|
|
|
| Expression and Purification of Zera Tag-induced Microparticles in Nicotiana benthamiana |
| 1, 1, 1, 1, |
|
|
|
|
Abstract Abstract Zera tag allowed its recombinant fusion antigens to form fully active protein microparticles in planta and markedly enhanced the immune response. Thus Zera tag-induced microparticles may be regarded as promising production and delivery vehicles for subunit vaccines. To test this hypothesis, Zera-GFP sequences were synthesized in vitro, and pJ Zera-GFP plant expression vector was constructed by subclone technology. The vectors were co-inoculated on Nicotiana benthamiana plants with pCBNoX HC-Pro vector, a plant binary vector expressing the RNA silencing suppressor of HC-Pro protein, through agroinfiltration. The epidermal cells expressing Zera-GFP in inoculated Nicotiana leaves were then surveyed by confocal laser scanning microscopy, and the relative expression level of Zera-GFP were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot respectively. All data were collected for the assessment of the potential of Zera tag-induced microparticles as delivery vehicles for subunit vaccines. And a simple purification technique for Zera tag-induced microparticles was established. The effect of Agrobacterium tumefaciens strains on the expression level of pJ Zera-GFP vector was tested firstly and the better results were obtained when using the EHA105 strains instead of GV3101strains. It was also found that Zera-GFP was expressed in the form of protein particles and they were not found to be toxic to the N. benthamiana tissues. The morphological characteristics of Zera-GFP particles in tobacco leaf cells were further observed. The particles were composed of small round spheres with different size and clear edge. The diameter of the sphere particles was between 0.5~2.5 μm, the spheres were close to each other and were closely clustered together. Based on the analysis of the factors such as particle size, morphology, uniformity and antigen content of Zera tag-induced particles, it is suggested that the particles had the potential to be a delivery vehicles of subunit vaccine. Then Zera-GFP particles were purified by sucrose density gradient and sucrose cushion centrifugation. The results showed that Zera-GFP particles could be purified from the inoculated tobacco leaves by both centrifugal technology and 60% sucrose cushion was more suitable for the purification of Zera-GFP particles. Otherwise, we speculated that the formation of Zera tag-induced particles in the plant cells was a dynamic process from soluble to aggregation. The results of this study provide a theoretical and practical basis for the study of the novel particulate vaccine delivery systems based on Zera tag.
|
|
Received: 20 March 2017
Published: 20 July 2017
|
|
|
|
| [1]Alvarez M L, Topal E, Martin F, et al.Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation[J].Plant Molecular Biology, 2010, 72(1):75-89[2]Anandalakshmi R, Pruss G J, Ge X, et al.A Viral Suppressor of Gene Silencing in Plants[J].Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(22):13079-13084[3]Bachmann M F, Jennings G T.Vaccine delivery: a matter of size,geometry,kinetics and molecular patterns[J].Nature Reviews Immunology, 2010, 10(11):787-796[4]Bobbala S, Hook S.Is There an Optimal Formulation and Delivery Strategy for Subunit Vaccines?[J].Pharmaceutical Research, 2016, 33(9):2078-2097[5]Bramwell V W, Perrie Y.Particulate delivery systems for vaccines: what can we expect?[J].Journal of Pharmacy & Pharmacology, 2006, 58(6):717-28[6]Chang J S, Choi M J, Cheong H S, et al.Development of Th1-mediated CD8+ effector T cells by vaccination with epitope peptides encapsulated in pH-sensitive liposomes[J].Vaccine, 2001, 19(27):3608-3614[7]Chen Y, Liu Y, Zhang G, et al.Human papillomavirus L1 protein expressed in Escherichia coli self-assembles into virus-like particles that are highly immunogenic[J].Virus Research, 2016, 0(220):97-103[8]Cleland J, Lim A, Daugherty A, et al.Development of a single-shot subunit vaccine for HIV-15. programmable in vivo autoboost and long lasting neutralizing response.