oru.sePublikationer
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
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
Örebro University, School of Science and Technology. (Örebro Life Science Center/Biokemi)
Evolutionsbiologiskt Centrum, Uppsala universitet.
Örebro University, School of Science and Technology. (Örebro Life Science Center/Biokemi)
Örebro University, School of Science and Technology. (Örebro Life Science Center/Biokemi)
Show others and affiliations
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. Vol. 66, no 1, 46-51 p.
National Category
Natural Sciences Chemical Sciences Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:oru:diva-5166DOI: 10.1016/j.pep.2008.12.015ISI: 000265346000008PubMedID: 19167502Scopus ID: 2-s2.0-63649152546OAI: oai:DiVA.org:oru-5166DiVA: diva2:158080
Available from: 2009-11-05 Created: 2009-01-29 Last updated: 2017-02-23Bibliographically approved
In thesis
1. 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
Open this publication in new window or tab >>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
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:nbn:se:oru:diva-8700 (URN)
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
2. Plant-produced STI vaccine antigens with special emphasis on HIV-1 p24
Open this publication in new window or tab >>Plant-produced STI vaccine antigens with special emphasis on HIV-1 p24
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Objective: To establish stable transgenic non-toxic plants as a platform for plant-based vaccine production as well as potential oral delivery system of vaccine antigens for sexually transmitted infections (STIs). The concept is to immunize the mucosal immune system present in the gut-associated lymphoid tissues (GALT). HIV-1 p24 subtype C protein has been used as the main antigen model, in parallel with an engineered unique chimeric MOMP antigen from Chlamydia trachomatis serovar E.

Methods: Chimeric MOMP and p24 vaccine antigens were successfully inserted into the nuclear genomes of Arabidopsis thaliana and Daucus carota via Agrobacterium-mediated gene transfer. The characteristics of the genetic inserts and corresponding mRNAs and recombinant proteins in planta were described using several methods, including northern, Southern, and western blotting, ELISA, and a commercial HIV Ag/Ab combination assay. Immunogenicity of the antigens was studied in mice models.

Results: Transgenes of both plant species expressing p24 or chimeric MOMP were successfully generated. Additional HIV-1 vaccine antigen candidates were introduced and the genetic inserts have been confirmed in Arabidopsis thaliana. The Arabidopsis thaliana expressing p24 and chimeric MOMP were demonstrated to be stable over generations and antigenicity analyses showed that plant-derived HIV-1 p24 and chimeric MOMP retained immunological epitopes when they were expressed in planta. Oral administration of transgenic plant material generated a priming effect of the immune competent cells present in the GALT, shown by the presence of antigen-specific-IgG in mice sera after boosting. Mice immunized with plant-derived HIV-1 p24 antigen were also analyzed for antigen-specific faecal IgA as well as cellular immune responses. However, detectable levels of the two latter immune responses were not observed. The Chlamydia trachomatis chimeric MOMP antigen was further evaluated for its potential as a vaccine antigen candidate, with positive results indicating a more rapid clearance of the Chlamydia trachomatis infection post immunization.

Conclusion: Stable non-toxic transgenic plants expressing either HIV-1 p24 or a novel  Chlamydia trachomatis chimeric MOMP antigens have successfully been developed. The two plant-produced STI vaccine antigens have in initial mice feeding studies provided important proof-of-concept for the oral vaccination approach. Now, immunization studies to expand, en-hance, and improve knowledge of the immune responses generated by the orally delivered transgenic plants are of high priority.

Place, publisher, year, edition, pages
Örebro: Örebro universitet, 2011. 86 p.
Series
Örebro Studies in Life Science, 8
Keyword
Plant-based vaccines, Arabidopsis thaliana, HIV-1, p24, GALT, mucosal immunity, Chlamydia trachomatis, MOMP, Daucus carota
National Category
Chemical Sciences
Research subject
Chemistry
Identifiers
urn:nbn:se:oru:diva-17242 (URN)978-91-7668-817-5 (ISBN)
Public defence
2011-10-28, Hörsal M, Örebro universitet, Fakultetsgatan 1, Örebro, 10:15 (English)
Opponent
Supervisors
Projects
Kemi/biokemi
Available from: 2011-09-13 Created: 2011-09-13 Last updated: 2011-10-18Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full textPubMedScopus

Search in DiVA

By author/editor
Lindh, IngridKalbina, IrinaSävenstrand, HelenaAndersson, SörenStrid, Åke
By organisation
School of Science and Technology
In the same journal
Protein Expression and Purification
Natural SciencesChemical SciencesBiochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 272 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf