استفاده از دو شاتل وکتور درکلون سازی و بیان ژن اینترلوکین 11 در باسیلوس سوبتیلیس
محورهای موضوعی : زیست فناوری میکروبی
آتنا آذرنوش
1
,
مجید مقبلی
2
,
فرشید کفیل زاده
3
,
محمد کارگر
4
,
هوشنگ جمالی
5
1 - گروه میکروبیولوژی دانشگاه آزاد اسلامی جهرم
2 - عضوهیئت علمی /دانشگاه آزاد اسلامی واحد دامغان/دامغان /ایران
3 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
4 - دانشگاه آزاد اسلامی، واحد جهرم، گروه میکروبیولوژی
5 - دانشگاه ازاد اسلامی جهرم
استاد یار
کلید واژه: باسیلوس سوبتیلیس, شاتل وکتور, اینترلوکین-11, pHT43, pMR12,
چکیده مقاله :
سابقه و هدف: در بیماران سرطانی که تحت شیمی درمانی قرار می‌گیرند پلاکت خونشان پایین می‌آید اینترلوکین 11، یک سایتوکین افزاینده پلاکت خون است. اینترلوکین 11 نوترکیب انسانی، تنها دارویی است که برای درمان اثرات ناشی از شیمی درمانی تأیید شده است. در این تحقیق از دو شاتل وکتور pHT43 و pMR12 جهت کلون کردن و بیان ژن اینترلوکین 11در باسیلوس سوبتیلیس استفاده شد و میزان بیان در این دو وکتور بررسی گردید.مواد و روش ها: در این مطالعه ژن اینترلوکین 11 به صورت ساختاری بسته با دو جایگاه برشBamHI وXbal با اندازه نهایی bp609 طراحی و سنتز شد. سپس این ژن بر روی شاتل وکتورهای pHT43 و pMR12 کلون و به باکتری باسیلوس سوبتیلیس WB600 انتقال یافت. میزان بیان پروتین نوترکیب اینترلوکین 11 بوسیله معرف بردفورد مورد بررسی قرار گرفت.یافته ها: با روشPCR وجود ژن اینترلوکین 11 را بر روی شاتل وکتورهای pHT43 و pMR12 نشان می‌دهد. میزان بیان توسط باکتری حاملpMR12-int11 پس از 9 ساعت انکوباسیون بیشترین میزان یعنی حدود µg/mL 75 و باکتری حامل pHT43-int11 پس از 4 ساعت، به میزانµg/mL 61/4 بوده است.نتیجه گیری: میزان بیان پروتین نوترکیب اینترلوکین 11 با استفاده از وکتور pMR12 نسبت به وکتور pHT43 بیشتر می‌باشد و تاکنون چنین میزان بیانی برای اینترلوکین 11 گزارش نشده است. باسیلوس سوبتیلیس قادر به بیان و تولید پروتین اینترلوکین 11 می‌باشد و می‌توان از آن به عنوان یک میزبان مناسب برای تولید دارو استفاده نمود.
Background & Objectives: The recombinant human IL-11 is the only approved medicine used for treating chemotherapy-induced side effects. Platelet count decreases (thrombocytopenia) in cancer patients who undergo chemotherapy. Interleukin-11 (IL-11) is a platelet increasing cytokine. This study aimed to use two shuttle vectors of pHT43 and pMR12 for cloning the IL-11 gene and expression of its protein in Bacillus. subtilis and the expression level in these two vectors was investigated.Materials & Methods: In this study, the IL-11 gene was designed and synthesized as a closed structure with two restriction sites for BamH1 and XbaI enzymes and a final length of 609 bp. Then the gene was cloned in two shuttle vectors of pHT43 and pMR12 and transferred to B. subtilis WB 600. The expression level of the recombinant IL-11 was evaluated with the Bradford incubation, the pMR12-int11-carrying bacteria expressed higher levels of the protein (75 µg/mL) than pHT43-int11-carrying bacteria.Results: The results of PCR indicated that the IL-11 gene existed in shuttle vectors pHT43 and pMR12. The expression of this protein was about 75 /g / mL using pMR12 vector, which is higher than the bacterium carrying pHT43-int11.Conclusion: The expression level of the recombinant IL-11 protein with pMR12 vector was higher than pHT43 vector. This amount has not been reported so far for IL-11. B. subtilis can express and produce IL-11 and can be used as a source for drug production
1994;15(2-1): 9-45.
