تأثیر بیان ژن P19 بر افزایش میزان رونویسی و تولید پروتئین نوترکیب پلاسمینوژن بافتی انسانی (rtPA) در گیاه توتون (Nicotiana benthamiana)

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه ژنتیک و به‌نژادی گیاهی، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

2 گروه بیوتکنولوژی کشاورزی، دانشکده کشاورزی، دانشگاه تربیت مدرس، تهران، ایران

3 تهران- گیشا- دانشگاه تربیت مدرس- دانشکده کشاورزی- گروه اصلاح نباتات و بیوتکنولوژی

چکیده

هدف: فعال‌کننده پلاسمینوژن بافتی (rtPA) یکی از مهم‌ترین داروها در درمان بیمارهای قلبی است. این دارو به‌صورت پروتئین نوترکیب در سیستم‌های بیانی تولید می‌شود که هزینه‌های تولید بسیار بالایی دارد. سیستم بیان موقت به‌دلیل بیان زیاد، سرعت ‌بالا، هزینه پایین و عدم تأثیرپذیری مکانی جهت بیان پروتئین بسیار مناسب می‌باشد. با این ‌وجود مشخص ‌شده است که خاموشی پس از رونویسی حاصل از کمپلکس RISC بر میزان بیان پروتئین نوترکیب تأثیر می‌گذارد. یکی از مهم‌ترین سرکوب کننده‏های خاموشی RNA شناخته شده در گیاهان، پروتئین P19 می‏باشد که از طریق میل ترکیبی زیادی که با siRNA دو رشته‏ای دارد به آن متصل می‌گردد و آن را تجزیه می‌کند و مانع خاموشی ژن می‌گردد. هدف از مطالعه حاضر بررسی تأثیر بیان هم‌زمان ژن P19 بر بیان ژن فعال‌کننده پلاسمینوژن بافتی (rtPA) در سطح رونویسی و پروتئین در گیاه توتون Nicotiana benthamiana بود.
مواد و روش‌ها: در تحقیق حاضر میزان بیان ژن rtPA در سطح رونویسی و پروتئین مورد بررسی قرار گرفت. در این تحقیق از تزریق هم‌زمان اگروباکتریوم حاوی ناقل دوتایی pCAMBIA1304-rtPA و اگروباکتریوم حاوی ناقل pCAMBIA1304-P19 در مقایسه با اگروباکتریوم حاوی تنها ناقل بیانی pCAMBIA1304-rtPA استفاده شد. نمونه­های برگی در روزهای 4، 7 و 10 روز پس از تلقیح با آگروباکتریوم تهیه شدند. سپس میزان رونویسی و پروتئین با استفاده از آزمون ReaTime PCR و الایزا محاسبه شد.
نتایج: نتایج آزمون Real Time PCR حاکی از افزایش 34 درصد میزان رونویسی ژن rtPA در حضور P19 نسبت به شاهد بود. بیشترین میزان رونویسی از ژن‌های P19 و rtPA باگذشت چهار روز از تلقیح گیاهان با اگروباکتریوم حاصل شد. نتایج الایزا نشان داد که میزان بیان پروتئین rtPA در حضور ژن P19 در روز هفتم و دهم پس از تلقیح به ترتیب 89 و 84 میکروگرم بر گرم وزن‌تر برگ بود که در مقایسه با شاهد به‌ترتیب به‌میزان 12 و 15 درصد بیشتر بود.
نتیجه‌گیری کلی: نتایج نشان داد که کاربرد P19 علاوه بر سرکوب خاموشی ژن، می‌تواند برای دست‌یابی به بیان در سطح بالا مؤثر باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Effect of P19 gene expression on transcription rate and production of recombinant human tissue plasminogen (rtPA) in tobacco (nicotiana benthamiana)

نویسندگان [English]

  • Yousef Sharafi 1
  • Mokhtar Jalali Javaran 2
  • Mohammad sadegh Sabet 3
1 Department of Genetics and Plant Breeding, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
2 Department of Agricultural Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
چکیده [English]

Objective
Tissue plasminogen activator is one of the most important drugs in the treatment of heart disease. This drug is produced in the expression system as a recombinant protein that has high production costs. Transient expression system is very suitable for protein expression because of its high expression, high speed, low cost and no spatial effect. Post-transcriptional silencing has been shown to affect expression levels. Therefore, the aim of this study was to investigate the effect of simultaneous expression of P19 silencing suppressor gene on transient expression of recombinant tissue plasminogen activator (rtPA) at transcriptional and protein levels in Nicotiana benthamiana.
 
