کلون سازی و بیان پروتین نوترکیب UreB-Omp18 از سویه ی ایرانی هلیکوباکتر پیلوری
محورهای موضوعی :
میکروب شناسی تشخیصی
حسن سیدحمزه
1
,
صفر فرج نیا
2
,
محمد کارگر
3
,
فرشید کفیل زاده
4
,
بهزاد برادران
5
1 - دانشگاه ازاد واحد جهرم دانشکده علوم پایه گروه زیست شناسی
2 - دانشگاه علوم پزشکی تبریز
3 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
4 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
5 - مرکز تحقیقات ایمونولوژی، دانشگاه علوم پزشکی تبریز، تبریز، ایران.
تاریخ دریافت : 1401/01/28
تاریخ پذیرش : 1401/05/05
تاریخ انتشار : 1401/06/15
کلید واژه:
هلیکوباکتر پیلوری,
ureB,
omp18,
pET-22b وسترن بلات,
چکیده مقاله :
سابقه و هدف: هلیکوباکتر پیلوری عامل عفونت های مزمن گوارشی شناخته شده است. درمیان روش های تشخیصی، تست سرولوژیک یک روش دردسترس با حساسیت قابل قبول است. اما اختصاصیت پایین، کاربرد آن را محدود می کند. هدف از این مطالعه طراحی، کلون سازی و بیان پروتین نوترکیب حاصل از دو ژنureB وomp18 از سویه بومی ایرانی می باشد که می تواند به‌عنوان یک الگو در طراحی کیت سرولوژیک با اختصاصیت بالا به منظور تشخیص عفونت هلیکوباکتر پیلوری به کار رود.مواد و روش ها: پس از استخراج DNA ژنومی هلیکوباکتر پیلوری، ژن های ureB وomp18 با واکنش PCR تکثیر شده و بعد از برش آنزیمی در وکتور بیانی pET-22b کلون گردید. بیان پروتین نوترکیب حاصله با کمک IPTG القا و با خلوص بالا با کروماتوگرافی میل-ترکیبی (Affinity Chromatography) تخلیص شد. خاصیت آنتی ژنی پروتین نوترکیب تخلیص شده با روش وسترن بلاتینگ تایید گردید.یافته ها: دو قطعه ی ژنی ureB وomp18 به ترتیب با سایزهای 597 و 479 جفت باز توسط PCR تکثیر یافته و به شکل یک قطعه ی هیبرید در وکتور pET-22b کلون گردید. بیان پروتین نوترکیب حاصل در باکتری E. coli BL21(DE3) به شکل یک قطعه ی حدود 60 کیلودالتونی در SDS-PAGE ظاهر گردیده و توسط ستون Ni-NTA تخلیص شد. نتایج وسترن بلات آنتی ژن کایمریک تخلیص شده با سرم بیماران مبتلا نشان دهنده ویژگی آنتی ژنیِ این پروتین نوترکیب بود.نتیجه گیری: در مطالعه ی حاضر برای اولین بار پروتین نوترکیب UreB- Omp18 از سویه بومی این باکتری تولید گردید که میتواند گزینه مناسبی برای طراحی کیت تشخیصی در منطقه باشد.
چکیده انگلیسی:
Background & Objectives: Helicobacter pylori (H. pylori) infection is accepted of chronic gastritis. Among the diagnostic methods, serological tests are widely available and relatively sensitive to detect H. pylori infection. However, the low specificity limits its application. The present study was aimed for designing, cloning and expression of UreB - Omp18 protein from Iranian H. pylori strain as a promising diagnostic candidate with high specificity.Materials & Methods: After extraction of genomic DNA from focal Helicobacter pylori strain, ureB and omp18 genes were amplified by primers designed for these genes by PCR reaction and cloned into pET-22b expression vector after enzymatic cleavage. The expression of the resulting recombinant protein was induced by IPTG and purified with high purity by affinity chromatography. The antigenic properties of the purified recombinant protein were confirmed by Western blotting.Results: In this study, two ureB and omp18 gene fragments were amplified by PCR as 597 and 479 bp fragments, respectively, and cloned as a hybrid fragment in the pET-22b vector. The expression of the recombinant protein in E. coli BL21 (DE3) appeared as a fragment of about 60 kDa on SDS-PAGE and was purified by Ni-NTA column. Western blot results of purified chimeric antigen with sera of H. pylori infected patients showed the antigenic properties of the recombinant protein.Conclusion: In the present study, for the first time, the recombinant UreB-Omp18 protein was produced from the native strain of Helicobacter pylori, which can be a suitable candidate for designing a Helicobacter pylori diagnostic kit in the region.
