fa بیان ژن کد کننده‌ی آنتی ژن BAB1-0278 باکتری بروسلا آبورتوس در باکتری پروبیوتیک لاکتوکوکوس لاکتیس پزشکی مولکولی و ژنتیک Molecular Medicine and Genetics پژوهشي اصیل Original Research <span style="font-size:11pt"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><b><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">زمینه و هدف: </span></span></b><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">آنتی‌ژن </span></span>&nbsp;<i><span dir="LTR" new="" roman="" style="font-family:" times=""> BAB1-0278</span></i><i><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">بروسلا آبورتوس </span></span></i><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">در ایجاد بیماری بروسلوز نقش دارد. این&nbsp; آنتی‌ژن،پروتئینی به نام </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">GcrA</span><span style="font-size:12.0pt"><span b="" style="font-family:" zar=""> را رمزگذاری می‌کند که نقش اساسی در رونویسی ژن‌های هدف در چرخه سلولی دارد. از این رو </span></span><i><span dir="LTR" new="" roman="" style="font-family:" times="">BAB1-0278</span></i><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> دارای پتانسیل‌های منحصر به فردی در استراتژی‌های درمانی علیه <i>بروسلا آبورتوس</i> می‌باشد.</span></span> <span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">هدف از مطالعه حاضر تولید باکتری <i>لاکتوکوکوس لاکتیس</i> مهندسی شده بیان کننده پروتئین </span></span><i><span dir="LTR" new="" roman="" style="font-family:" times="">BAB1-0278</span></i><span style="font-size:12.0pt"><span b="" style="font-family:" zar=""> <i>بروسلا آبورتوس</i> می‌باشد.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><b><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">مواد و روش‌ها:</span></span></b><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> طراحی سازواره ژنی حاوی یک توالی سیگنال پپتیدی درون ناقل بیانی مبتنی بر القا با نیسین </span></span><span b="" lang="FA" style="font-family:" zar="">(</span><span dir="LTR" new="" roman="" style="font-family:" times="">pN3z8148-Usp45-<i>BAB1-0278</i></span><span b="" lang="FA" style="font-family:" zar="">)</span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> صورت گرفت و سنتز آن</span></span> <span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">توسط شرکت </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">GENEray</span> <span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">انجام شد. پلاسمید نوترکیب در باکتری <i>اشریشیا کلی</i> </span></span>&nbsp;<span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">ترانسفورم شد.</span></span> <span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">ﺍﺳﺘﺨﺮﺍﺝ ﭘﻼﺳﻤﻴﺪ نوترکیب از باکتری‌های ترانسفورم شده صورت گرفت.<i> لاکتوکوکوس لاکتیس</i> توسط الکتروپوریشن هم با پلاسمید نوترکیب حاوی ژن هدف </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">pNZ8148-Usp45-<i>BAB1-0278</i></span> <span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">و هم با پلاسمید فاقد ژن هدف ترانسفورم شد. تایید بیان</span></span> <i><span dir="LTR" new="" roman="" style="font-family:" times="">BAB1-0278</span></i> <span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">توسط تکنیک‌های واکنش زنجیره پلیمراز معکوس </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">RT-PCR)</span><span b="" lang="FA" style="font-family:" zar="">) </span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">و سدیم دودسیل سولفات پلی آکریل آمید الکتروفورز ژل</span></span> &nbsp;<span dir="LTR" new="" roman="" style="font-family:" times="">(SDS-PAGE)</span><span style="font-size:12.0pt"><span b="" style="font-family:" zar=""> و وسترن بلات انجام شد.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><b><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">یافته‌ها:</span></span></b> &nbsp;<i><span dir="LTR" new="" roman="" style="font-family:" times="">BAB1-0278</span></i><span style="font-size:12.0pt"><span b="" style="font-family:" zar=""> <i>بروسلا آبورتوس</i> در <i>لاکتوکوکوس لاکتیس</i> بیان و به وسیله </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">RT-PCR</span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> مورد تایید قرار گرفت. تجزیه و تحلیل </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">SDS-PAGE</span><span style="font-size:12.0pt"><span b="" style="font-family:" zar=""> پروتئین‌های جدا شده از <i>لاکتوکوکوس لاکتیس</i> ترانسفورم شده با پلاسمید نوترکیب در مقایسه با</span></span> <i><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">لاکتوکوکوس لاکتیس</span></span></i><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> ترانسفورم نشده پروتئینی با وزن مولکولی </span></span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">۱۳</span></span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> کیلو دالتون </span></span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">را</span></span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">نشان داد.</span></span><span dir="LTR" style="font-size:12.0pt"><span style="font-family:"Times New Roman",serif"></span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><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="">نتایج </span></span><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="">، هضم آنزیم محدود کننده و تعیین توالی توسط شرکت </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">GENEray</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">نشان داد که </span></span><i><span dir="LTR" new="" roman="" style="font-family:" times="">BAB1-0278</span></i> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">به درستی در </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">pNZ8148-Usp45</span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">کلون شده است. </span></span><span dir="LTR" new="" roman="" style="font-family:" times="">SDS-PAGE</span><span b="" style="font-family:" zar=""> و </span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">وسترن بلات</span></span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> برای</span></span><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> آنالیز بیان پروتئین </span></span><i><span dir="LTR" new="" roman="" style="font-family:" times="">BAB1-0278</span></i><i><span lang="AR-SA" new="" roman="" style="font-family:" times=""> استفاده</span></i><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar=""> شد.