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1	Die Rolle von durch rhGM-CSF aktivierten Makrophagen bei der Immunabwehr von Glioblastomen im orthotopen C6-Tumormodell der Ratte / The role of macrophages activated by rhGM-CSF in the immune defense of glioblastoma in the rodent orthotopic C6 tumor model Jawork, Anna January 2022 (has links) (PDF) Die Immunabwehr des Patienten stellt eine Schlüsselrolle bei der spontanen Tumorregression dar. Bisher zählten zytotoxische CD8-positive T Zellen und natürliche Killerzellen zu den wichtigsten zellulären Vertretern der Tumorkontrolle. Im Tierversuch konnte jedoch kein signifikanter Einfluss dieser Zellen auf die spontane Regression nachgewiesen werden. Allerdings fand sich eine hohe Anzahl an Makrophagen im Tumorgewebe. In vorangegangenen Untersuchungen zeigte sich bei der Depletion der Makrophagen mittels Clodronate im Tiermodell der Ratte ein deutlich gesteigertes Tumorwachstum. In der hier durchgeführten Versuchsreihe wurde nun der Einfluss von Makrophagen auf das Tumorwachstum orthotop implantierter C6-Glioblastomsphäroide betrachtet. Dabei wurden die Makrophagen durch den Granulozyten-Makrophagen Kolonie-stimulierenden Faktor (rhGM-CSF, Leukine) aktiviert. 29 SD-Ratten wurden C6-Gliom-Sphäroide orthotop implantiert. 20 der Tiere wurden jeden zweiten Tag mit 1µg/100g Körpergewicht rhGSM-CSF s. c. behandelt. Neun Tiere dienten als Kontrollgruppe. Zur Verlaufsbeurteilung wurden an den Tagen 7, 14, 21, 28, 32 und 42 nach Implantation MRT-Untersuchungen (T1, T2 und 3D CISS-Sequenzen) durchgeführt. Die Tumorvolumina wurden mit Hilfe dieser MRT-Untersuchungen ermittelt. Die histologische Aufarbeitung beinhaltete HE-, CD68-Makrophagen-, CD8-positive T Zellen- sowie Ki-67 Proliferations- Färbungen in Paraffinschnitten von Gehirn, Tumor und Milz. In 15 der 20 behandelten Tiere entwickelten sich solide Tumoren. Am Tag 7 konnte lediglich bei zwei Tieren mittels MRT ein minimales Tumorwachstum nachgewiesen werden. In der Kontrollgruppe war bereits bei drei von neun Tieren minimales Tumorwachstum zu verzeichnen. Am Tag 14 zeigten sich bei 11 von 17 (65%) Tieren der Versuchsgruppe solide Tumoren. Drei der verbleibenden 15 Tiere zeigten am Tag 21 erstmalig Tumorwachstum. Im Gegensatz dazu konnte in der Kontrollgruppe bereits an Tag 14 bei allen Tieren ein Tumorwachstum nachgewiesen werden. In der GM-CSF Gruppe entwickelten sich die Tumoren später und erreichten mit einem Median von 134mm³ ein geringeres Volumen als in der Kontrollgruppe (262mm³). Das mediane Überleben war mit 35 Tagen in der Gruppe der behandelten Tiere signifikant länger als in der Kontrollgruppe mit 24 Tagen. Zudem wurden in der histologischen Aufarbeitung der Tumoren signifikant mehr Makrophagen im Tumorgewebe nachgewiesen. Die Stimulation der Makrophagen durch GM CSF im orthotopen C6 Glioblastommodell der Ratte führte zu einem beachtlich reduzierten und verzögerten Tumorwachstum. Die behandelten Tiere überlebten signifikant länger als die Tiere der Kontrollgruppe. Die aktuelle Datenlage bestätigt die bedeutende Rolle der angeborenen Immunabwehr durch Makrophagen in der Kontrolle des Tumorwachstums bei experimentellen Glioblastomen. Die Aktivierung der Makrophagen hatte einen deutlichen Einfluss auf das Tumorwachstum, wohingegen eine T Zell-Depletion nur einen geringen Einfluss darauf hatte. Makrophagen als Vertreter des angeborenen Immunsystems wurden bisher in ihrer Rolle der Tumorkontrolle unterschätzt. Es bedarf noch weiterer Untersuchungen, ob die Makrophagen in Zukunft, ohne die körpereigenen Zellen anzugreifen, zur wirkungsvollen Tumorbekämpfung herangezogen werden könnten. / The patient's immune defense represents a key role in spontaneous tumor regression. Until now, cytotoxic CD8-positive T cells and natural killer cells were considered to be one of the most important cellular representatives of tumor control. The aim of the present study was to investigate the influence of macrophages on tumor growth of orthotopically implanted C6 glioma spheroids. Macrophages were activated by granulocyte-macrophage colony-stimulating factor (rhGM-CSF, Leukine). 29 Sprague-Dawley rats were implanted C6 glioma spheroids orthotopically. 