کلون سازی و بیان ژن flab هلیکوباکتر پیلوری در سیستم یوکاریوتی به منظور تهیه واکسن
محورهای موضوعی : میکروب شناسی مولکولی
پویا خدادادی
1
,
محمد کارگر
2
,
مهدی بیژن زاده
3
,
عباس دوستی
4
,
شاپور آقایی
5
1 - گروه میکرویبولوژی، دانشگاه آزاد اسلامی، جهرم، ایران
2 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
3 - گروه ژنتیک پزشکی، دانشگاه جندی شاپور، دانشگاه عاوم پزشکی اهواز
4 - مرکز تحقیقات بیوتکنولوژی، دانشگاه آزاد اسلامی، شهرکرد
5 - گروه پزشکی داخلی، دانشگاه علوم پزشکی یاسوج
کلید واژه: RT-PCR, کلون سازی, واکسن نوترکیب, واژگان کلیدی: هلیکوباکتر پیلوری,
چکیده مقاله :
سابقه و هدف: داشتن فلاژل و تحرک از عوامل مهم در استقرار و بیماری زایی هلیکوباکتر پیلوری می باشد. flaB ، یکی از ژن های مهم کد کننده پروتئین تاژکی است که آنتی بادی تولید شده علیه آن نقش عمده ای در ایمنی زایی ایفا می کند. هدف از این پژوهش، ساخت سازواره ژنی حامل ژن flaB و بررسی بیان آن در سلول های یوکاریوتی به عنوان گزینه ای برای تولید واکسن می باشد.مواد و روش ها: کلون سازی و ساب کلونینگ ژن flaB در پلاسمیدهای pTZ57RT و pBudCE4.1 انجام شد. سپس صحت کلون های بدست آمده با روش های PCR، هضم آنزیمی دوگانه و توالی یابی تایید شد. پس از انتقال سازواره تایید شده به سلول های HDF، بیان ژن در سطح ترانس کریپتوم و پروتئوم به دو روش RT-PCR و SDS-PAGE بررسی شد.یافته ها: نتایج PCR نشان دهنده تکثیر قطعه 1563 جفت بازی مربوط به ژن flaB بود. کلون سازی ژن مورد نظر با استفاده از سه روش PCR، هضم آنزیمی دوگانه و توالی یابی تایید گردید. مشاهده باند های 1563 جفت بازی و 56 کیلودالتونی حاصل از RT-PCR و SDS-PAGE نشان دهنده بیان موفق این سازواره در سلول های HDF بود.نتیجه گیری: سازواره نوترکیب توانایی تولید پروتئین FlaB را در سلول های جانوری دارد، بنابراین با توجه به نتایج بدست آمده این سازواره می تواند به عنوان یک انتخاب به منظور تولید واکسن ژنی موثر علیه هلیکوباکتر پیلوری استفاده گردد.
Background & Objectives: Flagella and motility are important factors in bacterial colonization and pathogenicity. flaB is one of the important genes encoding the flagellar protein, which produces antibody against this gene play major role in immunogenesis. The aim of the present study was to make a flaB gene construct and to evaluate its expression in eukaryotic cells as a candidate for vaccine production. Materials & Methods: flaB cloning and subcloning was done in pTZ57RT and pBudCE4.1 plasmids. Then the accuracy of the obtained clones confirmed by PCR, double enzyme digestion and sequencing. After transferring the confirmed gene construct to the animal cells, the gene expression was checked at the level of transcriptome and proteome by RT-PCR and SDS-PAGE methods. Results: PCR results showed amplification of a1563 bp segment related to flaB gene. Desired gene cloning had confirmed by PCR, double enzyme digestion and sequencing. The observation of 1563 bp and 56 kDa bands from RT-PCR and SDS-PAGE indicates the successful expression of flaB in HDF cells.Conclusion: The recombinant gene construct can express the FlaB protein in animal cells. Therefore, according to the results, this construct can be used as a candidate to produce an effective gene vaccine against Helicobacter pylori.
1- Park HE, Park S, Nizamutdinov D, Seo JH, Park JS, Jun JS, Shin JI, Boonyanugomol W, Park JS, Shin MK, Baik SC. Antigenic Determinant of Helicobacter pylori FlaA for developing serological diagnostic methods in children. pathogens. 2022 Dec;11(12):1544.
2- Seong Kim J, Hoon Chang J, Il Chung S, Sun Yum J. Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility. Journal of bacteriology. 1999 Nov 15;181(22):6969-76.
3 - Khodadadi P, Kargar M, Bijanzadeh M, Doosti A, Aghaei S. Association of cagA, cagC, virB2, and vacA Subtypes of Helicobacter pylori with Adenocarcinoma Development in Iranian Patients. Jundishapur Journal of Microbiology. 2020 Jul 31;13(7).
4- Ghorbani-Dalini S, Kargar M, Doosti A, Najafi A. The relationship between Helicobacter pylori disease and bacterial count in stomach. Health. 2014;6(4):259–62.