منابع و مأخذ:
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Li M, Oshima T, Horikawa T, Tozawa K, Tomita T, Fukui H, et al. Systematic review with meta‐analysis: vonoprazan, a potent acid blocker, is superior to proton‐pump inhibitors for eradication of clarithromycin‐resistant strains of Helicobacter pylori. Helicobacter. 2018; 23(4): e12495.
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Paloheimo L, Tiusanen T, Suovaniemi O, SyrjÄnen K. Serological Biomarker Test (GastroPanel®) in the Diagnosis of Functional Gastric Disorders, Helicobacter pylori and Atrophic Gastritis in Patients Examined for Dyspeptic Symptoms. Anticancer Research. 2021; 41(2): 811-9.
Darma A, Nugroho BST, Yoanna V, Sulistyani I, Athiyyah AF, Ranuh RG, et al. Comparison of Helicobacter pylori stool antigen, salivary IgG, serum IgG, and serum IgM as diagnostic markers of H. pylori infection in children. Iranian journal of microbiology. 2019; 11(3): 206.
Graham DY, Miftahussurur M. Helicobacter pylori urease for diagnosis of Helicobacter pylori infection: A mini review. Journal of advanced research. 2018; 13:51-7.
Morihara F, Fujii R, Hifumi E, Nishizono A, Uda T. Effects of vaccination by a recombinant antigen ureB138 (a segment of the β-subunit of urease) against Helicobacter pylori infection. Journal of medical microbiology. 2007; 56(6): 847-53.
Khalilpour A, Santhanam A, Lee CW, Saadatnia G, Velusamy N, Osman S, et al. Antigenic proteins of Helicobacter pylori of potential diagnostic value. Asian Pacific Journal of Cancer Prevention. 2013; 14(3): 1635-42.
Blaser N, Backert S, Pachathundikandi SK. Immune cell signaling by Helicobacter pylori: impact on gastric pathology. Helicobacter pylori in Human Diseases. 2019: 77-106.
Miftahussurur M, Yamaoka Y. Diagnostic methods of Helicobacter pylori infection for epidemiological studies: critical importance of indirect test validation. BioMed research international. 2016; 2016.
Crowe J, Dobeli H, Gentz R, Hochuli E, Stiiber D, Henco K. 6xffis-ni-nta chromatography as a superior technique in recombinant protein expression/purification. Protocols for gene analysis. 1994: 371-87.
Khosroshahi SA, Farajnia S, Ghiamirad M, Tanomand A, Veisi K, Rahbarnia L, et al. Development and evaluation of a single domain antibody against human epidermal growth factor receptor (EGFR). Protein expression and purification. 2016; 120: 59-64.
Atkinson NS, Braden B. Helicobacter pylori infection: diagnostic strategies in primary diagnosis and after therapy. Digestive diseases and sciences. 2016; 61(1): 19-24.
Vakil N. The cost of diagnosing Helicobacter pylori infection. Alimentary pharmacology & therapeutics. 2001; 15: 10-5.
Shafaie E, Saberi S, Esmaeili M, Karimi Z, Najafi S, Tashakoripoor M, et al. Multiplex serology of Helicobacter pylori antigens in detection of current infection and atrophic gastritis-A simple and cost-efficient method. Microbial pathogenesis. 2018; 119: 137-44.
Yan J, Mao Y-F, Shao Z-X. Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients. World journal of gastroenterology: WJG. 2005; 11(3): 421.
Mony TJ, Kwon H-S, Won M-K, Kang Y-M, Lee S-H, Kim S-Y, et al. Anti-urease immunoglobulin (IgY) from egg yolk prevents Helicobacter pylori infection in a mouse model. Food and Agricultural Immunology. 2019; 30(1): 662-76.
