تأثیر پروتین نوترکیب IpaB در ایجاد پاسخ های ایمنی علیه شیگلا دیسانتری در خوکچه هندی
محورهای موضوعی :
میکروب شناسی کاربردی
سیداکبر آریان زاد
1
(دانشکده میکروبیولوژی، دانشگاه آزاد اسلامی واحد کرج)
مهدی زین الدینی
2
(دانشگاه صنعتی مالک اشتر، دانشکده شیمی و مهندسی شیمی، پژوهشکده علوم و فناوری زیستی)
اعظم حدادی
3
(دانشکده میکروبیولوژی دانشگاه آزاد اسلامی کرج)
شهرام نظریان
4
(مرکز تحقیقات زیست شناسی، دانشگاه امام حسین (ع))
رضا حسن ساجدی
5
(گروه بیوشیمی، دانشکده زیست شناسی، دانشگاه تربیت مدرس)
تاریخ دریافت : 1401/04/28
تاریخ پذیرش : 1401/08/20
تاریخ انتشار : 1401/09/15
کلید واژه:
شیگلا,
واکسن زیرواحد نوترکیب,
فاکتور بیماری‌زایی,
آزمایش سرنی,
پروتین IpaB,
چکیده مقاله :
سابقه و هدف: شیگلوزیس بیماری عفونی ناشی از شیگلا دیسانتری است و سالانه جان میلیون ها نفر را در سراسر جهان تهدید می کند. توسعه سویه های مقاوم به آنتی بیوتیک، واکسن ها را به عنوان اولویت اصلی سازمان بهداشت جهانی علیه بیماری قرار داده است. پروتین IpaB که بخشی از سیستم ترشحی نوع III (T3SS) در شیگلا است، می تواند پاسخ ایمنی مطلوبی در برابر باکتری ایجاد کند. هدف از این مطالعه ارزیابی ایمنی زایی پروتین نوترکیب حاوی نواحی ایمونوژن IpaB به عنوان گزینه واکسن زیر واحد نوترکیب علیه شیگلا دیسانتری است.مواد و روش ها: ژن کد کننده پروتین ایمونوژن در وکتور بیانی pET28a همسانه سازی و به میزبان باکتریایی E.coil سویه Rosetta (DE3) ترانسفورم شد و توسط IPTG القا گردید. پروتین خالص با استفاده از ستون کروماتوگرافی نیکل به دست آمد و برای ایمن زایی به خوکچه هندی تزریق شد. تیتر آنتی بادی تولید شده با استفاده از روش ELISA غیرمستقیم ارزیابی شد و در نهایت آزمون چالش حیوانی با آزمایش سرنی در خوکچه هندی انجام شد.یافته ها: باند 36 کیلو دالتون به عنوان پروتین IpaB در SDS-PAGE مشاهده شد که با وسترن بلات تایید شد. آنالیزهای ایمونولوژیکی تولید تیتر بالایی از آنتی بادی اختصاصی علیه IpaB (1:102400) را در خوکچه هندی واکسینه شده نشان داد. عدم وجود هرگونه کراتوکنژنکتیویت در خوکچه های هندی واکسینه شده در تست Sereny نشان دهنده سطح بالایی از محافظت در برابر شیگلا دیسانتری است.نتیجه گیری: یافته های این تحقیق نشان می دهد که پروتین نوترکیب تولید شده یک گزینه مناسب به عنوان واکسن برای مهار و درمان در برابر شیگلا دیسانتری است.
چکیده انگلیسی:
Background & Objectives: Shigellosis is an infectious disease caused by Shigella dysenteriae and threatens the lives of millions of people worldwide annually. IpaB protein, which is part of the Shigella type III secretion system (T3SS), it can elicit a favorable immune response against the bacterium. The aim of this study is experimental evaluation of the immunogenicity of a recombinant protein containing immunogenic regions of IpaB as a subunit recombinant vaccine candidate against Shigella dysenteriae. Material & Methods: The gene encoding immunogenic protein that cloned into pET28a expression vector was transformed in the bacterial host E.coil strain Rosetta (DE3) and induced by IPTG. The purified protein was achieved using nickel chromatography column and injected into guinea pigs for immunization. The produced antibody titer was assessed by indirect ELISA assay and finally animal challenge was performed using the Sereny test in guinea pigs.Results: A 36 KDa band as IpaB protein was observed in SDS-PAGE which was confirmed by western blot. The immunological analyses showed production of high titer of specific anti-IpaB antibody (1:102,400) in immunized Guinea pig. The absence of any keratoconjunctival inflammation in immunized guinea pigs in Sereny test indicated high level protection against virulent Shigella dysentriea.Conclusion: The results showed that IpaB can elicit high titer of antibody and protection against Shigella dysentriae in Guinea pigs.
