在烟草叶片中瞬时表达具有生物活性的重组人纤溶酶原激活剂
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摘要
目的探索在植物中瞬时高效表达重组人纤溶酶原激活剂(rhPA)的可行性,探寻低成本rhPA表达新途径。方法构建表达rhPA的烟草花叶病毒(TMV)表达载体pTMV rhPA-NSK和Zera融合标签植物表达载体pJ Zera-rhPA;农杆菌渗滤接种受体植物Nicotiana benthamiana和N. excelsiana叶片;观察叶片接种部位表型变化;Western blot和ELISA测定和分析rhPA在受体烟草中的表达情况及表达产物特点;纤维蛋白琼脂糖平板法评价rhPA的体外生物活性。结果病毒载体实验结果显示,pTMV rhPA-NSK病毒载体在N. benthamiana和N. excelsiana叶片接种部位产生不同程度的坏死症状;在坏死组织中依然存在完整的rhPA蛋白;N. benthamiana中rhPA的表达量略高于N. excelsiana(P<0.05),约占接种叶片可溶性总蛋白的0.6%;表达的rhPA具有体外溶栓活性。Zera融合标签实验结果显示,pJ Zera-rhPA表达载体在烟草叶片接种部位未造成坏死;Zera标签与其融合蛋白rhPA之间可能存在着易断裂位点;Zera标签蛋白微粒的形成是一种逐步积累聚集的过程;在微粒形成过程中,部分可溶性多肽可能被包裹进了微粒。结论在烟草中可以快速地表达具有生物学活性的rhPA,植物病毒表达系统为低成本rhPA的生产提供一种富有潜力的新平台。
Objective To assess the potential of transient expression of recombinant human plasminogen activator(rhPA) in plants as a cost-effective approach for recombinant rhPA production. Methods Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay(FAPA). Results Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves(P<0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein(or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles.Conclusion Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.
Objective To assess the potential of transient expression of recombinant human plasminogen activator(rhPA) in plants as a cost-effective approach for recombinant rhPA production. Methods Tobacco mosaic virus-based expression vector pTMV rhPA-NSK and plant binary expression vector pJ Zera-rhPA were constructed by in vitro sequence synthesis and subcloning. The two vectors were inoculated on either Nicotiana benthamiana or N. excelsiana leaves via agroinfiltration. The expression of recombinant rhPA in Nicotiana leaves was examined using Western blotting and ELISA, and the in vitro fibrinolysis activity of plant-produced rhPA was assessed by fibrin agarose plate assay(FAPA). Results Five to nine days after infiltration with an Agrobacterium inoculum containing pTMV rhPA-NSK, necrosis appeared in the infiltrated area on the leaves of both Nicotiana plants, but intact recombinant rhPA was still present in the necrotic leaf tissues. The accumulation level of recombinant rhPA in infiltrated N. benthamiana leaves was significantly higher than that in N. excelsiana leaves(P<0.05). The yield of recombinant rhPA was up to 0.6% of the total soluble protein(or about 60.0 μg per gram) in the fresh leaf biomass at 7 days post-inoculation. The plant-derived rhPA was bioactive to convert inactive plasminogen to active plasmin. No necrosis occurred in pJ Zera-rhPA-infiltrated leaves. The Zera-rhPA protein was partially cleaved between the site of Zera tag and rhPA sequence in both Nicotiana leaves. We speculated that the formation of Zera tags-induced particles in the plant cells was a dynamic process of progressive aggregation in which some of the soluble polypeptides were encapsulated in these particles.Conclusion Enzymatically active recombinant rhPA can be rapidly expressed in tobacco plants using the plant viral ampliconbased system, which offers a promising alternative for cost-effective production of recombinant rhPA.
引文
[1] Gurman P, Miranda OR, Nathan A, et al. Recombinant tissue plasminogen activators(rtPA):A review[J]. Clin Pharmacol Ther,2015, 97(3):274-85.
[2] Bagheri RK, Keihanian F, Ahmadi M, et al. Successful treatment of prosthetic pulmonary valve thrombosis with Reteplase:a case report[J]. Int Med Case Rep J, 2018, 11(3):205-8.
[3] Fathi-Roudsari M, Maghsoudi N, Maghsoudi A, et al. Autoinduction for high level production of biologically active Reteplase in Escherichia coli[J]. Protein Expres Purif, 2018, 151:18-22.
[4] Lucas BK, Giere LM, Demarco RA, et al. High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector[J]. Nucleic Acids Res, 1996, 24(9):1774-9.
[5] Majidzadeh-AK, Khalaj V, Fatemeh D, et al. Cloning and expression of functional Full-Length human tissue plasminogen activator in pichia pastoris[J]. Appl Biochem Biotechnol, 2010, 162(7):2037-48.
[6] He Z, Lu R, Zhang T, et al. A novel recombinant human plasminogen activator:Efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats[J]. PLoS One, 2018, 13(8):e0201788.
[7] Aflakiyan S, Sadeghi HM, Shokrgozar M, et al. Expression of the recombinant plasminogen activator(Reteplase)by a non-lytic insect cell expression system[J]. Res Pharm Sci, 2013, 8(1):9-15.
[8] Goojani HG, Javaran MJ, Nasiri J, et al. Expression and Large-Scale production of human tissue plasminogen activator(t-PA)in transgenic tobacco plants using different signal peptides[J]. Appl Biochem Biotechnol, 2013, 169(6):1940-51.
[9] Hidalgo D, Abdoli-Nasab M, Jalali-Javaran MA, et al.Biotechnological production of recombinant tissue plasminogen activator protein(reteplase)from transplastomic tobacco cell cultures[J]. Plant Physiol Biochem, 2017, 118(9):130-7.
[10]Hemayatkar M, Mahboudi F, Majidzadeh-A KA, et al. Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae[J]. Biotechnol J, 2010, 5(11, SI):1198-206.
[11]Peng J, Gao JT, Zhang YC, et al. Recombinant expression of rt-PA gene(encoding Reteplase)in gametophytes of the seaweed Laminaria japonica(Laminariales, Phaeophyta)[J]. Sci China C Life Sci, 2008, 51(12):1116-20.
[12]Hidalgo D, Sanchez R, Lalaleo L, et al. Biotechnological production of pharmaceuticals and biopharmaceuticals in plant cell and organ cultures[J]. Curr Med Chem, 2018, 25(30):3577-3596.
[13]Lindbo JA. TRBO:a high-efficiency Tobacco Mosaic virus RNAbased overexpression vector[J]. Plant Physiol, 2007, 145(4):1232-40.
[14]Mohammadi E, Seyedhosseini-Ghaheh H, Mahnam K, et al.Reteplase:Structure, Function, and Production[J]. Adv Biomed Res,2019, 8(1):19.
[15]Zamanlu M, Farhoudi M, Eskandani M, et al. Recent advances in targeted delivery of tissue plasminogen activator for enhanced thrombolysis in ischaemic stroke[J]. J Drug Target, 2018, 26(2):95-109.
[16]Ganapathy M. Plants as bioreactors-A review[J]. Adv Tech Biol Med, 2016, 4(161):2379-1764.
[17]Javaran VJ, Shafeinia A, Javaran MJ, et al. Transient expression of recombinant tissue plasminogen activator(rt-PA)gene in cucurbit plants using viral vector[J]. Biotechnol Lett, 2017, 39(4):607-12.
[18]Hefferon K. Plant virus expression vectors:a powerhouse for global health[J]. Biomedicines, 2017, 5(3):44.
[19]Bally J, Jung H, Mortimer C, et al. The rise and rise of Nicotiana benthamiana:a plant for all reasons[J]. Annu Rev Phytopathol,2018, 56:405-426.
[20]Gils M, Kandzia R, Marillonnet S, et al. High-yield production of authentic human growth hormone using a plant virus-based expression system[J]. Plant Biotechnol J, 2005, 3(6):613-20.
[21]Huang Z, Santi L, Lepore K, et al. Rapid,high-level production of hepatitis B core antigen in plant leaf and its immol/Lunogenicity in mice[J]. Vaccine, 2006, 24(14):2506-13.