-5Kargar M, Ghorbani-Dalini S, Doosti A, Najafi A. Five-year monitoring of considerable changes in tyrosine phosphorylation motifs of the Helicobacter pylori cagA gene in Iran. Journal of applied genetics. 2014 Aug;55:417-22.
6- LL, Oliveira MA, Goncalves MH, Chaves FK, Benigno TG, Gomes AD, et al. Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil.MemInst Oswaldo Cruz. 2014;109(8):1045–9. doi: 10.1590/0074-0276140279.
7-. Kargar M, Baghernejad M, Doosti A. Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains. Daru: Journal of Faculty of Pharmacy, Tehran University of Medical Sciences. 2010;18(2):137.
8- Heidari K, Kaboosi H, Jamali A, Ghaemi EA, Peyravii Ghadikolaii F. Prevalence of pathogenic genes cagA and vacA of helicobacter pylori isolated in patients with digestive disorders. Iranian Journal of Medical Microbiology. 2019 May 10;13(1):80-8.
9- Ishaq S, Nunn L. Helicobacter pylori and gastric cancer: a state of the art review. Gastroenterology and hepatology from bed to bench. 2015;8(Suppl1):S6.
10- Shiota S, Suzuki R, Yamaoka Y. The significance of virulence factors in Helicobacter pylori. Journal of digestive diseases. 2013 Jul;14(7):341-9.
11- Gu H. Role of Flagella in the Pathogenesis of Helicobacter pylori. Current microbiology. 2017 Jul;74:863-9.
12- Lertsethtakarn P, Ottemann KM, Hendrixson DR. Motility and chemotaxis in Campylobacter and Helicobacter. Annual review of microbiology. 2011 Oct 13;65:389-410
13- Kao CY, Sheu BS, Wu JJ. Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis. Biomedical journal. 2016 Feb 1;39(1):14-23.
14- Eslami E, Doosti A. Cloning and expression study of the hcpD gene of Helicobacter pylori. Journal of Ardabil University of Medical Sciences. 2017 Jan 1;17(1):46-57.
15- Doosti A, Ghasemi-Dehkordi P, Kargar M, Sharifi A. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (stx2) genes. Genetika. 2015;47(2):499-507.
16- Weel JF, van der Hulst RW, Gerrits Y, Roorda P, Feller M, Dankert J, et al. The interrelationship between cytotoxin-associated gene A, vacuolating cytotoxin, and Helicobacter pylori-related diseases. J Infect Dis. 1996; 173(5): 1171-1175.
17- Doosti A. Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E.coli. JMW. 2013 Jun; 5(3&4): 77-84. [Persian]
18- Sadeghi M, Doosti A. Cloning And Study Of Expression Of Helicobacter Pylori flaA gene In Eukaryotic System. ISMJ. 2017 Jul 10;20(3):245-56. [Persian]
19- Eaton KA, Suerbaum S, Josenhans C, Krakowka S. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 1996;64:2445e8.
20-Kheiri-Dastenaei S, Doosti A. Cloning and evaluation of expression of the Helicobacter pylori ureB gene by using pcDNA3. 1 (+) expression vector. JJUMS.2016 Aug 10;3(1):82-91.
21-Ansari H, Tahmasebi-Birgani M, Bijanzadeh M. DNA vaccine containing Flagellin A gene induces significant immune responses against Helicobacter pylori infection: An in vivo study. Iran J Basic Med Sci. 2021 Jun;24(6):796.
22- Yeh HY, Hiett KL, Line JE, Oakley BB, Seal BS. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins. Microbiological research. 2013 May 6;168(4):192-8.
23-Maleki M, Nassiri MR, Qazvini K. Cloning and Expression of Helicobacter Pylori cagA gene antigenic regions in E. coli. JFUMAY . 2016 Apr 10;6(1):113-9.[Persian]
24-Mehran MJ, Zendehbad SH, Malla SU. Cloning and expression of a partial UreA antigen for the production of vaccine against helicobacter pylori, the risk factor for gastric cancer. Asian J Pharm Clin Res. 2014 Jan 1;7(1):111-7.
_||_1- Park HE, Park S, Nizamutdinov D, Seo JH, Park JS, Jun JS, Shin JI, Boonyanugomol W, Park JS, Shin MK, Baik SC. Antigenic Determinant of Helicobacter pylori FlaA for developing serological diagnostic methods in children. pathogens. 2022 Dec;11(12):1544.
2- Seong Kim J, Hoon Chang J, Il Chung S, Sun Yum J. Molecular cloning and characterization of the Helicobacter pylori fliD gene, an essential factor in flagellar structure and motility. Journal of bacteriology. 1999 Nov 15;181(22):6969-76.
3 - Khodadadi P, Kargar M, Bijanzadeh M, Doosti A, Aghaei S. Association of cagA, cagC, virB2, and vacA Subtypes of Helicobacter pylori with Adenocarcinoma Development in Iranian Patients. Jundishapur Journal of Microbiology. 2020 Jul 31;13(7).