منابع و مأخذ:
Oany AR, Pervin T, Mia M, Hosein M, Shahnaij M, Mahmud S, Kibria KMK. Vaccinomics Approach for Designing Potential Peptide Vaccine by Targeting spp. Serine Protease Autotransporter Subfamily Protein SigA. J Immunol Res.2017; 2017:6412353.
Walker RI. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries. Vaccine. 2005; 23(26):3369 –85.
Livio S et al Shigella isolates from the global enteric multicenter study inform vaccine development. Clin Infect Dis. 2014; 59(7):933–41.
DuPont HL, Levine MM, Hornick RB, Formal SB.. Inoculum size in shigellosis and implications for expected mode of transmission. J Infect Dis. 1989;159(6):1126 –8.
Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H. Expression of cholera toxin B–proinsulin fusion protein in lettuce and tobacco chloroplasts–oral administration protects againstdevelopment of insulitis in non‐obese diabetic mice. Plant biotechnol j. 2007;5(4): 495-510.
Stensrud KF, Adam PR, La Mar CD, Olive AJ, Lushington GH, Sudharsan R, et al. Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip. J Biol Chem. 2008;283(27):18646-54.
Buysse JM, Stover CK, Oaks EV, Venkatesan M and Kopecko JD. Molecular Cloning of Invasion Plasmid Antigen (ipa) Genes from Shigella flexneri: Analysis of ipa Gene Products and Genetic Mapping. J bacteriol. 1987; 169(6)2561-9.
Guichon A, Hersh D, Smith MR and Zychlinsky AS. Structure-Function Analysis of the Shigella Virulence Factor IpaB. J bacteriol. 2001;183(4):1269-76.
Honari H, Amlashi I, Minaei ME, Safaei S. Immunogenicity in guinea pigs by IpaD –STxB recombinant protein. J Arak Med Uni. 2013; 16(73): 83-93.[In Persian].
Zhang L, Wang Y, Olive AJ, Smith ND, Picking WD, De Guzman RN, et al. Identification of the MxiH needle protein residues responsible for anchoring invasion plasmid antigen D to the type III secretion needle tip. J Bio Chem. 2007;282(44):32144-51.
Venkatesan MM, Buysse JM, Kopecko DJ. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A.1988;85(23):9317- 21.
Orrett FA.Prevalence of Shigella serogroups and their antimicrobial resistance pattern in southern Trinidad. j Health Popul Nutr. 2008;26(4):456-62.
Barnoy S, Jeong KI, Helm RF, Suvarnapunya AE, Ranallo RT ,Tzipori S , Venkatesan M M. Characterization of WRSs2 and WRSs3, new second-generation virG(icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity. Vaccine. 2010; 28(6):1642–54.
Katz DE, Coster TS, Wolf MK, Fernando C , Trespalacios FC, Cohen D, Robins G, Hartman AB , Venkatesan MM , Taylor DN , Hale T L . Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers. Infect Immun. 2004; 72(2):923–30.
Kotloff KL, Pasetti M F , Barry E M , Nataro J P ,Wasserman S S, Sztein M B , Picking W D , Levine M M. Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208. J Infect Dis. 2004;190:1745–54.
Phalipon A and Sansonetti PJ. Live Attenuated Shiglla flexneri Mutants as Vaccine Candidates Against Shigellosis and Vectors for Antigen Delivery. Biologicals. 1995; 23(2):125-134.
Walker RI. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries. Vaccine; 2005; 23(26): 3369-85.
McKenzie R, Walker RI , Nabors GS , Van De Verg L L , Carpenter C , Gomes G , Forbes E , Tian J H , Yang H H , Pace J L , Jackson W J , Bourgeois A L. Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial.Vaccine.2005; 24:(18):3735-45.
