fa همسانه‌سازی، بیان، تخلیص و بررسی فعالیت اندولیزین فاژ KP27 کلبسیلا پنومونیه در سیستم بیانی pET28a(+) سایر موارد Others... پژوهشي اصیل Original Research <span style="line-height:normal"><b><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:">زمینه و هدف:</span></span></b><span b="" lang="FA" mitra="" style="font-family:"> اندولیزین فاژ </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">KP27</span></span><span b="" lang="FA" mitra="" style="font-family:"> پروتئینی با جرم مولکولی 14/7 کیلو دالتون است که دارای فعالیت اندوپپتیدازی علیه دیواره سلولی باکتری&shy;ها می&shy;‌باشد. به‌دلیل وجود تحقیقات محدود روی اندولیزین </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">KP27</span></span><span b="" lang="FA" mitra="" style="font-family:"> و همچنین برتری آن در برابر شرایط محیطی سخت مثل دما و </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">pH</span></span><span b="" lang="FA" mitra="" style="font-family:"> هدف از این مطالعه تولید نوترکیب و بررسی فعالیت ضدباکتریایی آن علیه دو باکتری گرم منفی و مثبت <a name="_Hlk183591903">اشرشیاکلی و استافیلوکوک اورئوس </a>می‌&shy;باشد.</span></span><br> <span style="line-height:normal"><b><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:">روش‌ها: </span></span></b><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:">ژن اندولیزین </span></span><span dir="LTR" style="font-size:9.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times="">KP27</span></span></span><span style="background:white"><span b="" mitra="" style="font-family:"> بین دو آنزیم محدود&shy;کننده </span></span><i><span dir="LTR" style="font-size:9.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times="">NcoI</span></span></span></i><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:"> و </span></span><i><span dir="LTR" style="font-size:9.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times="">XhoI</span></span></span></i><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:"> در پلاسمید </span></span><i><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">pET-28a(+)</span></span></i><span b="" mitra="" style="font-family:"> همسانه&shy;سازی شد. تولید در باکتری </span><i><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">E. coli BL21 (DE3)</span></span></i><i> </i><span b="" lang="FA" mitra="" style="font-family:">و تخلیص توسط کروماتوگرافی </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">Ni²⁺-NTA</span></span><span b="" lang="FA" mitra="" style="font-family:"> سفاروز انجام شد. جهت بررسی فعالیت ضدباکتری اندولیزین از تست&shy;های فعالیت باکتری&shy;کشی، </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">MIC</span></span><span b="" lang="FA" mitra="" style="font-family:">، </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">MBC</span></span><span b="" lang="FA" mitra="" style="font-family:"> و فعالیت آنزیمی از تخریب دیواره سلولی استفاده شد. آنالیز آماری و رسم نمودارها توسط نرم&shy;افزار گراف پد پریسم انجام شد.<b><span style="background:white"></span></b></span></span><br> <span style="font-size:11pt"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:">یافته‌ها: </span></span></b><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:">اندولیزین </span></span><span dir="LTR" style="font-size:9.0pt"><span style="background:white"><span new="" roman="" style="font-family:" times="">KP27</span></span></span><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:"> با موفقیت </span></span><span b="" lang="FA" mitra="" style="font-family:">همسانه&shy;سازی <span style="background:white">و با راندمان بالا به مقدار </span>28<span style="background:white">/5 میلی‌گرم/لیتر محیط کشت تولید شد. میزان کشندگی آن </span>علیه باکتری&shy;های اشرشیاکلی و استافیلوکوک اورئوس در غلظت&shy;های بالاتر از 4 ماکروگرم/لیتر بیشتر از 50 درصد بود. میزان </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">MIC</span></span><span b="" lang="FA" mitra="" style="font-family:"> و </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">MBC</span></span><span b="" lang="FA" mitra="" style="font-family:"> آن به ترتیب </span>&nbsp;<span b="" lang="FA" mitra="" style="font-family:">16 و 64 ماکروگرم/لیتر برای اشرشیاکلی و 8 و 32 ماکروگرم/لیتر برای استافیلوکوک اورئوس به دست آمد. بررسی فعالیت تخریب دیواره سلولی اندولیزین حاکی از کاهش ۲۰ درصد کدورت محلول پس از نیم ساعت مجاورت با اندولیزین بود.&nbsp;</span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span lang="FA" style="background:white"><span b="" mitra="" style="font-family:">نتیجه‌گیری: </span></span></b><span b="" lang="FA" mitra="" style="font-family:">باتوجه‌به مقدار بیان و تولید بالای اندولیزین </span><span dir="LTR" style="font-size:9.0pt"><span new="" roman="" style="font-family:" times="">KP27</span></span><span b="" lang="FA" mitra="" style="font-family:"> و همچنین فعالیت بالای آن در برابر دو باکتری گرم منفی و گرم مثبت مورد مطالعه، می&shy;توان آن را گزینه مناسبی برای پژوهش&shy;های کاربردی و متعاقب آن صنعتی شدن به جای آنتی‌بیوتیک‌ها پیشنهاد داد.