Khalilpour A, Kazemzadeh-Narbat M, Tamayol A, Oklu R, Khademhosseini A. Biomarkers and diagnostic tools for detection of Helicobacter pylori. Applied microbiology and biotechnology. 2016; 100(11): 4723-34.
Voland P, Hafsi N, Zeitner M, Laforsch S, Wagner H, Prinz C. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori. Infection and Immunity. 2003; 71(7): 3837-43.
Talebkhan Y, Ebrahimzadeh F, Esmaeili M, Zamaninia L, Nahvijoo A, Khedmat H, et al. Helicobacter pylori Omp18 and its application in serologic screening of infection. Current microbiology. 2011; 62(1): 325-30.
Keikha M, Eslami M, Yousefi B, Ghasemian A, Karbalaei M. Potential antigen candidates for subunit vaccine development against Helicobacter pylori infection. Journal of cellular physiology. 2019; 234(12): 21460-70.
Raoufi E, Akrami H, Khansarinejad B, Abtahi H. Expression and antigenic evaluation of Helicobacter pylori UreB fragment. Jundishapur Journal of Microbiology. 2017;10(5).
Talebkhan Y, Ebrahimzadeh F, Esmaeili M, Zamaninia L, Nahvijoo A, Khedmat H, Fereidooni F, Mohagheghi MA, Mohammadi M .Helicobacter pylori Omp18 and its application in serologic screening of infection.Curr Microbiol. 2011 Jan;62(1):325-30. doi: 10.1007/s00284-010-9694.
Khalilpour A, Osman S, Yunus MH, Santhanam A, Vellasamy N, Noordin R. Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity.BMC Res Notes. 2014 Nov 18;7:809. doi: 10.1186/1756-0500-7-809.PMID: 25406411
Sambrook J. FEF, Maniatis T. Molecular Cloning, A Laboratory manual. 3 Ed. New York: Cold Spring Harbor Laboratory Press; 2001.
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Take S, Mizuno M, Ishiki K, Hamada F, Yoshida T, Yokota K, et al. Seventeen-year effects of eradicating Helicobacter pylori on the prevention of gastric cancer in patients with peptic ulcer; a prospective cohort study. Journal of gastroenterology. 2015; 50(6), 638-644.
Li M, Oshima T, Horikawa T, Tozawa K, Tomita T, Fukui H, et al. Systematic review with meta‐analysis: vonoprazan, a potent acid blocker, is superior to proton‐pump inhibitors for eradication of clarithromycin‐resistant strains of Helicobacter pylori. Helicobacter. 2018; 23(4): e12495.
Baj J, Korona-Głowniak I, Forma A, Maani A, Sitarz E, Rahnama-Hezavah M, et al. Mechanisms of the Epithelial–Mesenchymal Transition and Tumor Microenvironment in Helicobacter pylori-Induced Gastric Cancer. Cells. 2020; 9(4):1055.
Kao C-Y, Sheu B-S, Wu J-J. Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis. Biomedical journal. 2016; 39(1): 14-23.
Ricci AD, Rizzo A, Brandi G. DNA damage response alterations in gastric cancer: Knocking down a new wall. Future Medicine; 2021.
Skrebinska S, Mégraud F, Bessède E. Diagnosis of Helicobacter pylori infection. Helicobacter. 2018; 23: e12515.
Paloheimo L, Tiusanen T, Suovaniemi O, SyrjÄnen K. Serological Biomarker Test (GastroPanel®) in the Diagnosis of Functional Gastric Disorders, Helicobacter pylori and Atrophic Gastritis in Patients Examined for Dyspeptic Symptoms. Anticancer Research. 2021; 41(2): 811-9.
Darma A, Nugroho BST, Yoanna V, Sulistyani I, Athiyyah AF, Ranuh RG, et al. Comparison of Helicobacter pylori stool antigen, salivary IgG, serum IgG, and serum IgM as diagnostic markers of H. pylori infection in children. Iranian journal of microbiology. 2019; 11(3): 206.
Graham DY, Miftahussurur M. Helicobacter pylori urease for diagnosis of Helicobacter pylori infection: A mini review. Journal of advanced research. 2018; 13:51-7.
Morihara F, Fujii R, Hifumi E, Nishizono A, Uda T. Effects of vaccination by a recombinant antigen ureB138 (a segment of the β-subunit of urease) against Helicobacter pylori infection. Journal of medical microbiology. 2007; 56(6): 847-53.