Ravenscrot N, Haeuptle MA, Kowarik M, Fernandes FC, Carranza P, Brunner A, Steffen M, Wetter M,Keller S, Ruch C, Wacker M. Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli. Glycobiology. 2016;26(1):51-62.
Kaminski RW, Oaks EV. Inactivated and subunit vaccines to prevent shigellosis. Expert Rev Vaccines. 2009;8(12):1693-704.
Turbyfill KR, Kaminski RW, Oaks EV. Immunogenicity and efficacy of highly purified invasin complex vaccine from Shigella flexneri 2a. Vaccine.2008 ;26(10):1353-64.
Farhani, I.; nezafat, N.; Mahmoodi, SH.; Designing a novel multi-epitop peptide vaccine against pathogenic Shigella spp based immunoinformatics approaches. Int J Pept Res Ther. 2018;25(3).
Turbyfill KR, Hartman AB, Oaks EV.Isolation and characterization of Shigella flexneri invasin complex subunit vaccine. Infect Immun. 2000;68(12):6624-32.
Arianzad SA, Zeinoddini M, Haddadi A, Nazarian SH, Hasansajedi R. In Silicodesign of chimeric and immunogenic protein-containing IpaB and IpaD as a vaccine candidate against Shigella dysenteriae. Cuurent Proteomics.2020; 17(4):333-41.
Khademi F, Yousefi-Avarvand A, Derakhshan M, Zahra Meshkat Z, Mohsen Tafaghodi M, Ghazvini K, Ehsan Aryan E, Mojtaba Sankian M.. Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli. Rep Biochem Mol Biol.2017;6(1): 15–21.
Sambrook J, Russell DW. Preparation and transformation of competent E. coli using calcium chloride. Cold Spring Harbor Protocols. 2006 Jun 1;2006(1):pdb-rot3932.
Gaikwad SS, Lee HJ, Kim JY, Choi KS. Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein. Clinical and experimental vaccine research. 2019 Jan;8(1):27.
Sambrook J. Russell DW, Maniatis T. Molecular Cloning, A Laboratory manual. 3 Ed. New York: Cold Spring Harbor Laboratory Press; 2001.
Schägger H. Tricine–sds-page. Nature protocols. 2006 Jun;1(1):16.
Mahmood T, Yang P. Western Blot: Technique, Theory, and Trouble Shooting. N Am J Med Sci.2012 Sep; 4(9): 429–434.
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72, 248–254.
Panda CS, Riley LW, Kumari SN, Khanna KK, Prakash K.Comparison of alkaline phosphatase-conjugated oligonucleotide DNA probe with the Sereny test for identification of Shigella strains. J Clin Microbiol. 1990;28(9);2122-4.
Jneid B, Rouaix A, Feraudet-Tarisse C, Simon S. SipD and IpaD induce a cross-protection against Shigella and Salmonella infections. PLoS Negl Trop Dis. 2020; 28:14(5): e0008326.
Yang SC, Hung CF, Aljuffali IA, Fang JY.The roles of the virulence factor IpaB in Shigella spp: in the escapefrom immune cells and invasion of epithelial cells. Microbiol Res. 2015;181:43–51.
Barry E, Pasetti M, Sztein M, Fasano A, Kotloff K, Levine M. Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol. 2013;10(4): 245–255.
Turbyfill KR., Hartman AB, Oaks EV. Isolation and characterization of a Shigella flexneri invasin complex subunit vaccine. Infect Immun. 2000;68(12): 6624–32.
Martinez-Beccera FJ, Chen X, Dikenson NE, Choudhari SP, Harrison K, Clements JD, Picking WD. IpaB of Shigella spp. And its potential as a pan- shigella vaccine.Infect Immun.2013;81(12):4470-77.
Shannon J, Hein SJ, Diaz-McNair J, Andar AU, Drachenberg CB, Vandeverg L, Walker R, Picking WL,Pasetti MF. Intradermal Delivery of ShigellaIpaB and IpaD Type III Secretion Proteins: Kinetics of Cell Recruitment and Antigen Uptake, Mucosal and System Immunity, and Protection across Serotype. J Immun. 2014; 192(4):1630-40.
Arabshahi S, Nayeri Fasaei B, Derakhshande A, Novinrooz A. In silico desing of a novel chimeric Shigella IpaB fused to C terminal of Clostridum perfringens entrotoxin as a vaccine candidate. Bioengineered. 2018;9(1):170-77.