[J].Journal of Pharmaceutical Sciences, 1998, 87(12):1489-1495[9]Company N, Nadal A, La Paz J L, et al.The production of recombinant cationic α-helical antimicrobial peptides in plant cells induces the formation of protein bodies derived from the endoplasmic reticulum[J].New Biotechnology, 2014, 12(1):81-92[10]Gleba Y Y, Tusé D, Giritch A.Plant viral vectors for delivery by Agrobacterium.[J]. Current Topics in Microbiology & Immunology, 2014, 375:155-192. doi: 10.1007/82_2013_352[J].Current Topics in Microbiology & Immunology, 2014, 0(375):155-192[11]Hofbauer A, Melnik S, Tschofen M, et al.The Encapsulation of Hemagglutinin in Protein Bodies Achieves a Stronger Immune Response in Mice than the Soluble Antigen[J].Frontiers in Plant Science, 2016, 7(48):142-142[12]Holsters M, Waele D D, Depicker A, et al.Transfection and transformation of Agrobacterium tumefaciens[J].Molecular Genetics and Genomics, 1978, 163(2):181-187[13]Koppolu B, Zaharoff D A.The effect of antigen encapsulation in chitosan particles on uptake,activation and presentation by antigen presenting cells[J].Biomaterials, 2013, 34(9):2359-2369[14]Liu X, Li S, Li C, et al.Enhanced expression of the human CD14 protein in tobacco using a 22-kDa alpha -zein signal peptide[J].Plant Cell, Tissue and Organ Culture (PCTOC), 2013, 112(1):9-18[15]Llompart B, Lloptous I, Marzabal P, et al.Protein production from recombinant protein bodies[J].Process Biochemistry, 2010, 45(11):1816-1820[16]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].Microbial Cell Factories, 2006, 5(1):S42-S42[17]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-e19474[18]Minu J, Dolors L M, Margarita T, et al.Proteomic characterisation of endoplasmic reticulum-derived protein bodies in tobacco leaves[J].BMC Plant Biology, 2012, 12(1):36-36[19]O'Hagan D T, Rahman D, Mcgee J P, et al.Biodegradable microparticles as controlled release antigen delivery systems[J].Immunology, 1991, 73(2):239-239[20]Parlane N A, Grage K, Lee J W, et al.Production of a particulate hepatitis C vaccine candidate by an engineered Lactococcus lactis strain[J].Applied & Environmental Microbiology, 2011, 77(24):8516-22[21]Parlane N A, Grage K, Mifune J, et al.Vaccines Displaying Mycobacterial Proteins on Biopolyester Beads Stimulate Cellular Immunity and Induce Protection against Tuberculosis[J].Clinical & Vaccine Immunology Cvi, 2012, 19(1):37-44[22]Reed S G, Bertholet S, Coler R N, et al.New horizons in adjuvants for vaccine development[J].Trends in Immunology, 2009, 30(1):23-32[23]Sandiswa M, Elizabeth M, Pêra F F P G, et al.Production of H5N1 Influenza Virus Matrix Protein 2 Ectodomain Protein Bodies in Tobacco Plants and in Insect Cells as a Candidate Universal Influenza Vaccine[J].Frontiers in Bioengineering & Biotechnology, 2015, 3(Pt 8):197-120[24]Silva A L, Rosalia R A, Varypataki E, et al.Poly-(lactic-co-glycolic-acid)-based particulate vaccines: Particle uptake by dendritic cells is a key parameter for immune activation[J].Vaccine, 2015, 33(7):847-854[25]Singh M, Kazzaz J, Ugozzoli M, et al.Polylactide-co-glycolide microparticles with surface adsorbed antigens as vaccine delivery systems[J].Curr Drug Deliv, 2006, 3(1):115-120[26]Sun H X, Xie Y, Ye Y P.ISCOMs and ISCOMATRIXVaccine[J].Vaccine, 2009, 27(33):4388-4401[27]Torrent M, Llompart B, Lasserre-Ramassamy S, et al.Eukaryotic protein production in designed storage organelles[J].BMC Biology, 2009, 7(1):5-5[28]Torrent M, Lloptous I, Ludevid M D.Protein body induction: a new tool to produce and recover recombinant proteins in plants.[J]. Methods in Molecular Biology, 2009, 483:193-193. doi: 10.1007/978-1-59745-407-0_11[J].Methods in Molecular Biology, 2009, 0(483):193-193[29]Velásquez A C, Chakravarthy S, Martin G B.Virus-induced gene silencing (VIGS) in Nicotiana benthamiana and tomato[J].Journal of Visualized Experiments, 2009, 0(28):1292-1292[30]Virgili-López G, Langhans M, Bubeck J, et al.Comparison of membrane targeting strategies for the accumulation of the human immunodeficiency virus p24 protein in transgenic tobacco[J].International Journal of Molecular Sciences, 2012, 14(7):13241-13265[31]Virgilio M D, Marchis F D, Bellucci M, et al.The human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin[J].Journal of Experimental Botany, 2008, 59(10):2815-2815[32]Wroblewski T, Tomczak A, Michelmore R.Optimization of Agrobacterium‐mediated transient assays of gene expression in lettuce,tomato and Arabidopsis[J].Plant Biotechnology Journal, 2005, 3(2):259-273 |
|
|
|
