Design, selection and production of recombinant proteins for prevention of infectious diseases

Detta är en avhandling från Institutionen för biokemi och biokemisk teknologi

Författare: Marianne Hansson; Kth.; [1998]

Nyckelord: ;

Sammanfattning: The development of subunit vaccines is presently one of themain strategies for the generation of new vaccines againstinfectious diseases. The use of recombinant DNA techniques hasfacilitated the develoment of new strategies for constructionand production of subunit vaccines. This thesis describesvarious such techniques, including (i) new general methods forgene assembly applied to antigen-encoding genes, (ii) asuggested strategy for bioprocess improvement applied to theproduction of a malaria vaccine candidate, (iii) thedevelopment of novel live bacterial vaccine delivery systems,based on non-pathogenic staphylococci, and (iv) thein vitroselection of a new type of antigen-bindingprotein.Two different strategies forde novogene assembly have been developed. A method forassembly and polymerization of DNA fragments was developed,based on the class-IIS restriction enzymeBspMI. The central and the C-terminal repeat regions ofthePlasmodium falciparummalaria blood-stage antigenPf155/RESA were both polymerized separately (designated M5 andM3, respectively), using this method. The assembled genes weresuccessfully expressed inE. colias fusions to the divalent IgG-binding proteinZZ, from staphylococcal protein A, and the human serum albumin(HSA) binding protein BB from streptococcal protein G,respectively. Full-length fusion proteins wereaffinity-purified on IgG- and HSA-Sepharose, respectively.Three copies of a respiratorial syncytial virus (RSV) G proteinencoding fragment (designated G3) was also assembled using thismethod. For assembly of theP. falciparumblood-stage antigen Ag332 repeat region, asolid-phase gene construction method was developed. The desirednumber of repeats was built up in a controlled and stepwisemanner. Genes encoding two, three or four copies of an Ag332epitope were expressed inE. colias fusions to ZZ and BB. Both these methods forgene assembly, and in particular the solid-phase strategy, havefound broad applications in thede novoconstruction of genes.Expanded bed adsorption was, for the first time for arecombinant product, used to recover the secreted ZZ-M5 fusionprotein directly from crudeE. coliculture medium by anion-exchange chromatography.In a single step, more than 90% of the produced recombinantmalaria vaccine candidate was recovered. The study demonstratedthat the initial genetic design can allow integration of unitoperations and thus improve the downstream productionprocess.In the context of live bacterial vaccine delivery, twodifferent non-pathogenic food grade staphylococci,Staphylococcus xylosusandS. carnosus, have been investigated, and expressionsystems allowing efficient surface display of foreign antigenicdeterminants have been described. These systems were used todisplay the malarial antigen M3 onS. xylosusandS. carnosus,and the RSV antigen G3 onS. xylosus. New techniques to verify exposure of therecombinant proteins on the bacterial surface were alsodeveloped.S. xylosuscells displaying the RSV G3 protein were usedto immunize mice orally, resulting in the induction of G3specific serum antibodies that were sustained for more than 20weeks.Using phage-display technology, a binding protein (affibody)capable of specific recognition of an RSV vaccine candidate wasselectedin vitro. Biopanning of a phage-displayed combinatoriallibrary, based on the IgG-binding Z domain, against a 101 aminoacid region (G2nata) of the G protein of RSV subgroup A,resulted in the isolation of an affibody, ZRSV1, recognizingthe G proteins of both subgroup A and B RSV. The binding siteof the ZRSV1-affibody was mapped and found to be in a regionproposed to be involved in virus-to-cell attachment and inwhich protective epitopes have been described. The potentialuse of affibodies as diagnostic tools or devices forpassive-vaccination applications, is discussed.Key words:affibody, bacterial surface display, expandedbed adsorption, fusion protein, gene assembly,in vitroselection, live bacterial vector, phagedisplay,Plasmodium falciparum, respiratory syncytial virus,staphylococcal protein A,Staphylococcus carnosus,Staphylococcus xylosus, streptococcal protein G, subunitvaccine© Marianne Hansson, 1998

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