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
Swapan KN. Shigellosis. The Journal of Microbiology: 2005. 133-43.
Oludare O, Dequina N, Hiroshi Y, William L. AB Toxins: A paradigm switch from deadly to desirable. Toxins. 2010; 2(7):1612-45. doi: 10.3390/toxins2071612
Pina DG, Johannes L. Cholera and Shiga toxin B-subunits: Thermodynamic and structural considerations for function and biomedical applications. Toxicon. 2005; 45(4):389–93. doi: 10.1016/j.toxicon.2004.12.014
Bouter A, Delord B, Dransart E, Poirier C, Johannes L, van Effenterre D. Intracellular trafficking of Shiga-toxin-B-subunit-functionalized spherulites. Biology of the Cell. 2008; 100(12):717-25. doi: 10.1042/bc20080009
Janssen KP, Vignjevic, Boisgard R, Falguie T, Bousquet G, Decaudin D, et al. In vivo tumor targeting using a novel intestinal pathogen-based delivery approach. Cancer Research. 2006; 66(14):7230-36. doi: 10.1158/0008-5472.can-06-0631
Sani M. Botteaux A, Parsot C, Sansonetti P, Boekema EJ, Allaoui A. IpaD is localized at the tip of the shigella flexneri type iii secretion apparatus. Biochimica et Biophysica Acta. 2007; 1770(2):307-311. doi: 10.1016/j.bbagen.2006.10.007
Man A, Prieto G and Nicoletti C. Improving M-Cell-mediated transport across mucosal barriers. Immunology. 2004; 113(1):15-22. doi: 10.1111/j.1365-2567.2004.01964.x
Saadati M, Heiat M, Nazarian S, Barati B, Honari H, Doroudian M, et al. Cloning and expression of N-terminal region of IpaD from Shigella Dysenteriae in E. coli. Journal of Paramedical Sciences. 2010; 1(4):12-17.
Honari H, Amlashi I, Minaei ME, Safaei S. [Immunogenicity in guinea pigs by IpaD-STxB recombinant protein (Persian)]. Arak Medical University Journal. 2013; 16(73):83-93.
Honari H, Amlashi I, Minaei M. [Expression of Recombinant Proteins IpaD-STxB and Immunogenicity STxB in the Mice (Persian)]. Journal of Mazandaran University of Medical Sciences. 2014; 23(109):196-206
Honari H, Baranvand M, Minaei ME. [Immunogenicity Investigation of recombinant proteins (StxB) of Shigella dysenteriae type 1 in mice (Persian)]. Journal of Sabzevar University of Medical Sciences. 2015; 21(6):1103-1112.
Park JH, Saravanakumar G, Kim K, Kwon IC. Targeted delivery of low molecular drugs using chitosan and its derivatives. Advanced Drug Delivery Reviews. 2010; 62(1):28–41. doi: 10.1016/j.addr.2009.10.003
Makhlof A, Tozuka Y, Takeuchi H. Design and evaluation of novel pH-sensitive chitosan nanoparticles for oral insulin delivery. European Journal of Pharmaceutical Sciences. 2011; 42(5):445–51. doi: 10.1016/j.ejps.2010.12.007
Amini Y, Tebianian M, Mosavari N, Fasihi Ramandi M, Ebrahimi SM, Dabaghian M, et al. Preparation of ESAT-6 nanoparticles and evaluation of humoral immunity after intranasal administration. Journal of Shahid Sadoughi University of Medical Sciences. 2012; 20(5):605-14.
Danhier F, Ansorena E, Silva JM, Coco R, Le Breton A, Préat V. PLGA-based nanoparticles: An overview of biomedical applications. Journal of Controlled Release. 2012; 161(2):505–22. doi: 10.1016/j.jconrel.2012.01.043
Suh WH, Suslick KS, Stucky GD, Suh YH. Nanotechnology, nanotoxicology, and neuroscience. Progress in Neurobiology. 2009; 87(3):133–70. doi: 10.1016/j.pneurobio.2008.09.009
Ranjbar R, Soltan Dallal MM, Pourshafie MR, Aslani MM, Majdzadeh R. Serogroup Distribution of Shigella in Tehran. Iranian Journal of Public Health. 2004; 33(3):32-35.
Madanchi H, Honari H, Hesaraki H, Sayadnanesh A. Cloning and expression of stxB gene from shigella dysenteria typeI in E. coli Rosseta DE3. Genetics in the 3rd Millennium; 2012; 1:2641-2647.
Bernkop-Schnürch A, Dünnhaupt S. Chitosan-based drug delivery systems. European Journal of Pharmaceutics and Biopharmaceutics. 2012; 81(3):463–9. doi: 10.1016/j.ejpb.2012.04.007
Homayoni H, Ravandi SAH, Valizadeh M. Electrospinning of chitosan nanofibers: Processing optimization. Carbohydrate Polymers. 2009; 77(3):656–61. doi: 10.1016/j.carbpol.2009.02.008
Ranjbar R, Haghi-Ashtiani MT, Jonaidi Jafari N, and Abedini M, The prevalence and antimicrobial susceptibility of bacterial uropathogens isolated from pediatric patients. Iranian Journal of Public Health. 2009; 38(2):134-138.
Chia M-Y, Hsiao S-H, Chan H-T, Do Y-Y, Huang P-L, Chang HW, et al. The immunogenicity of DNA constructs co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus conjugated by GPGP linker in pigs. Veterinary Microbiology. 2010; 146(3-4):189–99. doi: 10.1016/j.vetmic.2010.05.007
Hromockyj AE, Maurelli AT. Identification of an Escherichia coli gene homologous to virR, a regulator of Shigella virulence. Journal of Bacteriology. 1989; 171(5):2879–81. doi: 10.1128/jb.171.5.2879-2881.1989
Magistris MT. Mucosal delivery of vaccine antigens and its advantages in pediatrics. Advanced Drug Delivery Reviews. 2006; 58(1):52–67. doi: 10.1016/j.addr.2006.01.002
Zhu C, Yu J, Yang Z, Davis K, Rios H, Wang B, et al. Protection against Shiga toxin-producing Escherichia coli infection by transcutaneous immunization with Shiga toxin subunit B. Clinical and Vaccine Immunology. 2007; 15(2):359–66. doi: 10.1128/cvi.00399-07
Gupta P, Singh MK, Singh Y, Gautam V, Kumar S, Kumar O, et al. Recombinant Shiga toxin B subunit elicits protection against Shiga toxin via mixed Th type immune response in mice. Vaccine. 2011; 29(45):8094–100. doi: 10.1016/j.vaccine.2011.08.040
Esmaeili A, Honari H, Hamedian M, Safaei S, Ghofrani M. Targeted cloning of GFP as a tracker and its fusion mediated by PRARR flexible linkers to CTB-STB chimerical vaccine genes. Genetics in the 3rd Millennium. 2012; 10(1):2657-2665.
Soane R, Frier M, Perkins A, Jones N, Davis S, Illum L. Evaluation of the clearance characteristics of bioadhesive systems in humans. International Journal of Pharmaceutics. 1999; 178(1):55–65. doi: 10.1016/s0378-5173(98)00367-6
Bacon A, Makin J, Sizer PJ, Jabbal-Gill I, Hinchcliffe M, Illum L, et al. Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens. Infection & Immunity 2000; 68(10):5764–70. doi: 10.1128/iai.68.10.5764-5770.2000