2. Tanaka.T, Kishimoto.T. The Biology and Medical Implications of Interleukin-6. Cancer
Immunol Res; 2(4) April 2014 chemistry. 1993;268(29) 21527-32.
3. Orazi A, Du X, Yang Z, Kashai M, Williams DA. Interleukin-11 prevents apoptosis and
accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy
and radiation. Laboratory investigation; a journal of technical methods and pathology. 1996;75
(1): 33-42.
4. Mehler MF, Rozental R, Dougherty M, Spray DC, Kessler JA. Cytokine regulation of neuronal
differentiation of hippocampal progenitor cells. Nature. 1993;362(6415):625.
5. Baumann H, Schendel P. Interleukin-11 regulates the hepatic expression of the same plasma
protein genes as interleukin-6. J. biol. chem. 1991;266(30): 2042-7.
6. Fukuda Y, Sassa S. Effect of interleukin-11 on the levels of mRNAs encoding heme oxygenase
and haptoglobin in human HepG hepatoma cells. Bioche. Bioph. Res. Commun. 1993;193(1):
297- 302.
7. Girasole G, Passeri G, Jilka RL, Manolagas SC. Interleukin-11: a new cytokine critical for
osteoclast development. J. clinic. invest. 1994;93(4): 1516-24.
8. Maier R, Ganu V, Lotz M. Interleukin-11, an inducible cytokine in human articular
chondrocytes and synoviocytes, stimulates the production of the tissue inhibitor of
metalloproteinases. J. biolog.1993 ;15;268(29):21527-32.
9. Peterson RL, Bozza MM, Dorner AJ. Interleukin-11 induces intestinal epithelial cell growth
arrest through effects on retinoblastoma protein phosphorylation. American j. patho. 1994;149
(3) 895-902.
10. Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner KJ, Dorner AJ.
Hematopoietic, immunomodulatory and epithelial effects of interleukin-11. Leukemia.
1999;13(9): 1307-15.
11. Kawashima I, Ohsumi J, Mita-Honjo K, Shimoda-Takano K, Ishikawa H, Sakakibara S, et al.
Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with
interleukin-11. FEBS letters. 1991;283(2):199-202.
12. Keller DC, Du XX, Srour EF, Hoffman R, Williams DA. Interleukin-11 inhibits adipogenesis
and stimulates myelopoiesis in human long-term marrow cultures. Blood. 1993;82(5):1428-35.
13. Montazeri-Najafabady N, Ghasemi Y, Ali Mobasher M, Ghasemian A, Rasoul-Amini S,
Ebrahimi. Codon Optimization, Cloning and Expression of Interleukin 11 in Two Different E.
coli. Sys.J.Pure App. Microb.2016;7(4):2717-2722.
14. McKinley D, Wu Q, Yang-Feng T, Yang YC. Genomic sequence and chromosomal location
of human interleukin-11 gene (IL11). Genomic.1993; 814(3),13.