20 of the animals were treated with 1μg/100g rhGSM-CSF s. c. every other day. Nine animals served as the control group. MRI examinations (T1, T2, and 3D CISS sequences) were performed on days 7, 14, 21, 28, 32, and 42 after implantation. Tumor volumes were determined using these MRI examinations. Histologic workup included HE, CD68, CD8, and Ki-67 staining in sections of brain and spleen. Tumors developed later and reached with a median of 126 mm³ a smaller size in the GM-CSF series compared to the controls with 150 mm³. Median survival was significantly longer in the treated group (35 days) compared with the control group (24 days). In addition, histological workup of the tumors showed significantly more macrophages in the tumor tissue. Stimulation of macrophages by GM-CSF in the rodent C6 glioma model resulted in reduced and delayed tumor growth. Treated animals survived significantly longer than in the control group. The current data confirm the important role of innate immune defense by macrophages in the control of tumor growth in experimental gliomas. Macrophage activation had a marked effect on tumor growth. Macrophages as representatives of the innate immune system have been underestimated in their role of tumor control. Glioblastoma multiforme Makrophagen Tumorwachstum Tiermodell ddc:610
2	Recombinant spider silk with antimicrobial properties Nilebäck, Linnea January 2013 (has links) 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. Spider silk antimicrobial peptides bactericidal enzymes recombinant production antibacterial assays Spindeltråd antimikrobiella peptider antibakteriella enzymer rekombinant produktion antibakteriella analyser
3	Kartläggning av bioproduktion i Sverige : Behov, hinder och drivkrafter Baczynska, Monika, Hafiz, Benjamin, MacCormack, Philip, Malmfors Sundheim, Hanna, Myhr, Nils, Skeppås, Madeleine January 2021 (has links) The market for drugs and treatments using biological products is rapidly growing. This is mainly due to the great potential that biological products have in comparison to traditional drugs. Biological products are composed of bigger molecules such as proteins and antibodies, and therefore allow a much more specific treatment. With the complexity of the molecules comes complexity in the upscaling of manufacturing. Start-up companies tend to lack knowledge of this process; consequently, outsourcing is often a requirement for the companies to grow. Outsourcing can be both risky and costly and the companies could benefit from having the skills in-house instead. This report provides information about needs, obstacles and driving forces regarding the future market for biological medicine and also outsourcing’s effect on the market. Desktop research was used to provide necessary information about the market and biological medicines, and also to find companies, research groups and investors that we mapped according to parameters, such as location and what field of medicine they engage in. Through phone and email contact, we gained further understanding about the actors we targeted. Desktop research and interviews were used to identify needs, obstacles and driving forces regarding the development of biological medicine. The result gives insight into where in Sweden the different candidates operate and it tells what pursuits and holdbacks they are facing when developing biological medicines. The major driving force is providing patients efficient treatments but also the opportunity to capitalise in a novel and successful type of industry. The major obstacle is first and foremost the great amount of money needed. Also, companies fail to grasp the many complex and time consuming steps in developing biological medicines. The interviews also showed that outsourcing is practically inevitable, especially for small companies, due to the difficulties of concentrating the competence in-house. In conclusion, the report shows that the benefits of biological medicine creates great driving forces for its development and that Sweden makes up a good climate for this. It also enlightens the importance of test beds to ensure that the future development relies on competence. Biologiska läkemedel kontraktstillverkning marknad kartläggning behov hinder drivkrafter bioproduktion mammalieceller testbäddar rekombinant produktion framtid expressionssystem läkemedelsutveckling Engineering and Technology Teknik och teknologier
