ارزیابی پاسخ ایمنی نسبت به پروتئین کایمریک نو ترکیب LFD1-PA4 باکتری باسیلوس آنتراسیس

نوع مقاله : مقاله کامل

نویسندگان

چکیده

پیشینه: سیاه زخم بیماری عفونی بسیار خطرناکی است که انسان و دام‌های اهلی را تحت تاثیر قرار می‌دهد. به منظور حفاظت انسان در برابر عفونت سیاه زخم، واکسن‌های موثر که بتوانند پاسخ ایمنی سریع و بلند مدت ایجاد کنند ضروری می‌باشد. ناحیه 4، آنتی ژن حفاظتی (PA) و ناحیه 1، فاکتور کشنده (LF)، دارای خاصیت آنتی ژنی بسیار قویی می‌باشند. هدف: در این مطالعه تجربی، با ترکیب این دو ناحیه، کاست ژنی lfD1-pa4 طراحی و در وکتور بیانی pET28a(+) همسانه سازی و به باکتری (DE3) PlysS E. coli BL21 منتقل گردید. روش کار: برای این منظور ژن pa4 با واکنش زنجیره‌ای پلیمراز (PCR) تکثیر و در ناقل pGEM-T Easy Vector همسانه سازی شد. هضم آنزیمی pGEM-pa4 و pGEM-lfD1 با آنزیم‌های XbaI و HindIII، انجام گرفت. واکنش الحاق توسط آنزیم T4 لیگاز انجام و کاست ژنی، lfD1-pa4، در pET28a زیر همسانه سازی و به باکتری E. coli BL21 (DE3) PlysS منتقل گردید. بیان و تخلیص پروتئین کایمریک با روش ‌الکتروفورز روی ژل پلی آکریل آمید سدیم دودسیل سولفات (SDS-PAGE) و وسترن بلاتینگ مورد تایید قرار گرفت. به منظور بررسی پاسخ ایمنی، پروتئین کایمریک (LFD1-PA4) و ترکیبی (LFD1+PA4) طی چهار نوبت به موش تزریق و بعد از جمع‌آوری نمونه‌های خون، میزان پادتن حاصل با آزمون سنجش ایمونوسوربنت متصل به آنزیم (ELISA) مورد ارزیابی قرار گرفت. نتایج: یافته‌های حاصل نشان می‌دهد که پروتئین‌های کایمریک و ترکیبی هر دو ایمونوژنیک می‌باشند با این حال پروتئین LFD1-PA4 سیستم ایمنی موش را به میزان بیشتری تحریک می‌نماید. نتیجه‌گیری: پروتئین کایمریک LFD1-PA4 پاسخ ایمنی قوی‌تر نسبت به پروتئین ترکیبی LFD1+PA4 ایجاد کرده و باعث پاسخ آنتی بادی نسبت به فاکتور کشنده (LF) و فاکتور ادم (EF) شد. بنابراین این امید می‌رود که این پروتئین کاندیدای واکسن سه ظرفیتی موثرتری جهت پیشگیری از بیماری سیاه زخم باشد.

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