fa بیان کاست ژنی SO6-STxB در باکتری اشرشیا کلی و بررسی تیترآنتی‌بادی بیولوژی (زیست شناسی) Biology پژوهشي Research <strong><span style="color:#0070C0;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">زمینه و هدف: </span></span></span></strong><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">یکی از راه&shy;های تقویت اثر واکسن&shy;ها استفاده از یاور&shy;ها می&shy;باشد. </span></span></span><em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">STxB</span></span></span></span></em> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">باکتری <em>شیگلا</em> دارای نقش یاوری و حاملی بوده و می&shy;توان با ترکیب کردن آنتی&shy;ژن&shy;های کاندیدای واکسن با این یاور به تولید واکسن مناسب پرداخت. غاسول</span> صابونی گیاهی</span> دارای فعالیت</span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> ان-گلیکوزیداز</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> می</span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">&shy;</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">باشد. ایزوفرم 6 این گیاه </span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">(</span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SO6</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">)</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">، آدنین </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">4324</span></span></span></span> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">را در توالی حفاظت شده </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">GAGA</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> در </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">28SrRNA</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> پورین&shy;زدایی کرده و سنتز پروتئین را مختل می&shy;کند. هدف این مطالعه</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">،</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> بیان</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> ژن</span></span></span> <em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SO6</span></span></span></span></em><em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">-STxB</span></span></span></span></em> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">در باکتری</span></span></span> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">اشرشیا کلی</span></span></span> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">و بررسی تیتر آنتی‌بادی آن در موش می&shy;باشد.</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"></span></span></span><br> <strong><span style="color:#0070C0;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">روش کار:</span></span></span></strong> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">در این مطالعه تجربی ژن </span></span></span><em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SO6</span></span></span></span></em><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> با جایگاه&shy;های آنزیمی </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">BamHI</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> و</span></span></span> <span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SalI</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> از پلاسمید </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">pUC57</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> جداسازی گردید و در وکتور بیانی </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">pET28a(+)</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> حاوی</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> ژن</span></span></span> <em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">STxB</span></span></span></span></em><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> زیرهمسانه&shy;سازی و به باکتری</span></span></span> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">اشرشیا کلی </span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">سویه </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">BL21(DE3)</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> تراریخت شد. بیان پروتئین نوترکیب </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SO6</span></span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">-STxB</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> توسط </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">IPTG</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> القا و تخلیص به وسیله کروماتوگرافی تمایلی ستون نیکل انجام گردید. تأیید پروتئین نوترکیب</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> با استفاده از وسترن بلات</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> انجام گرفت. موش</span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">&shy;</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">‌ها به صورت صفاقی با پروتئین تخلیص شده واکسینه شدند و تیتر </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">IgG</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> سرم به روش </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">ELISA</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> مورد سنجش قرار گرفت.</span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;"></span></span></span></span><br> <strong><span style="color:#0070C0;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">یافته&shy;ها:</span></span></span></strong><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> زیرهمسانه&shy;سازی ژن </span></span></span><em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">STxB</span></span></span></span></em><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">-</span></span></span><em><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SO6</span></span></span></span></em> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">در&shy;&shy; وکتور بیانی </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">pET28a(+)</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> به‌وسیله </span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">واکنش </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">PCR</span></span></span></span> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">و هضم آنزیمی، تائید شد. </span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">پروتئین نوترکیب 5/37 کیلودالتونی تولید شده به وسیله </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SDS-PAGE</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> و لکه&shy;گذاری وسترن تأیید شد. سپس آنتی&shy;بادی تولید شده از سرم موش جداسازی و توسط الایزا اندازه‌گیری گردید.</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"></span></span></span><br> <strong><span style="color:#0070C0;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">نتیجه&shy;گیری:</span></span></span></strong><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;"> آنتی&shy;ژن </span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">STxB</span></span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">-</span></span></span><span dir="LTR"><span style="color:black;"><span style="font-family:times new roman,serif;"><span style="font-size:10.0pt;">SO6</span></span></span></span> <span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">نوترکیب تخلیص شده می&shy;تواند برای تحقیقات ضد سرطانی و کاندید واکسن استفاده شود</span></span></span><span style="color:black;"><span style="font-family:b mitra;"><span style="font-size:10.0pt;">.</span></span></span> <strong>Background:</strong> One of the ways to strengthen the effect of vaccines is the use of adjuvant. STxB has a carrier role and can act as an adjuvant; thus it can be fused with vaccine candidate antigens in order to produce efficient vaccines. <em>Saponaria officinalis</em> is a plant that has shown N-glycosidase activity. <em>SO6</em> isoform of this plant, depurinates the adenine 4324 in the conserved sequence GAGA in 28SrRNA and disrupts protein synthesis. The aim of this study was expression of <em>SO6-STxB</em> gene in <em>Escherichia coli</em> (<em>E. coli)</em> and investigation of antibody titer in mice.<span dir="RTL"></span><br> <strong>Methods:</strong> In this study <em>SO6</em> gene with <em>Bam</em>HI and S<em>al</em>I restriction enzyme sites were isolated from pUC57 plasmid and subcloned into pET28a (+) -<em>STxB</em> expression vector and transferred to <em>E. coli</em> BL21 (DE3). Expression of<em> SO6-STxB</em> gene cassette was induced by IPTG and purified by nickel affinity chromatography. The recombinant protein was confirmed by western blotting. Mice were immunized intraperitoneally with purified protein and serum IgG titers were measured by ELISA.<span dir="RTL"></span><br> <strong>Results:</strong> Subcloning of <em>SO6&ndash;STxB</em> gene in pET28a (+) expression vector was confirmed by PCR and enzyme digestion reaction. A 37/5 kDa recombinant protein was confirmed by SDS-PAGE and western blotting. The antibody generated from mouse serum was isolated and confirmed by ELISA<span dir="RTL">.</span><br> <strong>Conclusion: </strong>Purified recombinant antigen STxB-SO6 can be used for research and be a suitable anti-cancer vaccine candidates.<br> &nbsp; STxB,شیگلا,غاسول صابونی,فعالیت N-glycosidase, SO6 STxB,Sigella,Saponaria Officinalis, N-glycosidase activity, SO6 52 60 http://rjms.iums.ac.ir/browse.php?a_code=A-10-3913-1&slc_lang=fa&sid=1 Masoud abdollahi مسعود عبدالهی ma.abdollahi102@yahoo.com 3900319475328460088788 3900319475328460088788 No Department of Biology, Biology Research Center, Imam Hosein University, Tehran گروه و مرکز تحقیقات زیست شناسی، دانشگاه جامع امام حسین (ع)، تهران، ایران Hossien Honari حسین هنری honari.hosein@gmail.com 3900319475328460088789 3900319475328460088789 Yes Imam Hosein University, Tehran Iran دانشگاه جامع امام حسین (ع)، تهران، ایران Seyed Masih A'temad Aubi سید مسیح اعتمادایوبی masih.etemadi@gmail.com 3900319475328460088790 3900319475328460088790 No Department of Biology, Biology Research Center, Imam Hosein University, Tehran گروه و مرکز تحقیقات زیست شناسی، دانشگاه جامع امام حسین (ع)، تهران، ایران