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Bestimmung der zellulären Ribosomenzahl bei der Influenza-Impfstoffherstellung in Säugerzellkultur

MPG-Autoren
http://pubman.mpdl.mpg.de/cone/persons/resource/persons86265

Büttner,  Robert
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Zitation

Büttner, R. (2011). Bestimmung der zellulären Ribosomenzahl bei der Influenza-Impfstoffherstellung in Säugerzellkultur. Bachelor Thesis, Otto-von-Guericke-Universität, Magdeburg.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0013-8BD5-F
Zusammenfassung
MDCK cells are routineously used as a host in cell culture based vaccine production since this cell line supports very efficient influenza virus replication. But despite the superior virus production characteristics of MDCK cells, product yields are relatively low, compared to other cell culture based bioprocesses like recombinant protein production. One strategy to optimize process yields is the identification and elimination of host cell associated bottle necks for virus replication. For example, virus replication completely depends on the host cell protein biosynthesis machinery. Limitations in the cellular protein synthesis capacity might therefore directly influence virus production and process yields, respectively. Ribosomes are a key component for cellular protein translation. Consequently we aimed to analyze the cellular ribosome content as a potential limiting factor for virus replication. In a first step, a Real-Time qRT-PCR based method from literature for the quantification of the cellular ribosome content in plant cells had to be adapted to adherent mammalian cells. The assay established here uses four artificial reference RNAs for the quantification of the absolute cellular ribosome copy number, which are added to cell lysates in defined concentrations. Subsequently 18S ribosomal RNA and reference RNAs are measured by Real-Time qRT-PCR and the absolute cellular ribosome copy number is calculated by creating a calibration curve from the reference RNAs. In this work, primers for the quantification of the reference RNAs and 18s RNA were designed and the reaction conditions optimized. To achieve comparable PCR efficiencies for all primer pairs, the primer concentrations were adjusted individually. Additionally, the specific sample dilution factors necessary for comparable PCR efficiencies of 18s RNA and reference RNAs, were determined. Last, a potential influence of the sample cell number and residual cellular DNA contamination on assay results was excluded. Next, the assay was applied for the quantification of the absolute ribosome copy number of MDCK cells in different growth phases. Exponentially growing MDCK cells contained 4.6 million ribosomes per cell whereas only 2.7 million ribosomes per cell were found for cells in the stationary phase. So the ribosome content differed significantly for MDCK cells in different growth phases. Finally, a first experiment concerning the influence of influenza virus infection on the cellular ribosome contend was conducted. Results indicate slightly higher ribosome content in infected cells, compared to uninfected controls. In summary, a reliable Real-Time qRT-PCR-based method for the quantification of the cellular ribosome content of adherent MDCK cells was established. The cellular ribosome copy number of MDCK cells was successfully determined and was found to be influenced by growth conditions and virus infection. Accordingly this work provides both the basis and motivation for advanced studies concerning the role of the cellular ribosome content for influenza virus replication.