4	Uttryck av ett nytt rekombinant protein Cp149 (HBV-kapsidprotein) modifierat med TfR apical domän / Expression of a new recombinant protein Cp149 (HBV capsid protein) modified with TfR apical domain Noorzai, Hamida January 2022 (has links) Hepatit-B är en leversjukdom som orsakas av hepatit-B-virus (HBV) vilket är en kapslad DNA-virus. Kapsidprotein (Cp) har stor betydelse i virusets livscykel exempelvis DNA-replikation, interaktion med värdceller och andra virala glykoproteiner. HBV, som många andra virus, tar sig in i cellen genom att binda till cellreceptorer. Transferrinreceptor är en välkänd receptor som mögliggör virus inträde i cellen genom att binda till virusproteiner, intraktionen sker i apikala domänen i TfR. Båda Cp149, kapsidsammansättnings domänen i Cp, och apikala domänen i TfR är betydelsefulla ändamål för utveckling av antivirala läkemedel. Syftet med arbetet var att klona och uttrycka olika varianter av ett nytt modifierat Cp149, där Cp149 har modifierats med AP01 (lösliga formen av apikala dömanen), och analysera intraktioner mellan proteinerna och viralt glykoprotein, MGP1. Modifierade proteingener klonades i plasmid (pET-11a) med hjälp av rekombinant DNA-teknik och användning av restriktionsenzymer NdeI och BamHI. Agarosgelelektrofores och DNA-sekvensering användes för att kontrollera förekomst av eftersökta DNA-sekvenser. Nya plasmider fördes över till bakterieceller, Escherichia coli, och proteinutrycket inducerades i bakteriecellerna genom kemiskbehandling. Framrenade proteiner från respektive provlösning analyserades med Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) och proteinernas funktion undersöktes med flödescytometri genom att besämma bindningsförmågan till MGP1, som uttrycktes på jästceller, i närvaro av TfR. Rekombinant plasmid innehållande proteingen kodande Cp149 för varianter A-D samt F lyckades att framställas. Resultatet från SDS-PAGE påvisade inga tydlyga protein-band och flödescytometri resultatet var svårt att bedömma, troligen då ytterliggare proteinupprening behövdes för att isolera kapsidproteinerna. Syftet med arbetet har erhållits delvis och fortsatt undersökningar på nya proteiner förslås. / Hepatitis B is one av the major worldwide health problems that is caused by enveloped DNA virus, Hepatitis B virus (HBV). HBV’s capsid protein (Cp) has an important role in the virus life cycle, för example DNA replication, intraction with host cells, and other viral glycoproteins. HBV, like many other viruses, enters the cells by binding to cell receptors. Transferrin receptor (TfR) is a well-known receptor that enables virus entry into the cell by binding to viral proteins. The interaction takes place in the apical domain of TfR. Both Cp149, the capsid’s composition domain of Cp, and the soluble form of the apical domain, AP01, from TfR are important builing parts to be explored as starting building blocks for the development of antiviral therapeutics. Modification of Cp149 with AP01 is an interesting combination to produce new protein-based drugs to prevent viral infections. The aim of this project was to clone and express six different variants of a AP01 modified Cp149 protein as a starting points to analyze interactions between the proteins and Machupo virus glykoprotein 1 (MGP1). All target DNA templates and plasmids (pET-11a) were digested with restriction enzymes, NdeI and BamHI, and ligated by T4 DNA ligase. Agarose gel electrophoresis and DNA sequencing were used as validation methods to confirm the presence of desired and corrected DNA sequences, respectively, during gene cloning. DNA transformation and induction of Escherichia coli cells was used to express the desired proteins. The purified proteins were validated for their binding ability to MGP1, expressed on yeast cells by flow cytometry in a competition assay with TfR. Recombinant plasmid including the expected DNA sequence encoding Cp149 for variants A-D and F was successfully produced. There was no clear detection of protein bands on Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) gel and flow cytometry results were difficult to interpret due to insufficient protein purification during ammonium sulphate percipitation. The purpose of the project has been obtained partially and more studies have to be carried out to produce pure proteins that can be used for further analysis. Gene cloning Recombinant DNA Transformation Protein expression SDS-PAGE Flow Cytometry Genkloning rekombinant DNA Transformering Proteinuttryck SDS-PAGE Flödescytometri Biomedical Laboratory Science/Technology