15. Shabani D, Kermani SH. Interleukins and tumor tissue death factor. The first international
conference on new findings in biotechnology.2017; https://civilica.com/doc/633058
16. Ye J, Wang Z, Ye D, et al. Increased interleukin-11 levels are correlated with cardiac events in
patients with chronic heart failure. Mediators Inflamm. 2019;15754-10
17. Cook S.A, Schafer S. Hiding in Plain Sight: Interleukin-11 Emerges as a Master Regulator of
Fibrosis, Tissue Integrity, and Stromal Inflammation. Annu. Rev. Med. 2020; 71:263–76
18. Yuchen Li, Qingxin Wu, Yuxin Jin and Qian Yang.Antiviral activity of interleukin-11
as a response to porcine epidemic diarrhea virus infection. Vet. Res .2019; 50:111
19. Widjaja AA, Dong J, Adami E, et al. Redefining interleukin 11 as a regeneration-limiting
hepatotoxin. bioRxiv 830018. https://doi.org/10. 2019; 1101/830018
20. Hirayama F, Katayama N, Neben S, Donaldson D, Nickbarg EB, Clark SC, et al. Synergistic
interaction between interleukin-12 and steel factor in support of proliferation of murine lymphohematopoietic progenitors in culture. Blood.1994;83(1):8-92
21. Walmsley M, Butler DM,Marinova-Mutafchieva L, et al. An anti-inflammatory role for interleukin11 in established murine collagen-induced arthritis. Immunology, 1998; 95(1):31–37.
22. Anguita J, Barthold SW, Samanta S, et al. Selective anti-inflammatory action of interleukin-11
in murine Lyme disease: arthritis decreases while carditis persists. J. Infect. Dis. 1999;179
(3):734–37
23. Elshabrawy HA, Volin MV, Essani AB, et al. IL-11 facilitates a novel connection between
RA joint fibroblasts and endothelial cells. Angiogenesis.2018; 21(2):215–28
24. Kuo D, Ding J, Cohn IS, et al. HBEGF+ macrophages in rheumatoid arthritis induce
fibroblast invasiveness. Sci. Transl. Med. 2019; 11(491): 85-87.
25. Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ. Expressing antimicrobial peptide
cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol.
2014;98(8):3651-8.
26. Rashidi M R, Moghbeli M. Construction of an expression shuttle vector for Escherichia coli
and Bacillus subtilis. J. microbial word. 2013, 3(16), 188-197. [In Persian]
27. Martinez M. A, Dezar C, Baigori M, Sineriz F. Simple method for plasmid mediated
transformation of different Bacillus species. Biotech. Tech.1999; 13(5):337-340.
28. Tepler I, Elias L, Smith JW, et al. A randomized placebo-controlled trial of recombinant
human interleukin- 11 in cancer patients with severe thrombocytopenia due to chemotherapy.
Blood., 1996, (87): 3607- 3614.
29. Hirayama F, Katayama N, Neben S, Donaldson D, Nickbarg EB, Clark SC, et al. Synergistic
interaction between interleukin-12 and steel factor in support of proliferation of murine
lymphohematopoietic progenitors in culture. Blood. 1994; 83(1):8-92
30. Liu N-W, Huang X, Liu S, et al. Elevated BNP caused by recombinant human interleukin-11
treatment in patients with chemotherapy-induced thrombocytopenia. Supp. Care Cancer.
2019;27(11):4293– 98.
31. Miao J, Wang J, Peng S, Tang P, Zou M, Duan J, Zhao C, Ma X. Expression of human
interleukin-11 cDNA in E. coli. 1995;38(10):1202-9.
32. Tan H, Dan G, Gong H, Cao L. Purification and characterization of recombinant truncated
human interleukin-11 expressed as fusion protein in Escherichia coli. Biotech. letters. 2005;
27(13): 905-910.
33. Hosseinian H, Borzamin B. L- asparaginase enzyme gene cloning research and evaluation in
Bacillus subtilis. Cell.Mol. Biotech. News .2014; 4(16), 61-66[In Persian].
34. Sadeghi A, Mahdieh M, Salimi S. Production of Recombinant Human Interleukin-11 (IL-11)
in Transgenic Tobacco (Nicotiana tabacum). J. Plant Biotech.2016;43(4):432-437.
35. Farajnia S, Hasanpour R, Lotfipour F. Cloning and expression of human IL-11 in E. coli .
Pharma.Sci. 2010, 15(4), 353- 359
_||_
1994;15(2-1): 9-45.
