Scientific Journal of Kurdistan University of Medical Sciences
مجله علمي دانشگاه علوم پزشكي كردستان
SJKU
General
http://sjku.muk.ac.ir
54
journal54
1560-652X
2345-4040
sjku
10.61186/sjku
en
jalali
1400
9
1
gregorian
2021
12
1
26
6
online
1
fulltext
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تولید ایمونوگلوبولین مرغی (IgY) علیه پروتئین نوترکیب LTB از اشرشیاکلی انتروتوکسیژنیک ( ETEC) و ارزیابی اثر محافظتی آن در مدل حیوانی
Production of Egg Yolk Immunoglobulin (IgY) Against Recombinant LTB of Enterotoxigenic Escherichia coli (ETEC) and Evaluation of Its Protective Effect in Animal Model
بیوتکنولوژی
Biotechnology
پژوهشي اصیل
Original Research
<strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">زمینه و هدف</span></span></strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">:</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> اشرشیاکلی انتروتوکسیژنیک</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">یکی از رایج</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ترین</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">عوامل باکتریایی مولد اسهال در جهان است</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">از مهم</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ترین</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">فاکتورهای حدت</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">زا </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">ETEC)</span></span></span> <span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">Enterotoxigenic Escherichia coli</span></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">)</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">، می</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">توان</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">به زیر</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">واحد </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">B</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">توکسین حساس به حرارت اشاره کرد که قسمت اتصالی توکسین را شکل می</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">دهد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">در</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">مطالعه پیش رو آنتی</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">بادی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">مرغی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">علیه پروتئین نوترکیب</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">Lymphotoxin Beta</span></span></span> <span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">(LTB)</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">تولید</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">و</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">اثر محافظتی آن در مدل حیوانی مورد آزمایش قرار گرفت</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">.</span></span><br>
<strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">مواد و روش</span></span></strong><strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span></strong><strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ها</span></span></strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">: 100 </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">میکروگرم</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">پروتئین</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">نوترکیب</span></span> <span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LTB</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">به</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">صورت</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">داخل</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">عضلانی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">به</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">مرغ</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ها</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">تزریق</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">شد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">تیتر</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">آنتی</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">بادی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">در</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">سرم</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">مرغ</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ها</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">اندازه</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">گیری</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">و تخم</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">مرغ</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ها</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">جمع</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">آوری</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">و</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">(IgY) Immunoglobulin Y </span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">ها</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">از</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">زرده</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">تخم</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">مرغ</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">با</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">کمک</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">سدیم</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">کلراید</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">تخلیص</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">و</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">تیتر</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">آنتی</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">بادی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">با</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">روش</span></span> <span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">ELISA</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">بررسی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">شد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">توانایی </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">IgY</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">در ممانعت از عملکرد</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LT</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">توکسین بر سلول</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">های</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">Y1</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">در رقت</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">های متفاوت </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">IgY</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">سنجیده شد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">به منظور بررسی اثر باکتری تیمار شده با </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">IgY</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">بر سلول</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">های اپیتلیال روده، از روش استاندارد لوپ ایلئال استفاده گردید</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">.</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;"></span></span></span><br>
<strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">یافته</span></span></strong><strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span></strong><strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">ها</span></span></strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">:</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> القاء بیان پروتئین منجر به تولید </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LTB</span></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> نوترکیب با وزن تقریبی 14 کیلو دالتون شد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">غلظت پروتئین تخلیص شده 5/4 میلیگرم بر میلی لیتر بود. با ایمنسازی مرغها تیتر آنتیبادی سرمی افزایش یافت. آنتیبادی مرغی علیه پروتئین </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LTB</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">با غلظت 12 میلی گرم بر میلیلیتر از زرده تخم مرغ تخلیص شد. </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">IgY</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">علیه</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">پروتئین</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">نوترکیب</span></span> <span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LTB</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">در</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">رقت</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> 125 </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">میکروگرم</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">بر</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">میلی</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">لیتر اثرات توکسین حساس به حرارت </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LT</span></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> بر سلولهای </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">Y1</span></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> را خنثی کرد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">در</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">تست</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">لوپ</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">ایلئال</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">خنثی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">سازی</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">توکسین</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">با</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">رقت</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> 1/5 </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">میلی</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">گرم</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">بر</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">میلی</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">لیتر</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">IgY</span></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">،</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">مانع</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">از</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">عملکرد</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">توکسین</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">در</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">روده</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">شد</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">. </span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">تجمع</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">مایع</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">درون</span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">لوپ 74/8درصد در مقایسه با لوپ های کنترل کاهش یافت.</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span><br>
<strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">نتیجه</span></span></strong><strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;"></span></span></strong><strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">گیری</span></span></strong><span style="font-family:B Zar;"><span style="font-size:12.0pt;">: نتایج به دست آمده بیانگر آن است که میتوان از آنتیبادی مرغی اختصاصی علیه پروتئین نوترکیب </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">LTB</span></span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;"> به عنوان آنتیبادی پیشگیرانه جهت جلوگیری از عملکرد توکسین حساس به حرارت باکتری </span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;">ETEC</span></span></span> <span style="font-family:B Zar;"><span style="font-size:12.0pt;">استفاده نمود</span></span><span style="font-family:B Zar;"><span style="font-size:12.0pt;">.</span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;"></span></span></span><span dir="LTR"><span style="font-family:Times New Roman,serif;"><span style="font-size:12.0pt;"></span></span></span>
<strong><span dir="LTR">ABSTRACT</span></strong><br>
<strong><span dir="LTR">Background and purpose:</span></strong><br>
Enterotoxigenic<em> Escherichia coli</em> is one of the most common bacterial causes of diarrhea worldwide.<br>
<span dir="LTR">One of the most important ETEC colonization factors is B subunit of the Heat Labile Enterotoxin, which forms the connecting part of the toxin. In the present study, egg yolk immunoglobulin (IgY) was produced and its effectiveness against recombinant LTB protein was tested on the animal model.</span><br>
<strong><span dir="LTR">Material and methods:</span></strong><br>
<span dir="LTR">Hens were injected intramuscularly with 100 μg of recombinant LTB protein. The antibody titer was estimated and the eggs were collected<ins cite="mailto:vaio" datetime="2020-04-06T09:54">.</ins></span> The water soluble part of the yolk was separated and the antibody titer was subsequently estimated by ELISA.Effecacy of different concentrations of IgY on nutralising the effect of LT toxin on Y1 cells was also studied. In order to investigate the effect of IgY-treated bacteria on intestinal epithelial cells, the standard Ileal loop technique was used.<br>
<strong><span dir="LTR">Results:</span></strong><br>
<span dir="LTR">Protein expression led to the production of recombinant LTB showing molecular weight of 11 kDa on SDS PAGE. Immunization of hens induced serum antibody rise. The purified antibody was 144 mg per 12 ml of egg yolk. Purified IgY exhibited significant effect against recombinant LTB proteinAt the concentration of 125 μg per ml, the IgY could prevent the effects of Heat Labile Enterotoxin on Y1 cells. In the Ileal loop test, 1.5 mg / ml IgY neutralized the toxin effect of LTB on the intestine. The accumulation of fluid in the test loops decreased by 74.8% compared to the untreated control loops.</span><br>
<strong><span dir="LTR">Conclusion:</span></strong><span dir="LTR"></span><br>
<span dir="LTR">The results showed that specific egg yolk immunoglobulin was effective against recombinant LTB protein and can be used as a preventive antibody to inhibit the Heat Labile Enterotoxin function of ETEC bacteria.</span>
اشیرشیاکلی انتروتوکسیژنیک, انتروتوکسین حساس به حرارت, ایمنی غیرفعال, ایمنوگلوبولین, فاکتوربیماریزایی
: Enterotoxigenic Escherichia coli, Heat labile enterotoxin, Passive immunity, Egg yolk immunoglobulin, Virulence factor
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http://sjku.muk.ac.ir/browse.php?a_code=A-10-5343-1&slc_lang=fa&sid=1
Maryam
Mafi
مریم
مافی
maryam.mafi95@gmail.com
5400319475328460057111
5400319475328460057111
No
Microbial Biotechnology, Departmant of Cell Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran.
دانشجوی کارشناسی ارشد بیوتکنولوژی میکروبی، گروه زیستشناسی، دانشکده علوم پایه، دانشگاه شاهد، تهران، ایران
latif
Mousavi gargari
سید لطیف
موسوی گرگری
slmousavi@shahed.ac.ir
5400319475328460057112
5400319475328460057112
Yes
Ph.D Biochemistry, Departmant of Cell Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran
دکتری تخصصی بیوشیمی، گروه زیستشناسی، دانشکده علوم پایه، دانشگاه شاهد، تهران، ایران.
shahram
nazarian
شهرام
نظریان
nazarian56@gmail.com
5400319475328460057113
5400319475328460057113
No
Ph.D. Microbiology, Biology Research Centere, Basic Science Faculty, Imam Hossein University, Tehran, Iran.
مرکز تحقیقات زیستشناسی، دانشکده و پژوهشکده علوم پایه، دانشگاه جامع امام حسین (ع)، تهران، ایران
fatemeh
mohammad khani
فاطمه
محمدخانی
fatemeh999m@yahoo.com
5400319475328460057114
5400319475328460057114
No
Ms.C Microbial Biotechnology, Departmant of Cell Biology, Faculty of Basic Sciences, Shahed University, Tehran, Iran.
دانشجوی کارشناسی ارشد بیوتکنولوژی میکروبی، گروه زیستشناسی، دانشکده علوم پایه، دانشگاه شاهد، تهران، ایران