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Development of transgenic Arabidopsis thaliana and Daucus carota plant producing the p24 capsid protein from HIV-1 subtype C for the use as an edible plant-based vaccine model
Örebro University, School of Science and Technology. Örebro University. (Biokemi)
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Place, publisher, year, edition, pages
Örebro, 2009. , 56 p.
Series
Licentiate theses in Life Science, 7
National Category
Biochemistry and Molecular Biology Biochemistry and Molecular Biology Natural Sciences Chemical Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:oru:diva-8700OAI: oai:DiVA.org:oru-8700DiVA: diva2:280174
Presentation
2009-04-29, Hörsal M, Örebro universitet, Örebro, 00:00 (English)
Opponent
Supervisors
Projects
Production of edible vaccines in plants
Available from: 2010-02-01 Created: 2009-12-08 Last updated: 2010-02-01Bibliographically approved
List of papers
1. Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs
Open this publication in new window or tab >>Production of the p24 capsid protein from HIV-1 subtype C in Arabidopsis thaliana and Daucus carota using an endoplasmic reticulum-directing SEKDEL sequence in protein expression constructs
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2009 (English)In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 66, no 1, 46-51 p.Article in journal (Refereed) Published
Abstract [en]

An optimized gene expression construct was designed in order to increase the accumulation of the HIV-1 subtype C p24 protein in Arabidopsis thaliana and carrot (Daucus carota) plants. An ER retention signal was introduced into the genetic construct generating a p24 protein containing a SEKDEL amino acid sequence at its C-terminus. Mature A. thaliana plants and carrot cells were transformed using Agrobacterium tumefaciens carrying the improved pGreen0229/p24_SEKDEL vector. Several transgenic plant lines were obtained from both plant species by growth on selective medium and confirmed by PCR. Transformed lines were analyzed for p24 protein content by western blotting using anti-p24-specific antibodies and by Southern blotting to establish the number of copies of the insert in the plant nuclear genome. To estimate the accumulation levels of p24 protein in the plants, ELISA was run using soluble plant extracts. By comparing these results with our previous findings, the ER retention signal increased the level of p24 protein 5-fold in the Arabidopsis thaliana plants. In carrot taproot, the content of p24_SEKDEL protein was approximately half of that in Arabidopsis on a fresh weight basis and was stable in planta for several months. However, on a total soluble protein basis, carrots produced considerable higher levels of the p24_SEKDEL protein than Arabidopsis.

Place, publisher, year, edition, pages
St. Louis: Academic Press, 2009
National Category
Natural Sciences Chemical Sciences Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:oru:diva-5166 (URN)10.1016/j.pep.2008.12.015 (DOI)000265346000008 ()19167502 (PubMedID)2-s2.0-63649152546 (Scopus ID)
Available from: 2009-11-05 Created: 2009-01-29 Last updated: 2017-02-23Bibliographically approved
2. Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses
Open this publication in new window or tab >>Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses
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2008 (English)In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 116, no 11, 985-994 p.Article in journal (Refereed) Published
Abstract [en]

Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not the least for use in developing countries. Therefore, we used Agrobacterium tumefaciens-mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV-1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR, four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by western blot analysis. The plants were shown to contain between 0.2 µg and 0.5 µg p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2-3, 2 and 1 T-DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T1 to the T4 generation and gave rise to the p24 protein in all cases, as verified by western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti-gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

Place, publisher, year, edition, pages
Oxford: Blackwell, 2008
National Category
Medical and Health Sciences Immunology in the medical area
Research subject
Immunology
Identifiers
urn:nbn:se:oru:diva-4631 (URN)10.1111/j.1600-0463.2008.00900.x (DOI)
Available from: 2008-10-14 Created: 2008-10-14 Last updated: 2017-03-27Bibliographically approved

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