[22]Huang Z, Lepore K, Elkin G, et al. High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system[J]. Plant Biotechnol J, 2008, 6(2):202-9.
[23]Giritch A, Marillonnet S, Engler C, et al. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors[J]. Proc Natl Acad Sci USA, 2006, 103(40):14701-6.
[24]Dorokhov YL, Sheveleva AA, Frolova OY, et al. Superexpression of tuberculosis antigens in plant leaves[J]. Tuberculosis, 2007, 87(3):218-24.
[25]Matoba N, Husk AS, Barnett BW, et al. HIV-1 neutralization profile and plant-based recombinant expression of actinohivin, an Env glycan-specific lectin devoid of T-cell mitogenic activity[J]. PLoS One, 2010, 5(6):e11143.
[26]Pinkhasov J, Alvarez ML, Rigano MM, et al. Recombinant plantexpressed tumour-associated MUC1 peptide is immol/Lunogenic and capable of breaking tolerance in muc1.tg mice[J]. Plant Biotechnol J, 2011, 9(9):991-1001.
[27]Nausch H, Mikschofsky H, Koslowski R, et al. Expression and subcellular targeting of human complement factor c5a in Nicotiana species[J]. PLoS One, 2012, 7(12):e53023.
[28]He J, Li P, Lai H, et al. A plant-produced antigen elicits potent immol/Lune responses against West Nile Virus in mice[J]. Biomed Res Int,2014(1):952865.
[29]Shamloul M, Trusa J, Mett V, et al. Optimization and utilization of agrobacterium-mediated transient protein production in nicotiana[J]. J Vis Exp, 2014, 86(86):e51204.
[30]Llompart B, Lloptous I, Marzabal P, et al. Protein production from recombinant protein bodies[J]. Process Biochem, 2010, 45(11):1816-20.
[31]Llop I, Torrent M, Marzabal P, et al. Zera?, a novel technology for stable accumulation and easy recovery of recombinant proteins in eukaryotic protein-production hosts[J]. Microb Cell Fact, 2006, 5(1):S42.
[32]Torrent M, Llompart B, Lasserre-Ramassamy S, et al. Eukaryotic protein production in designed storage organelles[J]. BMC Biol,2009, 7(1):5.
[33]Lloptous I, Ortiz M, Torrent M, et al. The expression of a xylanase targeted to ER-protein bodies provides a simple strategy to produce active insoluble enzyme polymers in tobacco plants[J]. PLoS One,2011, 6(4):e19474.
[34]Virgilio MD, Marchis FD, Bellucci M, et al. The human immol/Lunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin[J]. J Exp Bot, 2008, 59(10):2815.
[35]Virgili-López G, Langhans M, Bubeck J, et al. Comparison of membrane targeting strategies for the accumulation of the human immol/Lunodeficiency virus p24 protein in transgenic tobacco[J].Int J Mol Sci, 2012, 14(7):13241-65.
[36]Liu XX, Sun L, Li CL, et al. Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha-zein signal peptide[J]. Plant Cell Tissue Organ Cult, 2013, 112(1):9-18.
[37]Alvarez ML, Topal E, Martin F, et al. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation J]. Plant Mol Biol, 2009, 45(1):S36-7.
[38]Company N, Nadal A, La PL, et al. The production of recombinant cationicα-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum[J]. New Biotech, 2014, 12(1):81-92.
[39]Jacquet N, Navarre C, Desmecht D, et al. Hydrophobin fusion of an influenza virus hemagglutinin allows high transient expression in Nicotiana benthamiana,easy purification and immol/Lune response with neutralizing activity[J]. PLoS One, 2014, 9(12):e115944.
[40]Joseph M. Proteomic characterisation of endoplasmic reticulumderived protein bodies in tobacco leaves[J]. BMC Plant Biol, 2012,12(1):36-36.
[2] Bagheri RK, Keihanian F, Ahmadi M, et al. Successful treatment of prosthetic pulmonary valve thrombosis with Reteplase:a case report[J]. Int Med Case Rep J, 2018, 11(3):205-8.
[3] Fathi-Roudsari M, Maghsoudi N, Maghsoudi A, et al. Autoinduction for high level production of biologically active Reteplase in Escherichia coli[J]. Protein Expres Purif, 2018, 151:18-22.
[4] Lucas BK, Giere LM, Demarco RA, et al. High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector[J]. Nucleic Acids Res, 1996, 24(9):1774-9.
[5] Majidzadeh-AK, Khalaj V, Fatemeh D, et al. Cloning and expression of functional Full-Length human tissue plasminogen activator in pichia pastoris[J]. Appl Biochem Biotechnol, 2010, 162(7):2037-48.
[6] He Z, Lu R, Zhang T, et al. A novel recombinant human plasminogen activator:Efficient expression and hereditary stability in transgenic goats and in vitro thrombolytic bioactivity in the milk of transgenic goats[J]. PLoS One, 2018, 13(8):e0201788.
[7] Aflakiyan S, Sadeghi HM, Shokrgozar M, et al. Expression of the recombinant plasminogen activator(Reteplase)by a non-lytic insect cell expression system[J]. Res Pharm Sci, 2013, 8(1):9-15.
[8] Goojani HG, Javaran MJ, Nasiri J, et al. Expression and Large-Scale production of human tissue plasminogen activator(t-PA)in transgenic tobacco plants using different signal peptides[J]. Appl Biochem Biotechnol, 2013, 169(6):1940-51.
[9] Hidalgo D, Abdoli-Nasab M, Jalali-Javaran MA, et al.Biotechnological production of recombinant tissue plasminogen activator protein(reteplase)from transplastomic tobacco cell cultures[J]. Plant Physiol Biochem, 2017, 118(9):130-7.
[10]Hemayatkar M, Mahboudi F, Majidzadeh-A KA, et al. Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae[J]. Biotechnol J, 2010, 5(11, SI):1198-206.
[11]Peng J, Gao JT, Zhang YC, et al. Recombinant expression of rt-PA gene(encoding Reteplase)in gametophytes of the seaweed Laminaria japonica(Laminariales, Phaeophyta)[J]. Sci China C Life Sci, 2008, 51(12):1116-20.
[12]Hidalgo D, Sanchez R, Lalaleo L, et al. Biotechnological production of pharmaceuticals and biopharmaceuticals in plant cell and organ cultures[J]. Curr Med Chem, 2018, 25(30):3577-3596.
[13]Lindbo JA. TRBO:a high-efficiency Tobacco Mosaic virus RNAbased overexpression vector[J]. Plant Physiol, 2007, 145(4):1232-40.
[14]Mohammadi E, Seyedhosseini-Ghaheh H, Mahnam K, et al.Reteplase:Structure, Function, and Production[J]. Adv Biomed Res,2019, 8(1):19.
[15]Zamanlu M, Farhoudi M, Eskandani M, et al. Recent advances in targeted delivery of tissue plasminogen activator for enhanced thrombolysis in ischaemic stroke[J]. J Drug Target, 2018, 26(2):95-109.
[16]Ganapathy M. Plants as bioreactors-A review[J]. Adv Tech Biol Med, 2016, 4(161):2379-1764.
[17]Javaran VJ, Shafeinia A, Javaran MJ, et al. Transient expression of recombinant tissue plasminogen activator(rt-PA)gene in cucurbit plants using viral vector[J]. Biotechnol Lett, 2017, 39(4):607-12.
[18]Hefferon K. Plant virus expression vectors:a powerhouse for global health[J]. Biomedicines, 2017, 5(3):44.
[19]Bally J, Jung H, Mortimer C, et al. The rise and rise of Nicotiana benthamiana:a plant for all reasons[J]. Annu Rev Phytopathol,2018, 56:405-426.
[20]Gils M, Kandzia R, Marillonnet S, et al. High-yield production of authentic human growth hormone using a plant virus-based expression system[J]. Plant Biotechnol J, 2005, 3(6):613-20.
