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 u dijagnostici alergije je obećavajući koncept i naziva se dijagnostika razdvojena na komponente CRD (engl. component-resolved diagnosis). U tom smislu, neophodno je proceniti 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 (dot blot) i in vivo (kožne probe i aktivacija bazofila), dok je IgG reaktivnost pokazana u Western blotu i ELISA-i. Biološka aktivnost je ocenjena ispitivanjem antimikrobnog potencijala nCPI naspram tri soja fitopatogenih bakterija (A. tumefaciens, B. cepacia i u manjem 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 u plodu 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 (inhibicija proteazne aktivnosti papaina i rasta fitopatogenih gljiva) testovima. Na osnovu dobijenih rezultata jasno je da je CPI klinički relevantan alergen i kao takav trebalo bi da bude uključen u dijagnostiku alergije na kivi razdvojenu na komponente. Standardni imunoterapijski pristup u tretmanu alergije na hranu nije adekvatan zbog visokog rizika, pa je primena hipoalergenih molekula koji smanjeno vezuju IgE obećavajući koncept. Diverzifikacija gena za aktinidin rađena je lančanom reakcijom polimeraze sklone greškama epPCR (engl. error-prone PCR) koja je praćena kloniranjem biblioteke u Aga2 ekspresioni sistem za ekspresiju na površini ćelija kvasca. Nakon tri runde sortiranja, nasumično odabrani klonovi su dalje analizirani dot blotom i ELISA testom koristeći serum 6 osoba alergičnih na kivi U poređenju sa prirodnim i rekombinantnim aktinidinom, dva mutanata su pokazala smanjenu sposobnost aktivacije bazofila...

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