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Inverkan av positionella effekter, promotorer och terminatorer på proteinexpression, exemplifierat med multiprotein influenza-viruslika partiklar
KTH, School of Biotechnology (BIO).
2014 (Swedish)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesisAlternative title
Influence of positional effects, promoters and terminators on protein expression, exemplified by multi-protein influenza virus-like particles (English)
Abstract [en]

The existing seasonal influenza vaccine does not provide broad long-term protection against seasonal influenza and must be remanufactured yearly due to frequens mutations and reassortment of theinfluenza genes. A universal influenza vaccine with the ability to raise long lasting immunity is the focus of several studies, including the Edufluvac project. Edufluvac is based on virus-likeparticles, a modern recombinant platform wellsuited for vaccinatin applications. Redbiotec's rePAX® technology allows the generation of multivalent recombinant baculovirus which generatesvirus-like particles presenting multiple proteins on the surface in insect cell culture. Any effects oninsect cell culture protein expression brought on by the regulatory elements controlling each gene in the baculovirus, or by the genome position of the baculovirus genes, could affect the composition of the virus-like  particles. The ai of this thesis was to elicit a better understanding of the protein expression by analysing multi-protein influenza virus-like particles and virus-like particles encoding a reporter gene regulated by different promoter and terminator combinations. Different bivalent and tetravalent influenza gene bacmids were cloned as well as seven bacmids encoding a YFP gene regulated by different promoter and terminator combinations. Spodopera frugiperda cells weretransfected with the bacmids and harvested recombinant baculovirus was used to perform testexpressions in High-Five™ cells. The resulting protein expression levels from the bivalent andtetravalent recombinant baculovirus were analyzed and compared by Western blots and ELISA assays. The expression of YFP in infected Spodoptera and High-Five™ cells was monitored byfluorescence microscopy and measured with FACS to quantify protein expression differencesbetween the seven promoter and terminator combinations. Analysis of the bivalent constructs indicated that the order of the genes in a recombinant baculovirus does not affect the protein expression in High-Five™ cells. The analysis of the tetravalent constructs revelaed positionalvariations in expressin of the H1 and M1 genes, but the number of test expressions and recombinant baculovirus construct clones included in the analysis were not hogh enough to allow a definitive conclusion. Of the different promoter and terminator constructs highest mean fluorescence intensity was reached with the reference combination. The early promoter yielded mean fluorescent intensitites that were close to the values of the negative control in both cell lines. 

Place, publisher, year, edition, pages
Keyword [en]
Virus-like particles, influenza vaccine, baculovirus, insect cell culture, recombinant production
National Category
Engineering and Technology
URN: urn:nbn:se:kth:diva-163673OAI: diva2:801739
Available from: 2015-04-14 Created: 2015-04-10 Last updated: 2015-10-12Bibliographically approved

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