Izolovanje i karakterizacija prirodnih i rekombinantno dobijenih alergena iz kivija (Actinidia deliciosa Liang, Ferguson) za primenu u alergijskoj dijagnostici i terapiji

Abstract

Zamena alergenih ekstrakata pojedinačnim prirodnim i rekombinantnim alergenima udijagnostici alergije je obećavajući koncept i naziva se dijagnostika razdvojena nakomponente CRD (engl. component-resolved diagnosis). U tom smislu, neophodno jeproceniti alergeni potencijal svakog pojedinačnog kandidata za primenu u CRD.U ovoj tezi ocenjivane su biohemijske i alergene osobine cistein proteaznog inhibitora(nCPI) iz ploda zelenog kivija. Prečišćeni nCPI pokazao je IgE reaktivnosti in vitro (dotblot) i in vivo (kožne probe i aktivacija bazofila), dok je IgG reaktivnost pokazana uWestern blotu i ELISA-i. Biološka aktivnost je ocenjena ispitivanjem antimikrobnogpotencijala nCPI naspram tri soja fitopatogenih bakterija (A. tumefaciens, B. cepacia i umanjem obimu bakterije E. carotovora) kao i dva izolata fitopatogenih gljiva (A.radicina i B.cinerea), kako in vitro, tako i in vivo. Zbog slabe zastupljenosti nCPI uplodu kivija kodirajuća sekvenca nCPI je efikasno eksprimirana u Escherichia coli.Molekulska identičnost rCPI u odnosu na prirodni molekul pokazana je strukturnim(maseni otisak prsta) i imunohemijskim (imunoblot i ELISA) i biološkim (inhibicijaproteazne aktivnosti papaina i rasta fitopatogenih gljiva) testovima. Na osnovudobijenih rezultata jasno je da je CPI klinički relevantan alergen i kao takav trebalo bida bude uključen u dijagnostiku alergije na kivi razdvojenu na komponente.Standardni imunoterapijski pristup u tretmanu alergije na hranu nije adekvatan zbogvisokog rizika, pa je primena hipoalergenih molekula koji smanjeno vezuju IgE obećavajući koncept. Diverzifikacija gena za aktinidin rađena je lančanom reakcijompolimeraze sklone greškama epPCR (engl. error-prone PCR) koja je praćenakloniranjem biblioteke u Aga2 ekspresioni sistem za ekspresiju na površini ćelijakvasca. Nakon tri runde sortiranja, nasumično odabrani klonovi su dalje analizirani dotblotom i ELISA testom koristeći serum 6 osoba alergičnih na kivi U poređenju saprirodnim i rekombinantnim aktinidinom, dva mutanata su pokazala smanjenusposobnost aktivacije bazofila...

The replacement of fruit allergen extracts with component-resolved allergens seems tobe a promising concept. In this regard, the evaluation of allergenic properties of aparticular allergen candidate for component-resolved diagnosis needs to be performed.In this study evaluation of the biochemical and allergenic properties of cysteineproteinase inhibitor purified from green kiwifruit (nCPI) was performed. The IgEreactivity was detected both in vitro (dot blot) and in vivo (skin prick test and basophileactivation assay), while IgG rectivity in both Western blot analysis nd ELISA test.Biological activity was assesed by examining the growth inhibition of three plantpathogenic bacterial strains (A.tumefaciens, B. cepacia and, to a lesser extent, E.carotovora) and two phytopathogenic fungi (A. radicina and B. cinerea) both in vitroand in vivo. Due to low abundance of nCPI molecule in kiwifruit the coding sequence ofkiwi CPI was efficiently expressed in Escherichia coli. Molecular identity of rCPI to itsnatural counterpart has been clearly demonstrated in structural (mass fingerprint),immunological (immunoblot and ELISA inhibition) and biological sense (inhibition ofpapain proteolytic activity and antifungal activity). Based on the obtained results it isclear that nCPI is a clinically relevant kiwifruit allergen, and as such should be involvedin the component-resolved diagnosis of kiwifruit allergy.Standard immunotherapeutic approaches in food allergy are not adequate due tounacceptably high risk-to-benefit ratio, however hypoallergenic forms of molecules with reduced IgE binding potential seem to be promising concept. Diversification of thegene encoding the mature form of the actinidin was done via error prone PCR, followedby cloning of the library into the Aga2 yeast surface display expression system. Afterthree rounds of sorting by FACS, randomly picked colonies were further analyzed bydot blot and ELISA using pool of 6 kiwifruit allergic patients. Compared to both naturaland recombinant wild type actinidin two of the mutants exhibited diminished ability toactivate basophils..