</span></span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span style="direction:rtl"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><span b="" lang="FA" mitra="" style="font-family:"><span style="color:black"></span></span></span></span></span></span></span></span></span> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:150%"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Background and Aim: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Endolysin of phage KP27 is a protein with a molecular mass of 14.7 kDa, which has endopeptidase activity against bacterial cell walls. Due to limited research on endolysin KP27 and its superiority against harsh environmental </span></span></span><span style="font-size:10.0pt"><span style="background:white"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">conditions</span></span></span></span><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times=""> such as temperature and pH, the aim of this study is recombinant production and investigation of its antibacterial activity against two gram-negative and positive bacteria</span></span></span><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span style="font-family:"B Mitra""></span></span></span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:150%"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Methods: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">KP27 endolysin gene was cloned between two restriction enzymes <i>NcoI</i> and <i>XhoI</i> in plasmid <i>pET-28a(+)</i>. <i>E. coli BL21 (DE3)</i> was used for expression and production and Purification was performed by Ni²⁺-NTA sepharose chromatography. Bactericidal activity tests, MIC and MBC against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> bacteria and cell wall degradation were used to investigate the antibacterial and enzymatic activities of endolysin, respectively. Statistical analysis and generation of graphs were achieved by Graphpad Prism software.</span></span></span><b><span style="font-size:10.0pt"><span style="line-height:150%"><span style="font-family:"Times New Roman","serif""></span></span></span></b></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:150%"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Results: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Endolysin KP27 was successfully cloned and produced with high efficiency in the amount of 28.5 mg/L of culture medium. Bactericidal activity against Escherichia coli and Staphylococcus aureus bacteria was over 50% at the concentrations of 4 &micro;g/ml. For <i>Escherichia coli</i>, MIC and MBC were calculated 16 and 64 </span></span></span><span style="font-size:10.0pt"><span style="line-height:150%">&micro;</span></span><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">g/ml while for&nbsp; <i>Staphylococcus aureus</i> they were 8 and 32 </span></span></span><span style="font-size:10.0pt"><span style="line-height:150%">&micro;</span></span><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">g/ml, respectively. Evaluating the cell wall degradation activity of endolysin indicated a 20% decrease in the turbidity of the solution after half an hour incubation with endolysin.</span></span></span><span dir="RTL" lang="FA" style="font-size:10.0pt"><span style="line-height:150%"><span style="font-family:"B Mitra""></span></span></span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:150%"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><b><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">Conclusion</span></span></span></b><b><span new="" roman="" style="font-family:" times="">: </span></b><span new="" roman="" style="font-family:" times="">Considering the </span><span style="font-size:10.0pt"><span style="line-height:150%"><span new="" roman="" style="font-family:" times="">high</span></span></span><span new="" roman="" style="font-family:" times=""> expression and production as well as its high activity against both Gram-negative and Gram-positive bacteria, endolysin KP27 can be suggested as a convenient candidate for further applied research and subsequent industrialization instead of antibiotics.</span></span></span></span></span></span></span><br> <span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><span lang="FA" style="font-size:10.0pt"><span new="" roman="" style="font-family:" times=""><span style="color:black">&lrm;</span></span></span><b><span style="font-size:10.0pt"><span new="" roman="" style="font-family:" times=""><span style="color:black"></span></span></span></b></span></span></span></span></span></span><span style="font-size:11pt"><span style="text-justify:kashida"><span style="text-kashida:0%"><span style="line-height:normal"><span style="unicode-bidi:embed"><span sans-serif="" style="font-family:Calibri,"><span style="font-size:10.0pt"><span new="" roman="" style="font-family:" times=""><span style="color:black"> </span></span></span><span dir="RTL" lang="FA" style="font-size:10.0pt"><span b="" mitra="" style="font-family:"><span style="color:black"></span></span></span></span></span></span></span></span></span><br> &nbsp; اندولیزین KP27, باکتریوفاژ, پروتئین نوترکیب, دیواره سلولی, مقاومت آنتی­ بیوتیکی ​​​​​​​ Endolysin KP27, Bacteriophage, Recombinant protein, Cell wall, Antibiotic resistance, 137 148 http://ijpp.phypha.ir/browse.php?a_code=A-10-39-1&slc_lang=fa&sid=1 Zeynab Zare زینب زارع 10031947532846006501 10031947532846006501 No Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran بخش بیوشیمی، دانشکده دامپزشکی، دانشگاه ارومیه، ارومیه، ایران Mehdi Imani مهدی ایمانی 10031947532846006502 10031947532846006502 Yes Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran بخش بیوشیمی، دانشکده دامپزشکی، دانشگاه ارومیه، ارومیه، ایران Safiyeh Aghazadeh صفیه آقازاده 10031947532846006503 10031947532846006503 No Department of Biochemistry, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran بخش بیوشیمی، دانشکده دامپزشکی، دانشگاه ارومیه، ارومیه، ایران