. The purpose of this research is to clone a gene construct that has Tir virulence factors.</span></span><br> <span style="line-height:normal"><b><span new="" roman="" style="font-family:" times="">Materials and Methods:<i> </i></span></b><span new="" roman="" style="font-family:" times="">Specific primer design for <i>tir</i> gene was performed using oligo softwares and gene amplification was performed by PCR reaction on a DNA template.</span> <span new="" roman="" style="font-family:" times="">After the enzyme digestion, the gene was inserted into expression vector pET28a; the construct was transformed to the expression host. After confirmation of gene cloning, the Tir recombinant protein was expressed by induced IPTG induction. The western blot analysis was carried out to confirm Tir protein.</span> <span new="" roman="" style="font-family:" times="">The gene of <i>tir</i> was cloned inside pET28a vector.</span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span new="" roman="" style="font-family:" times="">Results:<i> </i></span></b><span new="" roman="" style="font-family:" times="">Cloning of tir gene in pET28a vector was performed appropriately between desired sites and after induction protein, the Tir recombinant protein showed appropriate and significant expression. The antibody used in Western blot was also able to properly detect Tir.</span><b><span new="" roman="" style="font-family:" times="">Conclusion: </span></b><span new="" roman="" style="font-family:" times="">After the induction, the protein of Tir showed a remarkable expression of the recombinant protein.</span> <span new="" roman="" style="font-family:" times="">As a result,</span> <span new="" roman="" style="font-family:" times="">this structure can be used to produce Tir protein for immunological studies against <i>E.coli</i> O157: H7.</span><span style="font-family:"Times New Roman","serif""></span></span></span></span><br> &nbsp; اشرشیاکلی O157:H7, پروتئین نوترکیب, همسان‌سازی, Tir, . Iau Science E.coli O157: H7, Recombinant protein, Cloning, Tir, Iau Science. 25 34 http://ncmbjpiau.ir/browse.php?a_code=A-10-1456-2&slc_lang=fa&sid=1 Shilla Phalsaphi شیلا فلسفی 100319475328460012824 100319475328460012824 No Department of Biochemistry, Pharmaceutical Sciences Branch, Faculty of New Technologies, Islamic Azad University, Tehran, Iran. گروه بیوشیمی ، دانشکده فناوری های نوین، واحد علوم دارویی ، دانشگاه آزاد اسلامی، تهران، ایران Jafar Amani جعفر امانی jafar.amani@gmail.com 100319475328460012825 100319475328460012825 No Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran انستیتو سیستم بیولوژی و مسمومیت ها، مرکز تحقیقات میکروبیولوژی کاربردی، دانشگاه علوم پزشکی بقیه الله (عج)، تهران، ایران Seyed Ali Mirhosseini سیدعلی میرحسینی ali.mirh@gmail.com 100319475328460012826 100319475328460012826 Yes Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran انستیتو سیستم بیولوژی و مسمومیت ها، مرکز تحقیقات میکروبیولوژی کاربردی، دانشگاه علوم پزشکی بقیه الله (عج)، تهران، ایران