 
Material and methods
To serve this purpose, the expression proportion of injected Agrobacterium tumefaciens containing a binary vector pCAMBIA1304-rtPA with agrobacterium containing pCAMBIA1304-P19 have been studied comparison with the expression level from Agrobacterium containing only the binary vector pCAMBIA1304-rtPA. Leaf samples were prepared on 4, 7, and 10 day post-inoculation with Agrobacterium. Transcription and then protein levels were calculated using the Real Time PCR and ELISA tests.
 
Results
The results of Real Time PCR test showed that rtPA transcript increased in the presence of P19. Also, 4 days after plant inoculation, the highest transcript levels were obtained from p19 and rtPA genes. ELISA results showed that the expression of rtPA protein in the presence of P19 was 89 and 84 µg.g-1 leaf weight at the 7 and 10 day after inoculation, respectively. This expression was 12 and 15% higher than of when agrobacterium-containing pCAMBIA1304-rtPA vector alone was used, respectively.
 
 Conclusion
The results showed that the use of transient expression method could be a suitable method for rtPA protein production.

کلیدواژه‌ها [English]

  • Agrobacterium
  • Transient Expression
  • Molecular Farming
  • Silencing Suppressor
Abdoli-Nasab M, Jalali-Javaran M, Cusidó RM et al. (2013) Expression of the truncated tissue plasminogen activator (K2S) gene in tobacco chloroplast. Mol Biol Rep 40, 5749–58.
Alvarez ML, Pinyerd HL, Topal E, Cardineau GA (2008) P19-dependent and P19-independent reversion of F1-V gene silencing in tomato. Plant Mol Biol 68, 61–79.
Arzola L, Chen J, Rattanaporn K et al. (2011) Transient co-expression of post-transcriptional gene silencing suppressors for increased in planta expression of a recombinant anthrax receptor fusion protein. Int J Mol Sci 12, 4975–4990.
Asgari M, Javaran MJ., Moieni A et al. (2014) Production of Human Tissue Plasminogen Activitor (tPA) In Cucumis sativus. Prep Biochem Biotech 44, 182-192.
Baruah DB, Dash RN, Chaudhari M, Kadam S (2006) Plasminogen activators: a comparison. Vasc Pharmacol 44, 1-9.
Boivin EB, Lepage É, Matton DP et al. (2010) Transient expression of antibodies in suspension plant cell suspension cultures is enhanced when co‐transformed with the tomato bushy stunt virus p19 viral suppressor of gene silencing. Biotechnol Prog 26, 1534-43.
Browne MJ, Tyrrell AWR, Chapman CG et al (1985) Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells. Gene 33, 279–284.
Burgyán J, Havelda Z (2011) Viral suppressors of RNA silencing. Trends in J Plant Sci 16, 265–272.
Csorba T, Kontra L, Burgyán J (2015) Viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence. J VIROL 479, 85-103.
Dodd I, Jalalpour S, Southwick W et al. (1986) Large scale, rapid purification of recombinant tissue-type plasminogen activator. Febs Lett 209, 13–17.
Dunoyer P, Lecellier C-H, Parizotto EA et al (2004) Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. The Plant Cell Online 16, 1235–1250.
Eamens A, Wang MB, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol 147, 456-68.
Fann CH, Guirgis F, Chen G et al. (2000) Limitations to the Amplification and Stability of Human Tissue-Type Plasminogen Activator Expression by Chinese Hamster Ovary Cells. Biotechnol Bioeng 2, 204–212.
Flemmig M, and Melzig MF (2012) Serine‐proteases as plasminogen activators in terms of fibrinolysis. J Pharm Pharmacol 64, 1025-1039.
Garabagi F, Gilbert E, Loos A et al. (2012) Utility of the P19 suppressor of gene-silencing protein for production of therapeutic antibodies in Nicotiana expression hosts. Plant Biotechnol J 10, 1118–1128.
Goodin MM, Dietzgen RG, Schichnes D et al. (2002) pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J 31, 375-383.
Goojani HG, Javaran MJ, Nasiri J et al. (2013) Expression and large-scale production of human tissue plasminogen activator (t-PA) in transgenic tobacco plants using different signal peptides. Appl Biochem biotechnol 169, 1940-1951.