2. Tanaka.T, Kishimoto.T. The Biology and Medical Implications of Interleukin-6. Cancer
Immunol Res; 2(4) April 2014 chemistry. 1993;268(29) 21527-32.
3. Orazi A, Du X, Yang Z, Kashai M, Williams DA. Interleukin-11 prevents apoptosis and
accelerates recovery of small intestinal mucosa in mice treated with combined chemotherapy
and radiation. Laboratory investigation; a journal of technical methods and pathology. 1996;75
(1): 33-42.
4. Mehler MF, Rozental R, Dougherty M, Spray DC, Kessler JA. Cytokine regulation of neuronal
differentiation of hippocampal progenitor cells. Nature. 1993;362(6415):625.
5. Baumann H, Schendel P. Interleukin-11 regulates the hepatic expression of the same plasma
protein genes as interleukin-6. J. biol. chem. 1991;266(30): 2042-7.
6. Fukuda Y, Sassa S. Effect of interleukin-11 on the levels of mRNAs encoding heme oxygenase
and haptoglobin in human HepG hepatoma cells. Bioche. Bioph. Res. Commun. 1993;193(1):
297- 302.
7. Girasole G, Passeri G, Jilka RL, Manolagas SC. Interleukin-11: a new cytokine critical for
osteoclast development. J. clinic. invest. 1994;93(4): 1516-24.
8. Maier R, Ganu V, Lotz M. Interleukin-11, an inducible cytokine in human articular
chondrocytes and synoviocytes, stimulates the production of the tissue inhibitor of
metalloproteinases. J. biolog.1993 ;15;268(29):21527-32.
9. Peterson RL, Bozza MM, Dorner AJ. Interleukin-11 induces intestinal epithelial cell growth
arrest through effects on retinoblastoma protein phosphorylation. American j. patho. 1994;149
(3) 895-902.
10. Schwertschlag US, Trepicchio WL, Dykstra KH, Keith JC, Turner KJ, Dorner AJ.
Hematopoietic, immunomodulatory and epithelial effects of interleukin-11. Leukemia.
1999;13(9): 1307-15.
11. Kawashima I, Ohsumi J, Mita-Honjo K, Shimoda-Takano K, Ishikawa H, Sakakibara S, et al.
Molecular cloning of cDNA encoding adipogenesis inhibitory factor and identity with
interleukin-11. FEBS letters. 1991;283(2):199-202.
12. Keller DC, Du XX, Srour EF, Hoffman R, Williams DA. Interleukin-11 inhibits adipogenesis
and stimulates myelopoiesis in human long-term marrow cultures. Blood. 1993;82(5):1428-35.
13. Montazeri-Najafabady N, Ghasemi Y, Ali Mobasher M, Ghasemian A, Rasoul-Amini S,
Ebrahimi. Codon Optimization, Cloning and Expression of Interleukin 11 in Two Different E.
coli. Sys.J.Pure App. Microb.2016;7(4):2717-2722.
14. McKinley D, Wu Q, Yang-Feng T, Yang YC. Genomic sequence and chromosomal location
of human interleukin-11 gene (IL11). Genomic.1993; 814(3),13.
