|
|
Plant virus-based biotechnology
Vaculík, Petr
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
|
|
|
Plant virus-based biotechnology
Vaculík, Petr ; Čeřovská, Noemi (advisor) ; Ryšánek, Pavel (referee) ; Petrzik, Karel (referee)
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
|
|
|
Plant virus-based biotechnology
Vaculík, Petr
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
|
|
|
Heterologous expression of the E7 oncoprotein from human papillomavirus HVP16
Lidický, Ondřej ; Hudeček, Jiří (advisor) ; Mach, Otakar (referee)
Production of vaccines and pharmaceutical proteins in plants is a promising nascent technology with a great potential to provide high-quality, safe and non-expensive production and delivery platform. In this work we studied the experimental vaccine against human papillomavirus based on modified plant pathogen - Potato virus X (PVX). The experimental vaccine is based on PVX virus particles decorated with genetically fused HPV-E7 oncoprotein. These chimeric virus particles should be able to activate strong and specific cellular immune response. However the modification of the PVX coat protein with such relatively large fused protein might influence its ability to form particles. In this work we have characterized some properties of such chimeric virus particles like solubility or ability infect host plant. (In Czech)
|
|
|
Mutational analysis of Potato virus X (PVX) coat protein
Werschallová, Markéta ; Ryšánek, Pavel (advisor) ; Jan, Jan (referee)
The thesis deals with the mutational analysis of conserved amino acids of Potato virus X coat protein (PVX CP). The importance of selected amino acids for the spread of the virus in the plant should be determined. Nicotiana benthamiana was selected as an experimental plant. Mutations of the PVX CP were based on the comparison of PVX CP amino acid sequence with the sequence of Papaya mosaic virus coat protein (PapMV CP), the only representative of potexvirus, which includes PVX, with the described crystal structure of the CP. The importance of certain amino acids for interaction of coat protein subunits PapMV CP,CP and PapMV CP,RNA and thus for virus particles formation was experimentally determined. The available amino acid sequences of isolates and strains of PVX CP were obtained from the National Center for Biotechnology Information (NCBI) database, compared with each other and alsowith the sequence of PVX CP used in the Laboratory of Virology, Institute of Experimental Botany of the CAS. Codons encoding conserved phenylalanine and lysine at positions 33 and 118 in the PVX CP amino acid sequence were mutated using methods of molecular biology.
Five constructs of PVX CP mutants were prepared, Two deletion mutants of the PVX CP N-terminus which were created in the vector derived from PVX (pGR106) in which the cDNA sequence of PVX is integrated, the remaining 3 point mutants were prepared only as a product of SOE PCR. The reporter gene GFP for monitoring of infection and spread of mutants in plant tissue was cloned into pGR106 carrying the deletion mutants of the N-terminus of PVX CP (deletion of 2.-32. or 2.-33. amino acid). Both mutants are able to move only in a short distance from infected cells to adjacent cells within the inoculated N. benthamiana leaf. These two deletion mutants showed difference in the speed and in the extent of the GFP signal spread. Deletion mutant still possessing the codon for F33 showed faster onset of the GFP signal and was able to spread more rapidly to surrounding cells in comparison with deletion mutant, where the codon for F33 was removed. Other mutants carrying the point mutations were also prepared: the deletion mutant of the codon for F33 (deletion F33), the substitution mutant, in which the codon for phenylalanine at position 33 was replaced by the codon for alanine (F33A) and substitution mutant, where the codon for lysine at position 118 was replaced by the codon for alanine (K118A). Unfortunately, all three point mutants could not be cloned into the vector pGR106, therefore, the evaluation of their spread in N. benthamiana plants was not possible.
Based on obtained results it is possible to conclude that the amino acid F33 is important for the intercellular movement within the neighboring cells. To assess the importance of the amino acids F33 and K118 in the systemic infection, it would be necessary to evaluate also the point mutants deletion F33, F33A, and K118.
|
guest ::
