Immobilization of biomolecures with antigenic properties in polymeric substrates for the production of diagnostic system and examination of Brucella melitensis biotypes

Καθήλωση βιομορίων με αντιγονικές ιδιότητες σε πολυμερικούς φορείς για την παραγωγή διαγνωστικού συστήματος και τον έλεγχο των ελληνικών βιοτύπων της Brucella melitensis

Immobilization of biomolecures with antigenic properties in polymeric substrates for the production of diagnostic system and examination of Brucella melitensis biotypes

Noutsios, Georgios

Νούτσιος, Γεώργιος

PhD Thesis

2009

The goals of this dissertation are the invention of new molecular methods and techniques for the diagnosis of brucellosis and the finding of new molecular markers that differentiate the contagious field strains from the vaccine strain Rev-1. The techniques that have been used for this purpose were the PCR, PCRRFLP, the Hypervariable Octrameric Oligonucleotide Finger print method, the DNA sequencing, the Denaturant Gradient Gel Electrophoresis analysis and production of antibodies against antigens of Br. melitensis. The samples of our study comprised of 44 field strains of Br. melitensis (whole blood, serum or tissue) originated exclusively from sheep and goats of Greek provinces, three reference strains of Br. melitensis (biovars 1, 2 and 3) and the vaccine strain Rev-1 that was kindly relinquished from the Department of Greek Veterinary services. All samples were tested with classical biochemical methods, were identified to be strains of Br. melitensis and ten different regions of their genonome were analyzed with PCR. We were able to identify and differentiate the attenuated vaccine strain Rev-1 from the contagious field strains. The fingerprinting method was implemented for genotyping of Br. melitensis and differentiation of strain Rev-1. The genotyping was based on the assessment of the number of complete repetitions of the nucleotide sequence AGGGCAGT, that has been found in eight loci of the Br. melitensis genome. In addition, the PCR-RFLP technique was successfully used for studying of the gene omp2, where there were found two homologous copies of the gene with different sites for the restriction enzyme PstI. Locus omp2 was also analyzed with DGGE. It was proved that the omp2 gene bares a polymorphism that can be used as an effective tool for the differentiation of vaccine strain Rev-1 from the natural contagious field strains of Br. melitensis. The above results were also validated with sequencing technique. It was confirmed that there is single site mutation of vaccine strain Rev-1. Lipopolysaccharides which are found in the surface of the extracellular membrane are the major component of Brucella antigen. LPS from different contagious field strains of Br. melitensis and Rev-1 vaccine strain were isolated. LPS is comprised of lipid A which is an oligosaccharide region that contains two types of aminoglycosides and other characteristic fatty acids, core polysaccharide which contains glucose, mannose and quinovosamine and O-antigen. The last component of LPS, O-antigen, is considered to be the most immunogenic. There has been elimination of the lipid A part of the LPS by using the precipitation protocol of magnesium chloride in ethanol solution. LPS deprived of lipid A from all samples were then electrophoresed in polyacrylamide gels and some major differences in their components’ construction were observed. In addition, the capillary zone electrophoresis has been used to achieve an in-depth analysis of the composition of LPS. There were found many saccharides that were common in the LPS of all strains but there were also found a few chemical compounds that were present in contagious field strains and absent in Rev-1 strain. Due to the fact that capillary zone electrophoresis is not a preparative technique, HPLC has been recruited so as to analyse the composition of LPS. This technique yielded more quantitative differences than quality differences as far as the components of LPS are concerned. LPS from contagious field strains and from vaccine strain Rev-1 have been injected subcutaneously into rabbits so as to produce polyclonal antibodies. After a few boosts of antigen stimulus, there have been produced three different antibodies against the LPS of contagious field strains and an antibody against LPS of vaccine strain Rev-1. These antibodies were tested with the immunoblot technique against the antigens of contagious field strains. The produced antibodies were found to be specific for the LPS of Br. melitensis since they did not bind with antigens of other bacteria that also have similar structure of LPS in the outer membrane of their cells. In addition, it was shown that the anti Rev-1 antibody binds with the LPS of Rev-1, whereas in cases of field strains it does not bind with certain parts of antigen. It seems that the anti Rev-1 is very promising not only for direct and easy diagnosis of brucellosis but also for differentiating animals that are vaccinated with Rev-1 from the ones that are infected with natural field strains of Br. melitensis. The produced antibodies can be immobilized into non-motile surfaces, such as polymers, so as to produce an enzyme-linked immunosorbent assay.

Χημεία

Φυσικές Επιστήμες

Μέθοδος δακτυλικών αποτυπωμάτων

Polyclonal antibodies of LPS

Βρουκέλλωση αιγοπροβάτων

Hyper-variable octameric oligonucleotide finger prints

Denaturant gradient gel electrophoresis

Lipopolysaccharides are the major antigens

Conserved single nucleotide mutation

Φυσικές Επιστήμες

Αντιγονική δράση λιποπολυσακχαριτών

Chemical Sciences

Brucellosis of sheep and goats

Μελιταίος πυρετός

Πολυκλωνικά αντισώματα έναντι των λιποπολυσακχαριτών

Ηλεκτροφόρηση σε διαβαθμισμένη συγκέντρωση αποδιατακτικών ουσιών

Χημεία

Πολυμορφισμός γονιδίου omp2

Polymorphism of omp2 gene

Τριχοειδής ηλεκτροφόρηση λιποπολυσακχαριτών

Brucella melitansis

Natural Sciences

Capillary zone electrophoresis of lipopolysaccharides

Ελληνική γλώσσα

Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης (ΑΠΘ)

Aristotle University Of Thessaloniki (AUTH)

Αριστοτέλειο Πανεπιστήμιο Θεσσαλονίκης (ΑΠΘ). Σχολή Θετικών Επιστημών. Τμήμα Χημείας. Τομέας Οργανικής Χημείας και Βιοχημείας. Εργαστήριο Βιοχημείας

Εθνικό Κέντρο Τεκμηρίωσης και Ηλεκτρονικού Περιεχομένου (ΕΚΤ)

Εθνικό Αρχείο Διδακτορικών Διατριβών

Συλλογή ΕΑΔΔ