</span></span> <i><span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">لاکتوکوکوس لاکتیس</span></span></i> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">ترانسفورم شده می‌تواند گام مهمی جهت </span></span><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">تحقیقات</span></span> <span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">واکسن خوراکی علیه <i>بروسلا آبورتو</i></span></span><i><span lang="FA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">س</span></span></i> &nbsp;<span lang="AR-SA" style="font-size:12.0pt"><span b="" style="font-family:" zar="">باشد. </span></span><span lang="AR-SA" style="font-family:"Arial",sans-serif"></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span calibri="" style="font-family:"><span arial="" style="font-family:"><span style="color:black"></span></span></span></span></span></span></span><br> &nbsp; <span style="font-size:11pt"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">Background and Aim: </span></span></b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">The <i>BAB1-0278</i> has an essential role in </span></span><i><span style="font-size:12.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times=""><span style="color:black">abortus</span></span></span></span></i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""> infection. This antigen encodes a protein called GcrA, which plays an essential role in the transcription of target genes in the cell cycle. Thus, <i>BAB1-0278</i> has unique potential in therapeutic strategies against </span></span><i><span style="font-size:12.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times=""><span style="color:black">B. abortus</span></span></span></span></i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">. The purpose of this research is to produce engineered <i>Lactococcus lactis</i> bacteria expressing the <i>BAB1-0278</i> protein of </span></span><i><span style="font-size:12.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times=""><span style="color:black">B. abortus</span></span></span></span></i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">.<b><i></i></b></span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">Materials and Methods:</span></span></b> <span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">The gene construction design contains a signal peptide sequence in the nisin-based expression vector (pN3z8148-Usp45-<i>BAB1-0278</i>) and is synthesized by GENEray. </span></span><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">The recombinant plasmid transformed into <i>Escherichia coli </i>bacteria. The recombinant plasmid was extracted from transformed bacteria.</span></span><em><span style="font-size:12.0pt"><span style="background:white"><span style="color:black"> L. lactis</span></span></span></em><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""> transformed by electroporation both with the recombinant plasmid containing the target gene pNZ8148-Usp45-<i>BAB1-0278</i> and the plasmid without the target gene. <i>BAB1-0278</i> expression was confirmed by reverse polymerase chain reaction (RT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot techniques.</span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">Results:</span></span></b> <i><span style="font-size:12.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times=""><span style="color:black">B. abortus</span></span></span></span></i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""> BAB1-0278 was expressed in </span></span><em><span style="font-size:12.0pt"><span style="background:white"><span style="color:black">L. lactis</span></span></span></em><b> </b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">and confirmed by RT-PCR. SDS-PAGE analysis of proteins isolated from </span></span><em><span style="font-size:12.0pt"><span style="background:white"><span style="color:black">L. lactis</span></span></span></em><b> </b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">transformed with recombinant plasmid, compared to untransformed </span></span><em><span style="font-size:12.0pt"><span style="background:white"><span style="color:black">L. lactis</span></span></span></em><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">, showed a protein with a molecular weight of 13 kDa.</span></span></span></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span calibri="" style="font-family:"><b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">Conclusion:</span></span></b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times=""> The results of PCR, restricted</span></span> <span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">enzyme digestion, and sequencing by GENEray revealed that <i>BAB1-0278</i> was cloned correctly in pNZ8148-Usp45. SDS-PAGE and Western blot were used to analyze <i>BAB1-0278</i> protein expression. Transformed </span></span><em><span style="font-size:12.0pt"><span style="background:white"><span style="color:black">L. lactis</span></span></span></em><b> </b><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">can be a crucial step for oral vaccine research against </span></span><i><span style="font-size:12.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times=""><span style="color:black">B. abortus</span></span></span></span></i><span style="font-size:12.0pt"><span new="" roman="" style="font-family:" times="">.</span></span></span></span></span><br> <br> &nbsp; بروسلا آبورتوس, BAB1-0278, لاکتوکوکوس لاکتیس, واکسن, پروبیوتیک, الکتروپوریشن Brucella abortus, BAB1-0278, Lactococcus lactis, Vaccine, Probiotic, Electroporation 1 13 http://sjku.muk.ac.ir/browse.php?a_code=A-10-7193-1&slc_lang=fa&sid=1 donya kazemi دنیا کاظمی Donyaakazemii@yahoo.com 5400319475328460070319 5400319475328460070319 No Ph.D. Candidate, Dept. of Biology, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran دانشجوی دکتری. گروه زیست شناسی، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران abbas doosti عباس دوستی abbasdoosti@yahoo.com 5400319475328460070320 5400319475328460070320 Yes Professor, Department of Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran استاد، مرکز تحقیقات بیوتکنولوژی، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران mostafa Shakhsi niaee مصطفی شخصی نیایی niaee_m@yahoo.com 5400319475328460070321 5400319475328460070321 No Assistant Professor, Department of Biotechnology Research Center, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran استادیار، گروه زیست شناسی، واحد شهرکرد، دانشگاه آزاد اسلامی، شهرکرد، ایران