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.

Immobilizing antimicrobial substances onto biocompatible materials is an important approach for the design of novel, functionalized medical devices. By choosing antimicrobial substances from innate immune systems, the risk for development of resistance in pathogenic microbes is lower than if conventional antibiotics are used. Combining natural antimicrobial peptides and bactericidal enzymes with strong and elastic spider silk through recombinant protein technology would enable large-scale production of materials that could serve as functionalized wound dressings. Herein, fusion proteins with the engineered spider silk sequence 4RepCT and five different antimicrobial substances were constructed using two different strategies. In the first, the fusion proteins had a His-tag as well as a solubility-enhancing domain N-terminally to the antimicrobial agent during expression. The tags were cleaved and separated from the target protein during the purification process. The other approach provided a His-tag but no additional solubility domain. The antimicrobial agents included in the work were a charge engineered enzyme and four antimicrobial peptides herein called Peptide A, Peptide B, Peptide C and Peptide D. Four out of five fusion proteins could be expressed in Escherichia coli without exhibiting noticeable toxicity to the host. However, most target proteins were found in the non-soluble fraction. For D-4RepCT, neither soluble nor non-soluble proteins were identified. An operating strategy for expression and purification of antimicrobial spider silk proteins was developed, where the construct system providing the solubility-enhancing domain N-terminally to the antimicrobial sequence, and long time expression at low temperatures is a promising approach. The fusion proteins A-4RepCT and C-4RepCT could be produced in adequate amounts, and they proved to possess the ability to assemble into stable fibers. When incubating solutions of Escherichia coli on the functionalized silk material A-4RepCT, it showed to decrease the number of living bacteria in solution, in contrary to wild-type 4RepCT on which bacteria continued to proliferate. Initial studies of the viability of bacteria adhered to the surface of the functionalized spider silk are so far inconclusive. A larger sample size, complementary experiments and methodology optimization is needed for a proper assessment of antibacterial properties. However, preliminary results for the development of antimicrobial spider silk are positive, and the approach elaborated in this work is believed to be applicable for the construction of functional spider silk with a wide range of natural antimicrobial agents for future wound healing applications.