نوع مقاله : مقاله پژوهشی

نویسندگان

1 کارشناس ارشد، گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه جامع امام حسین (ع)، تهران، ایران.

2 دانشیار، گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه جامع امام حسین (ع)، تهران، ایران.

چکیده

اهداف عفونت حاد روده‌ای که توسط باکترهای شیگلا و اشرشیاکلی ایجاد می‌شود به عنوان عامل بیوتروریستی شناخته شده است. پروتئین IpaD و STx نقش مهمی در تهاجم و بیماری‌زایی شیگلاها دارد. با ممزوج‌کردن IpaD با STxB می‌توان کاندیدای واکسن مناسب تهیه کرد. در این مطالعه ایمنی‌زایی پروتئین‌های نوترکیب نانوذره‌ای STxB-IpaD ممزوجی و STxB به صورت خوراکی و تزریقی در موش بررسی شده است.
مواد و روش ها در این مطالعه تجربی، از وکتورهای (+)pET28a دارای ژن‌های stxB-ipaD و stxB استفاده شد که به درون باکتریE.coli BL21 DE3 ترانسفورم شدند. این باکتری روی محیط آنتی‌بیوتیک رشد داده شد و با روش PCR مستقیم و بیان پروتئین و ژل SDS-PAGE تأیید شد. پروتئین‌های نوترکیب توسط ستون نیکل تخلیص و توسط ژل SDS-PAGE و ایمنوبلاتینگ تأیید شدند. پروتئین‌های نوترکیب STxB-IpaD و STxB با روش ژله‌ای‌شدن یونی با پلیمر کیتوسان نانویی شدند و تصویربرداری آن با میکروسکوپ الکترونی نگاره (SEM) انجام گرفت. تجویز خوراکی و تزریقی آنتی‌ژن‌های نانوذره‌ای STxB-IpaD و STxB در چهار نوبت متوالی به موش‌های سوری انجام و تیتر آنتی‌بادی و ایمنی‌زایی آن‌ها بررسی شد.
یافته ها با انجام آزمایش الایزا تیتر آنتی‌بادی IgG در حالت تزریقی مشاهده شد، ولی درحالت خوراکی مشاهده نشد. ممکن است به علت ازبین‌رفتن ساختار نانوذره و آنتی‌ژن توسط محیط اسیدی معده و آنزیم تریپسین باشد. موش‌های ایمن‌شده با پروتئین‌های نوترکیب STxB و STxB-IpaD با روش ژله‌ای‌شدن یونی نانویی توانستند به ترتیب تا هفت و ده برابر LD50 شیگا توکسین E.coli O157:H7 را تحمل کنند.
نتیجه گیری می‌توان از نانوذره پروتئینی STxB-IpaD و STxB به عنوان اجوانت تزریقی برای ایمنی‌زایی در برابر شیگا توکسین E.coli O157:H7 استفاده کرد.

کلیدواژه‌ها

عنوان مقاله [English]

Evaluation of Immunogenicity of Chitosan Nanoparticles Containing STxB and STxB-IpaD Antigens of Shigella Dysenteriae Type 1 in Mice

نویسندگان [English]

  • Mahdi Baranvand 1
  • Hosein Honari 2

1 MSc., Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.

2 Associate Professor, Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran.

چکیده [English]

Background The intestinal infection caused by Shigella and Escherichia coli is known as a bioterrorist agent. IpaD and STx proteins play an important role in the invasion and angiogenesis by Shigella. Therefore, IpaD with STxB can be appropriate candidates for a safety vaccine. In this study the immunogenicity of STxB and combined STxB-IpaD nanocapsule recombinant proteins has been examined in the form of oral and injection in mice.
Methods & Materials In this experimental study, pET28a(+) vectors containing stxB and stxB-ipaD genes were transformed into E. coli BL21 DE3 bacteria. These bacteria were grown on antibiotic medium and were confirmed by direct PCR, and protein expression and SDS-PAGE gel. Recombinant proteins purified by nickel column and SDS-PAGE gel and were confirmed by immunoblotting. STxB and STxB-IpaD recombinant proteins become nanoparticles by inotropic gelation method with chitosan polymer and its picture was taken by Scanning Electronic Microscope (SEM). STxB and STxB-IpaD nanocapsule antigens prescript in the form of oral and injection four time to mice and their antibodies titer and immunogenicity were monitored.
Results By performing ELISA test, IgG antibody titer was detected by injection method but not in oral method, maybe due to loss of nanoparticle structure and antigens in acidic environment and trypsin enzyme of stomach. Immunized mice’s with STxB and STxB-IpaD recombinant proteins with inotropic gel method were able to tolerance order up to 7 and 10 times the E. coli O157: H7 Shiga toxin LD50.
Conclusion STxB and STxB-IpaD protein nanoparticles can be used as safety injection adjuvant for immunogenesis against the E. coli O157: H7 Shiga toxin.

کلیدواژه‌ها [English]

  • Shigella dysenteriae I
  • STxB STxB-IpaD
  • Chitosan nanoparticles
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