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Characteristics and gene expression levels of plant transformants modified by one related constructs shearing a gene for proteinase inhibitor.
BEZUNK, František
Tobacco plants were transformed by bacteria Agrobacterium tumefaciens bearing a plasmid construct with a fusion gene SPI2:GFP and selectable marker NPTII coding for resistance of transformants to antibiotic kanamycin. SPI2:GFP gene was created by a fusion of SPI2- (for serine protease inhibitor) and GFP gene (coding for a green fluorescent protein - GFP) sequences. Transformed tobacco plants were selected on a medium with kanamycin. Positively selected plants were selfed and segregation ratios for NPTII determined for each of genotypes. The presence and function of transgenic DNA was verified by methods of PCR, RT-PCR, Western blot and using the stereomicroscopic fluorescence study of transformants of T0 and T1 generations. This Transgenic genotypes of tobacco Nicotiana tabacum, cv. Petit Havana, SR1 WT showing the expression of the fused gene SPI2:GFP on a level of mRNA and GFP protein were obtained. Thesis was perfomed with a support of the project No. M106030 of Ministry of Education, Youth and Sports of the Czech Republic.
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The use of plants for the expression of Human papillomavirus vaccine
Dlabalová, Lucie ; Moravec, Tomáš (advisor) ; Fischer, Lukáš (referee)
Papillomaviruses are causing various diseases from skin warts to the lesions leading to malignant tumours and are widespread among people. For this reason, the current research is trying to develop methods for the production of inexpensive and effective vaccines against both Papillomaviruses and against all other infectious diseases. Currently animal and microbial expression systems are most frequently used for the production of biopharmaceuticals which have several drawbacks and their capacity is limited. This opens up the doors for plants - potentially very efficient producers of biopharmaceuticals. Currently there is rapid development towards the optimization and improvement of the results of plant expression systems and establishing the best and safest methods of their use. This paper summarizes and compares the advantages and disadvantages of different methods of plant transformation, leading either to stable production of the protein of interest in transgenic plants or to transient expression of recombinant virus infecting non-transgenic plants. Furthermore it analyzes the most appropriate plant species, which provide high yields combined with a transformation method and ease of cultivation, describes few basic ways of optimizing expression levels and outlines the future of plant expression systems.
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