Izolovanje i karakterizacija prirodnih i rekombinantno dobijenih alergena iz kivija (Actinidia deliciosa Liang, Ferguson) za primenu u alergijskoj dijagnostici i terapiji
Isolation and characterisation of natural and recombinant allergens from green kiwifruit (Actinidia deliciosa Liang, Ferguson) for allergy diagnosis and therapy
Докторанд
Popović, Milica M.Ментор
Gavrović-Jankulović, MarijaЧланови комисије
Atanasković-Marković, MarinaJankov, Ratko M.
Prodanović, Radivoje
Dimitrijević, Ljiljana
Метаподаци
Приказ свих података о дисертацијиСажетак
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 Escheri...chia 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..
Факултет:
Универзитет у Београду, Хемијски факултетДатум одбране:
07-12-2012Пројекти:
- Алергени, антитела, ензими и мали физиолошки значајни молекули: дизајн, структура, функција и значај (RS-MESTD-Basic Research (BR or ON)-172049)