4- Ghorbani-Dalini S, Kargar M, Doosti A, Najafi A. The relationship between Helicobacter pylori disease and bacterial count in stomach. Health. 2014;6(4):259–62.
-5Kargar M, Ghorbani-Dalini S, Doosti A, Najafi A. Five-year monitoring of considerable changes in tyrosine phosphorylation motifs of the Helicobacter pylori cagA gene in Iran. Journal of applied genetics. 2014 Aug;55:417-22.
6- LL, Oliveira MA, Goncalves MH, Chaves FK, Benigno TG, Gomes AD, et al. Helicobacter pylori strains of asymptomatic children from a high-risk gastric cancer area in northeastern Brazil.MemInst Oswaldo Cruz. 2014;109(8):1045–9. doi: 10.1590/0074-0276140279.
7-. Kargar M, Baghernejad M, Doosti A. Role of NADPH-insensitive nitroreductase gene to metronidazole resistance of Helicobacter pylori strains. Daru: Journal of Faculty of Pharmacy, Tehran University of Medical Sciences. 2010;18(2):137.
8- Heidari K, Kaboosi H, Jamali A, Ghaemi EA, Peyravii Ghadikolaii F. Prevalence of pathogenic genes cagA and vacA of helicobacter pylori isolated in patients with digestive disorders. Iranian Journal of Medical Microbiology. 2019 May 10;13(1):80-8.
9- Ishaq S, Nunn L. Helicobacter pylori and gastric cancer: a state of the art review. Gastroenterology and hepatology from bed to bench. 2015;8(Suppl1):S6.
10- Shiota S, Suzuki R, Yamaoka Y. The significance of virulence factors in Helicobacter pylori. Journal of digestive diseases. 2013 Jul;14(7):341-9.
11- Gu H. Role of Flagella in the Pathogenesis of Helicobacter pylori. Current microbiology. 2017 Jul;74:863-9.
12- Lertsethtakarn P, Ottemann KM, Hendrixson DR. Motility and chemotaxis in Campylobacter and Helicobacter. Annual review of microbiology. 2011 Oct 13;65:389-410
13- Kao CY, Sheu BS, Wu JJ. Helicobacter pylori infection: An overview of bacterial virulence factors and pathogenesis. Biomedical journal. 2016 Feb 1;39(1):14-23.
14- Eslami E, Doosti A. Cloning and expression study of the hcpD gene of Helicobacter pylori. Journal of Ardabil University of Medical Sciences. 2017 Jan 1;17(1):46-57.
15- Doosti A, Ghasemi-Dehkordi P, Kargar M, Sharifi A. Generation of divalent DNA vaccine based on p39 and shiga-like toxin 2 (stx2) genes. Genetika. 2015;47(2):499-507.
16- Weel JF, van der Hulst RW, Gerrits Y, Roorda P, Feller M, Dankert J, et al. The interrelationship between cytotoxin-associated gene A, vacuolating cytotoxin, and Helicobacter pylori-related diseases. J Infect Dis. 1996; 173(5): 1171-1175.
17- Doosti A. Cloning of the gene encoding neurotoxin heavy chain of Clostridium botulinum in E.coli. JMW. 2013 Jun; 5(3&4): 77-84. [Persian]
18- Sadeghi M, Doosti A. Cloning And Study Of Expression Of Helicobacter Pylori flaA gene In Eukaryotic System. ISMJ. 2017 Jul 10;20(3):245-56. [Persian]
19- Eaton KA, Suerbaum S, Josenhans C, Krakowka S. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 1996;64:2445e8.
20-Kheiri-Dastenaei S, Doosti A. Cloning and evaluation of expression of the Helicobacter pylori ureB gene by using pcDNA3. 1 (+) expression vector. JJUMS.2016 Aug 10;3(1):82-91.
21-Ansari H, Tahmasebi-Birgani M, Bijanzadeh M. DNA vaccine containing Flagellin A gene induces significant immune responses against Helicobacter pylori infection: An in vivo study. Iran J Basic Med Sci. 2021 Jun;24(6):796.
22- Yeh HY, Hiett KL, Line JE, Oakley BB, Seal BS. Construction, expression, purification and antigenicity of recombinant Campylobacter jejuni flagellar proteins. Microbiological research. 2013 May 6;168(4):192-8.
23-Maleki M, Nassiri MR, Qazvini K. Cloning and Expression of Helicobacter Pylori cagA gene antigenic regions in E. coli. JFUMAY . 2016 Apr 10;6(1):113-9.[Persian]
24-Mehran MJ, Zendehbad SH, Malla SU. Cloning and expression of a partial UreA antigen for the production of vaccine against helicobacter pylori, the risk factor for gastric cancer. Asian J Pharm Clin Res. 2014 Jan 1;7(1):111-7.