Camacho A, Irache J, Gamazo C. Recent progress towards development of Shigella vaccine. Expert Rev Vaccines.2013;12:43–55.
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Oany AR, Pervin T, Mia M, Hosein M, Shahnaij M, Mahmud S, Kibria KMK. Vaccinomics Approach for Designing Potential Peptide Vaccine by Targeting spp. Serine Protease Autotransporter Subfamily Protein SigA. J Immunol Res.2017; 2017:6412353.
Walker RI. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries. Vaccine. 2005; 23(26):3369 –85.
Livio S et al Shigella isolates from the global enteric multicenter study inform vaccine development. Clin Infect Dis. 2014; 59(7):933–41.
DuPont HL, Levine MM, Hornick RB, Formal SB.. Inoculum size in shigellosis and implications for expected mode of transmission. J Infect Dis. 1989;159(6):1126 –8.
Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H. Expression of cholera toxin B–proinsulin fusion protein in lettuce and tobacco chloroplasts–oral administration protects againstdevelopment of insulitis in non‐obese diabetic mice. Plant biotechnol j. 2007;5(4): 495-510.
Stensrud KF, Adam PR, La Mar CD, Olive AJ, Lushington GH, Sudharsan R, et al. Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip. J Biol Chem. 2008;283(27):18646-54.
Buysse JM, Stover CK, Oaks EV, Venkatesan M and Kopecko JD. Molecular Cloning of Invasion Plasmid Antigen (ipa) Genes from Shigella flexneri: Analysis of ipa Gene Products and Genetic Mapping. J bacteriol. 1987; 169(6)2561-9.
Guichon A, Hersh D, Smith MR and Zychlinsky AS. Structure-Function Analysis of the Shigella Virulence Factor IpaB. J bacteriol. 2001;183(4):1269-76.
Honari H, Amlashi I, Minaei ME, Safaei S. Immunogenicity in guinea pigs by IpaD –STxB recombinant protein. J Arak Med Uni. 2013; 16(73): 83-93.[In Persian].
Zhang L, Wang Y, Olive AJ, Smith ND, Picking WD, De Guzman RN, et al. Identification of the MxiH needle protein residues responsible for anchoring invasion plasmid antigen D to the type III secretion needle tip. J Bio Chem. 2007;282(44):32144-51.
Venkatesan MM, Buysse JM, Kopecko DJ. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A.1988;85(23):9317- 21.
Orrett FA.Prevalence of Shigella serogroups and their antimicrobial resistance pattern in southern Trinidad. j Health Popul Nutr. 2008;26(4):456-62.
Barnoy S, Jeong KI, Helm RF, Suvarnapunya AE, Ranallo RT ,Tzipori S , Venkatesan M M. Characterization of WRSs2 and WRSs3, new second-generation virG(icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity. Vaccine. 2010; 28(6):1642–54.
Katz DE, Coster TS, Wolf MK, Fernando C , Trespalacios FC, Cohen D, Robins G, Hartman AB , Venkatesan MM , Taylor DN , Hale T L . Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers. Infect Immun. 2004; 72(2):923–30.
Kotloff KL, Pasetti M F , Barry E M , Nataro J P ,Wasserman S S, Sztein M B , Picking W D , Levine M M. Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208. J Infect Dis. 2004;190:1745–54.
Phalipon A and Sansonetti PJ. Live Attenuated Shiglla flexneri Mutants as Vaccine Candidates Against Shigellosis and Vectors for Antigen Delivery. Biologicals. 1995; 23(2):125-134.
Walker RI. Considerations for development of whole cell bacterial vaccines to prevent diarrheal diseases in children in developing countries. Vaccine; 2005; 23(26): 3369-85.
McKenzie R, Walker RI , Nabors GS , Van De Verg L L , Carpenter C , Gomes G , Forbes E , Tian J H , Yang H H , Pace J L , Jackson W J , Bourgeois A L. Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial.Vaccine.2005; 24:(18):3735-45.
Ravenscrot N, Haeuptle MA, Kowarik M, Fernandes FC, Carranza P, Brunner A, Steffen M, Wetter M,Keller S, Ruch C, Wacker M. Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli. Glycobiology. 2016;26(1):51-62.