15. Shabani D, Kermani SH. Interleukins and tumor tissue death factor. The first international
conference on new findings in biotechnology.2017; https://civilica.com/doc/633058
16. Ye J, Wang Z, Ye D, et al. Increased interleukin-11 levels are correlated with cardiac events in
patients with chronic heart failure. Mediators Inflamm. 2019;15754-10
17. Cook S.A, Schafer S. Hiding in Plain Sight: Interleukin-11 Emerges as a Master Regulator of
Fibrosis, Tissue Integrity, and Stromal Inflammation. Annu. Rev. Med. 2020; 71:263–76
18. Yuchen Li, Qingxin Wu, Yuxin Jin and Qian Yang.Antiviral activity of interleukin-11
as a response to porcine epidemic diarrhea virus infection. Vet. Res .2019; 50:111
19. Widjaja AA, Dong J, Adami E, et al. Redefining interleukin 11 as a regeneration-limiting
hepatotoxin. bioRxiv 830018. https://doi.org/10. 2019; 1101/830018
20. Hirayama F, Katayama N, Neben S, Donaldson D, Nickbarg EB, Clark SC, et al. Synergistic
interaction between interleukin-12 and steel factor in support of proliferation of murine lymphohematopoietic progenitors in culture. Blood.1994;83(1):8-92
21. Walmsley M, Butler DM,Marinova-Mutafchieva L, et al. An anti-inflammatory role for interleukin11 in established murine collagen-induced arthritis. Immunology, 1998; 95(1):31–37.
22. Anguita J, Barthold SW, Samanta S, et al. Selective anti-inflammatory action of interleukin-11
in murine Lyme disease: arthritis decreases while carditis persists. J. Infect. Dis. 1999;179
(3):734–37
23. Elshabrawy HA, Volin MV, Essani AB, et al. IL-11 facilitates a novel connection between
RA joint fibroblasts and endothelial cells. Angiogenesis.2018; 21(2):215–28
24. Kuo D, Ding J, Cohn IS, et al. HBEGF+ macrophages in rheumatoid arthritis induce
fibroblast invasiveness. Sci. Transl. Med. 2019; 11(491): 85-87.
25. Luan C, Zhang HW, Song DG, Xie YG, Feng J, Wang YZ. Expressing antimicrobial peptide
cathelicidin-BF in Bacillus subtilis using SUMO technology. Appl Microbiol Biotechnol.
2014;98(8):3651-8.
26. Rashidi M R, Moghbeli M. Construction of an expression shuttle vector for Escherichia coli
and Bacillus subtilis. J. microbial word. 2013, 3(16), 188-197. [In Persian]
27. Martinez M. A, Dezar C, Baigori M, Sineriz F. Simple method for plasmid mediated
transformation of different Bacillus species. Biotech. Tech.1999; 13(5):337-340.
28. Tepler I, Elias L, Smith JW, et al. A randomized placebo-controlled trial of recombinant
human interleukin- 11 in cancer patients with severe thrombocytopenia due to chemotherapy.
Blood., 1996, (87): 3607- 3614.
29. Hirayama F, Katayama N, Neben S, Donaldson D, Nickbarg EB, Clark SC, et al. Synergistic
interaction between interleukin-12 and steel factor in support of proliferation of murine
lymphohematopoietic progenitors in culture. Blood. 1994; 83(1):8-92
30. Liu N-W, Huang X, Liu S, et al. Elevated BNP caused by recombinant human interleukin-11
treatment in patients with chemotherapy-induced thrombocytopenia. Supp. Care Cancer.
2019;27(11):4293– 98.
31. Miao J, Wang J, Peng S, Tang P, Zou M, Duan J, Zhao C, Ma X. Expression of human
interleukin-11 cDNA in E. coli. 1995;38(10):1202-9.
32. Tan H, Dan G, Gong H, Cao L. Purification and characterization of recombinant truncated
human interleukin-11 expressed as fusion protein in Escherichia coli. Biotech. letters. 2005;
27(13): 905-910.
33. Hosseinian H, Borzamin B. L- asparaginase enzyme gene cloning research and evaluation in
Bacillus subtilis. Cell.Mol. Biotech. News .2014; 4(16), 61-66[In Persian].
34. Sadeghi A, Mahdieh M, Salimi S. Production of Recombinant Human Interleukin-11 (IL-11)
in Transgenic Tobacco (Nicotiana tabacum). J. Plant Biotech.2016;43(4):432-437.
35. Farajnia S, Hasanpour R, Lotfipour F. Cloning and expression of human IL-11 in E. coli .
Pharma.Sci. 2010, 15(4), 353- 359