Khalilpour A, Santhanam A, Lee CW, Saadatnia G, Velusamy N, Osman S, et al. Antigenic proteins of Helicobacter pylori of potential diagnostic value. Asian Pacific Journal of Cancer Prevention. 2013; 14(3): 1635-42.
Blaser N, Backert S, Pachathundikandi SK. Immune cell signaling by Helicobacter pylori: impact on gastric pathology. Helicobacter pylori in Human Diseases. 2019: 77-106.
Miftahussurur M, Yamaoka Y. Diagnostic methods of Helicobacter pylori infection for epidemiological studies: critical importance of indirect test validation. BioMed research international. 2016; 2016.
Crowe J, Dobeli H, Gentz R, Hochuli E, Stiiber D, Henco K. 6xffis-ni-nta chromatography as a superior technique in recombinant protein expression/purification. Protocols for gene analysis. 1994: 371-87.
Khosroshahi SA, Farajnia S, Ghiamirad M, Tanomand A, Veisi K, Rahbarnia L, et al. Development and evaluation of a single domain antibody against human epidermal growth factor receptor (EGFR). Protein expression and purification. 2016; 120: 59-64.
Atkinson NS, Braden B. Helicobacter pylori infection: diagnostic strategies in primary diagnosis and after therapy. Digestive diseases and sciences. 2016; 61(1): 19-24.
Vakil N. The cost of diagnosing Helicobacter pylori infection. Alimentary pharmacology & therapeutics. 2001; 15: 10-5.
Shafaie E, Saberi S, Esmaeili M, Karimi Z, Najafi S, Tashakoripoor M, et al. Multiplex serology of Helicobacter pylori antigens in detection of current infection and atrophic gastritis-A simple and cost-efficient method. Microbial pathogenesis. 2018; 119: 137-44.
Yan J, Mao Y-F, Shao Z-X. Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients. World journal of gastroenterology: WJG. 2005; 11(3): 421.
Mony TJ, Kwon H-S, Won M-K, Kang Y-M, Lee S-H, Kim S-Y, et al. Anti-urease immunoglobulin (IgY) from egg yolk prevents Helicobacter pylori infection in a mouse model. Food and Agricultural Immunology. 2019; 30(1): 662-76.
Khalilpour A, Kazemzadeh-Narbat M, Tamayol A, Oklu R, Khademhosseini A. Biomarkers and diagnostic tools for detection of Helicobacter pylori. Applied microbiology and biotechnology. 2016; 100(11): 4723-34.
Voland P, Hafsi N, Zeitner M, Laforsch S, Wagner H, Prinz C. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori. Infection and Immunity. 2003; 71(7): 3837-43.
Talebkhan Y, Ebrahimzadeh F, Esmaeili M, Zamaninia L, Nahvijoo A, Khedmat H, et al. Helicobacter pylori Omp18 and its application in serologic screening of infection. Current microbiology. 2011; 62(1): 325-30.
Keikha M, Eslami M, Yousefi B, Ghasemian A, Karbalaei M. Potential antigen candidates for subunit vaccine development against Helicobacter pylori infection. Journal of cellular physiology. 2019; 234(12): 21460-70.
Raoufi E, Akrami H, Khansarinejad B, Abtahi H. Expression and antigenic evaluation of Helicobacter pylori UreB fragment. Jundishapur Journal of Microbiology. 2017;10(5).
Talebkhan Y, Ebrahimzadeh F, Esmaeili M, Zamaninia L, Nahvijoo A, Khedmat H, Fereidooni F, Mohagheghi MA, Mohammadi M .Helicobacter pylori Omp18 and its application in serologic screening of infection.Curr Microbiol. 2011 Jan;62(1):325-30. doi: 10.1007/s00284-010-9694.
Khalilpour A, Osman S, Yunus MH, Santhanam A, Vellasamy N, Noordin R. Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity.BMC Res Notes. 2014 Nov 18;7:809. doi: 10.1186/1756-0500-7-809.PMID: 25406411
Sambrook J. FEF, Maniatis T. Molecular Cloning, A Laboratory manual. 3 Ed. New York: Cold Spring Harbor Laboratory Press; 2001.