5	Identification of changes in biomarkers relevant for breast cancer biology occurring in a novel 3D-Biosilk model Ståhl, Emmy January 2021 (has links) Bröstcancer är den vanligaste formen av cancer som drabbar kvinnor. Det är en heterogen och komplex sjukdom som består av flera undergrupper, var och en med distinkt morfologi och kliniska implikationer [1]. För att modellera och studera cellbiologi, vävnadsmorfologi, molekylära mekanismer och läkemedels effekter används cellkulturer [2]. Idag är tvådimensionella (2D) modeller fortfarande den mest använda metoden för att odla celler in vitro [3]. En nackdel med 2D-modeller är att mikromiljön i dessa modeller inte imiterar in vivo strukturen av tumörer och vävnader, då de saknar tre dimensionella (3D) cell-cell och cellextracellulär matrix (ECM) interaktioner [2]. På grund av nackdelarna med 2D-modeller, har 3D-modeller blivit mer intressanta som alternativ för att lösa behovet av en pålitlig preklinisk modell för läkemedelstestning och för studier av cancerbiologi. För att utveckla ett redskap som är relevant för cancerforskning etablerar professor My Hedhammars laboratorium en 3D-modell av bröstcancer. I en sådan ny modell används Biosilk som byggnadsställning för att odla odödliga cellinjer som är representativa för de tre huvudklasserna av bröstcancer (i.e. MCF-7 (luminal-lik), SKBR-3 (HER2-överuttryckt) och MDAMB- 231 (trippel-negativ)). Eftersom transkriptions signaturer kan användas för att klassificera och studera bröstcancer är det viktigt att undersöka om och hur tillväxt i 3D-Biosilk kan påverka genuttrycksprofiler. Hypotesen som testades i denna studie var om cellkulturer i 3DBiosilk kan ha signifikanta skillnader i uttryck av biomarkörer, relevanta för bröstcancerbiologi, vid jämförelse av samma cellinje kultiverad i 2D. För att testa detta utvärderades kvalitén och reproducerbarheten av 3D-Biosilk konstruktionen med hjälp av olika kvalitetstester. Strukturen granskades med brightfield mikroskopi, arean av konstruktionen mättes med ImageJ, infärgning med phalloidin bekräftade cellnärvaro och cellvidhäftning till modellen. Alamar blue utfördes för att bedöma den cellulära metaboliska aktiviteten i modellen. Förändringarna av målgenernas genuttryck undersöktes med kvantitativ omvänd transkription PCR (RT-qPCR) och detta påvisade en statistiskt signifikant skillnad i genuttrycket beroende på om cellerna odlats i 2D- eller 3D-Biosilk modeller. I cellinje MDA-MB-231 hittades tre gener, i cellinje SKBR-3 hittades två gener och i cellinje MCF-7 hittades fyra gener. Genuttrycket för en av dessa gener i cellinje MCF-7, som var kultiverad i 3D-Biosilk, var nedreglerad (i.e. ZO-1). Detta kunde valideras på proteinnivå med immunofluorescens. Sammanfattningsvis, celler odlade i 3D-Biosilk visar på en mer aggressiv fenotyp. / Breast cancer is the most common cancer among women. It is a heterogenous and complex disease composed of several subtypes, each with distinct morphological and clinical implications [1]. To model and study cell biology, tissue morphology, molecular mechanisms and drug actions, cell cultures are canonically used [2]. Today two-dimensional (2D) models are still widely the preferred method for culturing cells in vitro [3]. A drawback with 2D models is that the microenvironment in these models does not mimic the in vivo structure of tumors and tissues, lacking three-dimensional (3D) cell-cell and cell-extracellular matrix (ECM) interactions [2]. Due to the disadvantages of 2D models, 3D cultures have become an increasingly interesting alternative to solve the need for a reliable preclinical model for drug testing and the study of cancer biology. To develop a relevant tool for cancer research, the laboratory of professor My Hedhammar is currently establishing a 3D model of breast cancer. In such novel model, Biosilk is used as scaffold to grow immortalized cell lines representative of the three major classes of breast cancer (i.e. MCF-7 (luminal-like), SKBR-3 (HER2-overexpression) and MDA-MB-231 (triplenegative)). Since transcriptional signatures can be used to classify and study breast cancers, it is important to investigate if and how growth in 3D-Biosilk can impact gene expression profiles. The hypothesis tested in this study was that cells cultured in 3D-Biosilk have differences in expression of biomarkers relevant to breast cancer biology, when compared to the same cell lines cultured in 2D. To examine this, 3D-Biosilk models were created and evaluated to ensure their quality and reproducibility, for instance, the scaffold structure was monitored by brightfield microscopy, the construct’s area was measured with ImageJ, staining with phalloidin confirmed the presence of cells as well as their attachment to the construct, and Alamar blue was used to assess the cellular metabolic activity. Differences in gene expression of target genes were investigated using reverse transcription quantitative PCR (RTqPCR), which revealed statistically significant changes depending on whether the cells were cultivated in 2D or a 3D-Biosilk model. For cell line MDA-MB-231 three genes were found, for SKBR-3 two genes were found and for MCF-7 four genes were found. The expression of one gene which was found downregulated in MCF-7 cultured in 3D-Biosilk (i.e. ZO-1) was validated at protein level by immunofluorescence. In conclusion, cultivating cells in 3D-Biosilk indicates a more aggressive phenotype. 3D-Biosilk breast cancer 3D cell culture recombinant spider silk ZO-1 3D-Biosilk bröstcancer 3D cellkultur rekombinant spindeltråd ZO-1 Biochemistry and Molecular Biology Biokemi och molekylärbiologi
6	Flödescytometrisk undersökning av inbindning mellan designade topdomänen från transferrinreceptorn till virala glykoproteiner för potentiell användning inom läkemedelsframtagning / Flow cytometric investigation of binding between the designed top domain of the transferrin receptor to viral glycoproteins for potential use in drug development Rydell, Emma January 2022 (has links) Machupovirus är ett virus som kan orsaka hemorragisk feber hos människor. Efter utvärdering av bindning mellan designade proteiner och virala glykoproteiner skulle proteinerna potentiellt kunna användas vid framtagning av ett proteinbasserat läkemedel mot hemorragisk feber. Syftet med studien var att efter riktad evolution och framrening av optimerade varianter av proteinet AP01 undersöka inbindningen till virala glykoproteiner mellan designade AP01 proteiner och transferrinreceptorn med hjälp av flödescytometrisk undersökning. Den fysiologiska nivån av järn i kroppen upprätthålls av transferrin (Tf) och transferrinreceptorn (TfR), ett transmembranprotein bestående av tre domäner. TfR apikala domän används av glykoprotein 1 (MGP1) och Plasmodium vivax för att ta sig in i celler genom receptormedierad endocytos. Med rekombinant genteknik kan rekombinanta plasmider skapas där en gen av intresse ligeras in i en plasmid med hjälp av DNA-ligas. I studien skapades rekombinanta plasmider pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 och S3.6 som transformerades till E. coli. Erhållna resultat från sekvensering visade att samtliga sex AP01-gener hade ligerats i vektorn men sekvensering av rekombinanta plasmider visade att endast pET29b+/AP01 S2.1, S2.2, S2.3 och S3.6 hade nukleotidsekvens utan mutationer. Proteinuttryck inducerades innan proteiner renades fram med immobilized metal ion affinity chromatography (IMAC). Den uppskattade molekylvikten hos de framrenade proteinerna var 18 kDa som bestämdes med sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) vilket överrenstämde med den teoretiska molekylvikten. Flödescytometri användes för att undersöka inbindningsförmågan mellan de uttryckta proteinerna och glykoprotein 1 (MGP1). Interaktionsbindningen mellan de designade proteinerna och MGP1 är bättre än interaktionen mellan originalgen AP01 och MGP1. De designade proteinerna visar på en svag effekt i den utförda ”competition assay” som gjorts vilket kan förklaras med en ej optimal struktur hos de designade proteinerna eller närvaro av BSA. / Machupovirus is a virus that can cause hemorragic fever in humans. After evaluating the binding between designed proteins and viral glycoproteins, the proteins could potentially be used in the development of a protein-based drug for hemorrhagic