Kaminski RW, Oaks EV. Inactivated and subunit vaccines to prevent shigellosis. Expert Rev Vaccines. 2009;8(12):1693-704.
Turbyfill KR, Kaminski RW, Oaks EV. Immunogenicity and efficacy of highly purified invasin complex vaccine from Shigella flexneri 2a. Vaccine.2008 ;26(10):1353-64.
Farhani, I.; nezafat, N.; Mahmoodi, SH.; Designing a novel multi-epitop peptide vaccine against pathogenic Shigella spp based immunoinformatics approaches. Int J Pept Res Ther. 2018;25(3).
Turbyfill KR, Hartman AB, Oaks EV.Isolation and characterization of Shigella flexneri invasin complex subunit vaccine. Infect Immun. 2000;68(12):6624-32.
Arianzad SA, Zeinoddini M, Haddadi A, Nazarian SH, Hasansajedi R. In Silicodesign of chimeric and immunogenic protein-containing IpaB and IpaD as a vaccine candidate against Shigella dysenteriae. Cuurent Proteomics.2020; 17(4):333-41.
Khademi F, Yousefi-Avarvand A, Derakhshan M, Zahra Meshkat Z, Mohsen Tafaghodi M, Ghazvini K, Ehsan Aryan E, Mojtaba Sankian M.. Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli. Rep Biochem Mol Biol.2017;6(1): 15–21.
Sambrook J, Russell DW. Preparation and transformation of competent E. coli using calcium chloride. Cold Spring Harbor Protocols. 2006 Jun 1;2006(1):pdb-rot3932.
Gaikwad SS, Lee HJ, Kim JY, Choi KS. Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein. Clinical and experimental vaccine research. 2019 Jan;8(1):27.
Sambrook J. Russell DW, Maniatis T. Molecular Cloning, A Laboratory manual. 3 Ed. New York: Cold Spring Harbor Laboratory Press; 2001.
Schägger H. Tricine–sds-page. Nature protocols. 2006 Jun;1(1):16.
Mahmood T, Yang P. Western Blot: Technique, Theory, and Trouble Shooting. N Am J Med Sci.2012 Sep; 4(9): 429–434.
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72, 248–254.
Panda CS, Riley LW, Kumari SN, Khanna KK, Prakash K.Comparison of alkaline phosphatase-conjugated oligonucleotide DNA probe with the Sereny test for identification of Shigella strains. J Clin Microbiol. 1990;28(9);2122-4.
Jneid B, Rouaix A, Feraudet-Tarisse C, Simon S. SipD and IpaD induce a cross-protection against Shigella and Salmonella infections. PLoS Negl Trop Dis. 2020; 28:14(5): e0008326.
Yang SC, Hung CF, Aljuffali IA, Fang JY.The roles of the virulence factor IpaB in Shigella spp: in the escapefrom immune cells and invasion of epithelial cells. Microbiol Res. 2015;181:43–51.
Barry E, Pasetti M, Sztein M, Fasano A, Kotloff K, Levine M. Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol Hepatol. 2013;10(4): 245–255.
Turbyfill KR., Hartman AB, Oaks EV. Isolation and characterization of a Shigella flexneri invasin complex subunit vaccine. Infect Immun. 2000;68(12): 6624–32.
Martinez-Beccera FJ, Chen X, Dikenson NE, Choudhari SP, Harrison K, Clements JD, Picking WD. IpaB of Shigella spp. And its potential as a pan- shigella vaccine.Infect Immun.2013;81(12):4470-77.
Shannon J, Hein SJ, Diaz-McNair J, Andar AU, Drachenberg CB, Vandeverg L, Walker R, Picking WL,Pasetti MF. Intradermal Delivery of ShigellaIpaB and IpaD Type III Secretion Proteins: Kinetics of Cell Recruitment and Antigen Uptake, Mucosal and System Immunity, and Protection across Serotype. J Immun. 2014; 192(4):1630-40.
Arabshahi S, Nayeri Fasaei B, Derakhshande A, Novinrooz A. In silico desing of a novel chimeric Shigella IpaB fused to C terminal of Clostridum perfringens entrotoxin as a vaccine candidate. Bioengineered. 2018;9(1):170-77.
Camacho A, Irache J, Gamazo C. Recent progress towards development of Shigella vaccine. Expert Rev Vaccines.2013;12:43–55.
