کلون سازی و بیان پروتین نوترکیب UreB-Omp18 از سویه ی ایرانی هلیکوباکتر پیلوری
الموضوعات :
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.
تاريخ الإرسال : 16 الأحد , رمضان, 1443
تاريخ التأكيد : 28 الأربعاء , ذو الحجة, 1443
تاريخ الإصدار : 10 الثلاثاء , صفر, 1444
الکلمات المفتاحية:
هلیکوباکتر پیلوری,
ureB,
omp18,
pET-22b وسترن بلات,
ملخص المقالة :
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.
المصادر:
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.
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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.
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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.