[21]Huang Z, Santi L, Lepore K, et al. Rapid,high-level production of hepatitis B core antigen in plant leaf and its immol/Lunogenicity in mice[J]. Vaccine, 2006, 24(14):2506-13.
[22]Huang Z, Lepore K, Elkin G, et al. High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system[J]. Plant Biotechnol J, 2008, 6(2):202-9.
[23]Giritch A, Marillonnet S, Engler C, et al. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors[J]. Proc Natl Acad Sci USA, 2006, 103(40):14701-6.
[24]Dorokhov YL, Sheveleva AA, Frolova OY, et al. Superexpression of tuberculosis antigens in plant leaves[J]. Tuberculosis, 2007, 87(3):218-24.
[25]Matoba N, Husk AS, Barnett BW, et al. HIV-1 neutralization profile and plant-based recombinant expression of actinohivin, an Env glycan-specific lectin devoid of T-cell mitogenic activity[J]. PLoS One, 2010, 5(6):e11143.
[26]Pinkhasov J, Alvarez ML, Rigano MM, et al. Recombinant plantexpressed tumour-associated MUC1 peptide is immol/Lunogenic and capable of breaking tolerance in muc1.tg mice[J]. Plant Biotechnol J, 2011, 9(9):991-1001.
[27]Nausch H, Mikschofsky H, Koslowski R, et al. Expression and subcellular targeting of human complement factor c5a in Nicotiana species[J]. PLoS One, 2012, 7(12):e53023.
[28]He J, Li P, Lai H, et al. A plant-produced antigen elicits potent immol/Lune responses against West Nile Virus in mice[J]. Biomed Res Int,2014(1):952865.
[29]Shamloul M, Trusa J, Mett V, et al. Optimization and utilization of agrobacterium-mediated transient protein production in nicotiana[J]. J Vis Exp, 2014, 86(86):e51204.
[30]Llompart B, Lloptous I, Marzabal P, et al. Protein production from recombinant protein bodies[J]. Process Biochem, 2010, 45(11):1816-20.
[31]Llop I, Torrent M, Marzabal P, et al. Zera?, a novel technology for stable accumulation and easy recovery of recombinant proteins in eukaryotic protein-production hosts[J]. Microb Cell Fact, 2006, 5(1):S42.
[32]Torrent M, Llompart B, Lasserre-Ramassamy S, et al. Eukaryotic protein production in designed storage organelles[J]. BMC Biol,2009, 7(1):5.
[33]Lloptous I, Ortiz M, Torrent M, et al. The expression of a xylanase targeted to ER-protein bodies provides a simple strategy to produce active insoluble enzyme polymers in tobacco plants[J]. PLoS One,2011, 6(4):e19474.
[34]Virgilio MD, Marchis FD, Bellucci M, et al. The human immol/Lunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin[J]. J Exp Bot, 2008, 59(10):2815.
[35]Virgili-López G, Langhans M, Bubeck J, et al. Comparison of membrane targeting strategies for the accumulation of the human immol/Lunodeficiency virus p24 protein in transgenic tobacco[J].Int J Mol Sci, 2012, 14(7):13241-65.
[36]Liu XX, Sun L, Li CL, et al. Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha-zein signal peptide[J]. Plant Cell Tissue Organ Cult, 2013, 112(1):9-18.
[37]Alvarez ML, Topal E, Martin F, et al. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation J]. Plant Mol Biol, 2009, 45(1):S36-7.
[38]Company N, Nadal A, La PL, et al. The production of recombinant cationicα-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum[J]. New Biotech, 2014, 12(1):81-92.
[39]Jacquet N, Navarre C, Desmecht D, et al. Hydrophobin fusion of an influenza virus hemagglutinin allows high transient expression in Nicotiana benthamiana,easy purification and immol/Lune response with neutralizing activity[J]. PLoS One, 2014, 9(12):e115944.
[40]Joseph M. Proteomic characterisation of endoplasmic reticulumderived protein bodies in tobacco leaves[J]. BMC Plant Biol, 2012,12(1):36-36.