fever. The aim of the study was to investigate the binding to viral glycoproteins between designed AP01 proteins and the transferrin receptor after directed evolution and purification of optimized variants of the AP01 protein by means of flow cytometric examination. The physiological level of iron in the body is maintained by transferrin (Tf) and the transferrin receptor (TfR), a transmembrane protein consisting of three domains. The apical domain of TfR is used by glycoprotein 1 (MGP1) and Plasmodium vivax to enter cells through receptor mediated endocytosis. With recombinant DNA technology, recombinant plasmids can be created where a gene of interest is ligated into a plasmid using DNA ligase. In this study, recombinant plasmids pET29b+/AP01 S2.1, S2.2, S2.3, S3.3, S3.4 and S3.6 were created and transformed into E. coli. Sequencing results showed that all six AP01 genes had been ligated into the vector but sequencing of recombinant plasmids showed that only endast pET29b+/AP01 S2.1, S2.2, S2.3 and S3.6 had nucleotid sequence without mutations. Protein expression was induced before proteins were purified by immobilized metal ion affinity chromatography (IMAC). The estimated molecular weight of the purified proteins was 18 kDa as determined by sodium dodecyl sulfate – polyacrylamid gel electrophoresis (SDS-PAGE) which was consistent with the theoretical molecular weight. Flow cytometry was used to examine the binding ability between the expressed proteins and glycoprotein 1 (MGP1). The interaction binding between the designed proteins and MGP1 is better than the interaction between the original gene AP01 and MGP1. The designed proteins show a weak effect in the “competition assay” preformed, wich can be explained by a non-optimal structure how the designed proteins or the presence of BSA. Transferrin receptor recombinant DNA techniques protein purification yeast surface display flow cytometry Transferrinreceptor rekombinant genteknik proteinupprening yeast surface display flödescytometri Biochemistry and Molecular Biology Biokemi och molekylärbiologi
7	Stabil och antibiotikafri läkemedelsproduktion i rekombinant Escherichia coli Benevides, Kristina, Broström, Oscar, Elison Kalman, Grim, Swenson, Hugo, Vlassov, Andrei, Ågren, Josefin January 2017 (has links) Den här rapporten presenterar ett antibiotikafritt, stabilt och kromosombaserat expressionssystem för läkemedelsproduktion i Escherichia coli på beställning av företaget Affibody AB. E. coli-stammen BL21(DE3) valdes som värdorganism för expressionssystemet. Systemet består av en genkassett som innehåller en T7-promotor, en 5′-UTR från genen ompA och en terminatorsekvens från RNA-operonet rrnB. Fyra kopior av genkassetten ska integreras i pseudogenerna caiB, yjjM, hsdS och yjiV. En datormodell som modellerar det egentliga kopietalet i cellerna har skapats i mjukvaran MATLAB, vilket visar att det uppskattas vara maximalt 32 kopior av genkassetten per cell på grund av replikation av kromosomen. Ett högt pH i fermentorn; att använda fed-batch och blandade kolhydratkällor; och att använda stammen BL21(DE3) minskar acetatproduktionen i cellen. En lägre acetatproduktion kan leda till en högre produkthalt. En proteinutbytesmodell för mjukvaran MATLAB har konstruerats för att uppskatta koncentrationen av Affibody®-molekylen i en E. coli cell. copynumber copy-number copy number origin of replication rekombinant proteinexpression läkemedel E. coli antibiotikafri antibiotika expressionssystem expression system escherichia coli kromosom kromosombaserad kromosomal plasmidfri plasmid T7-promotor T7 promotor ompA rrnB autoinduktion auto induktion matlab kopietalmodell utbytemodell stabil pH pseudogen pseudo gen caiB yjjM hsdS yjiV fermentor batch fed-batch kontinuerlig fed batch fermentation Affibody affibody-molekyl affibody-molekyler affibodymolekyl affibodymolekyler bioreaktor acetat acetatbildning acetatproduktion acetatreduktion acetatreducering peptidläkemedel peptider peptid T7-system operator lac-operator operon 5'-UTR 3'-UTR sekundärstruktur mRNA T1 T2 terminator termineringssekvens optimering lac-operon repressor inducering BL21(DE3) BL21 B-stam B stam modell kopietal kopie tal OriC Pharmaceutical Biotechnology Läkemedelsbioteknik

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