Hahn BS, Sim JS, Kim HM et al. (2009) Expression and characterization of human tissue-plasminogen activator in transgenic tobacco plants. Plant Mol Biol Rep 27, 209-216.
Hsieh YC, Omarov RT, Scholthof HB (2009) Diverse and newly recognized effects associated with short interfering RNA binding site modifications on the Tomato bushy stunt virus p19 silencing suppressor. J Virol 83, 2188–2200.
Jalanko A, Pirhonen J, Pohl G, Hansson L (1990) Production of human tissue-type plasminogen activator in different mammalian cell lines using an Epstein-Barr virus vector. J Biotechnol 15, 155–168.
Jinek M, Doudna JA (2009) A three-dimensional view of the molecular machinery of RNA interference. Nature 457, 405-12.
Kohnert U, Rudolph R, Verheijen JH et al. (1992) Biochemical properties of the kringle 2 and protease domains are maintained in the refolded t-PA deletion variant BM 06.022. Protein Eng 5, 93-100.
Li J, Park E, Arnim AG, Nebenführ A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 15, 1–15.
Lindbo JA (2007) High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors. BMC biotechnol 7, 52-63.
Liu Z, Kearney CM (2010) A tobamovirus expression vector for agroinfection of legumes and Nicotiana. J Biotechnol 147, 151–159.
Livak KJ, Thomas DS (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25, 402-408.
Lombardi R, Circelli P, Villani ME et al. (2009) High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus. BMC Biotechnol pp, 96.
Masoumi Asl A, Jalali-Javaran M, Mahboudi F, Alizadeh H (2010) Cloning and expression of tissue plasminogen activator (t-pa) gene in tobacco plants. Sci Res Essays pp, 917-922.
Obukowicz MG, Gustafson ME, Junger KD et al. (1990) Secretion of active kringle-2-serine protease in Escherichia coli. Biochem 29, 9737–9745.
Omar A (2013) Effect of a silencing suppressor gene towards the expression of VP2 protein of highly virulent infectious bursal disease virus in tobacco. J Biosci Bioeng 2, 159–166.
Park JW, Faure-Rabasse S, Robinson MA et al. (2004) The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein. J Virol 323, 49–58.
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29, e45-e45.
Pogue GP, Vojdani F, Palmer KE et al. (2010) Production of pharmaceutical‐grade recombinant aprotinin and a monoclonal antibody product using plant‐based transient expression systems. Plant biotechnol J 8, 638-654.
Ranby M (1982) Studies on the kinetics of plasminogen activation by tissue plasminogen activator. BBA-Protein Struct Mol Enzymol 704, 461-469.
Saxena P, Hsieh YC, Alvarado VY et al. (2011) Improved foreign gene expression in plants using a virus‐encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotechnol J 9, 703-712.
Soleimani M, Davudi N, Fallahian F, Mahboudi F (2006) Cloning of Tissue Plasminogen Activator cDNA in Nonpathogenic Leishmania. Yakhteh Med J 8, 196–203.
Thomas DR, Walmsley AM (2014) Improved expression of recombinant plant-made hEGF. Plant Cell Rep 33, 1801–1814.
Voinnet O, Rivas S, Mestre P, Baulcombe D (2003) Retracted: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33, 949-56.
Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 3, 259–73.
Xu J, Dolan MC, Medrano G et al. (2012) Green factory: Plants as bioproduction platforms for recombinant proteins. Biotechnol Adv 30, 1171–1184.
Zangi M, Ofoghi H, Amini-Bayat Z, Ehsani P (2016) Utility of P19 Gene-Silencing Suppressor for High Level Expression of Recombinant Human Therapeutic Proteins in Plant Cells. Res Mol Med 4, 35-40.
Zhou B, Zhao X, Kawabata S, Yuhua L (2009) Transient expression of a foreign gene by direct incorporation of DNA into intact plant tissue through vacuum infiltration. Biotechnol Lett 31, 1811–1815.
WHO (2018). The top 10 causes of death: World Health Organization; [Availablefrom: https://www.who.int/en/news-room/fact-sheets/detail/the-top-10-causes-of-death.