LIPOPROTEIN-LIPAZA

Dominik Gaser
Na!rtovanje in razvoj peptidnih ligandov lipaz z metodo bakteriofagnega prikaza Lipases peptide ligands design and development through phage display technology
Doktorska disertacija
Ljubljana, 2010
Mentor: prof. dr. Borut trukelj Somentorica: doc. dr. Mojca Lunder Poro!evalca: izr. prof. dr. Samo Kreft prof. dr. Peter Ma!ek
Dekan: izr. prof. dr. Stanislav Gobec
Izjava Izjavljam, da je priloena doktorska disertacija samostojno delo, ki sem ga izvedel pod mentorstvom prof. dr. Boruta truklja in doc. dr. Mojce Lunder na Katedri za farmacevtsko biologijo Fakultete za farmacijo Univerze v Ljubljani.
Dominik Gaser
Ljubljana, 2010
Kazalo
KAZALO
POVZETEK........................................................................................................................................................... 1 ABSTRACT ........................................................................................................................................................... 2 SEZNAM OKRAJAV ......................................................................................................................................... 3 STANDARDNE OKRAJAVE ZA NUKLEOTIDE...................................................................................................... 4 UVOD ..................................................................................................................................................................... 5 POMEN PEPTIDOV PRI RAZVOJU NOVIH ZDRAVILNIH U"INKOVIN ................................................................... 5 PEPTIDNE KNJINICE ......................................................................................................................................... 6 I. BIOLOKE PEPTIDNE KNJINICE ................................................................................................................... 7 II. BAKTERIOFAGNE PREDSTAVITVENE KNJINICE .......................................................................................... 7 ZNA"ILNOSTI PEPTIDNIH LIGANDOV, ODKRITIH S PRESEJANJEM BIOLOKIH KNJINIC .............................. 13 METABOLIZEM MA"OB .................................................................................................................................. 14 LIPAZE .............................................................................................................................................................. 15 LIPOPROTEIN-LIPAZA ...................................................................................................................................... 17 FIZIOLOKA VLOGA ...................................................................................................................................... 17 GENSKI ZAPIS ZA LIPOPROTEIN-LIPAZO......................................................................................................... 19 SINTEZA LIPOPROTEIN-LIPAZE ...................................................................................................................... 19 STRUKTURA LIPOPROTEIN-LIPAZE ................................................................................................................ 20 MESTA IZRAANJA LIPOPROTEIN-LIPAZE ...................................................................................................... 21 LIPOPROTEIN-LIPAZA IN BOLEZENSKA STANJA ............................................................................................. 22 APOLIPOPROTEINI C........................................................................................................................................ 30 APOLIPOPROTEIN C1 .................................................................................................................................... 30 APOLIPOPROTEIN C2 .................................................................................................................................... 31 APOLIPOPROTEIN C3 .................................................................................................................................... 31 LIPOPROTEIN-LIPAZA KOT TAR"A U"INKOVIN .............................................................................................. 32 NAMEN DELA.................................................................................................................................................... 33 MATERIALI IN METODE ............................................................................................................................... 34 MATERIALI....................................................................................................................................................... 34 APARATURE.................................................................................................................................................. 34 LABORATORIJSKI MATERIALI ........................................................................................................................ 35 REAGENTI ..................................................................................................................................................... 36 PUFRI IN RAZTOPINE ..................................................................................................................................... 38 GOJI"A........................................................................................................................................................ 44 KOMPETENTNE CELICE ................................................................................................................................. 45 BAKTERIOFAGNI PREDSTAVITVENI KNJINICI ............................................................................................... 46 PROTITELESA ................................................................................................................................................ 46 PLAZMIDI...................................................................................................................................................... 46 ZA"ETNI OLIGONUKLEOTIDI ......................................................................................................................... 46 METODE ........................................................................................................................................................... 48 BAKTERIOFAGNI PRIKAZ ............................................................................................................................... 48 TEST ELISA ................................................................................................................................................. 58 IZOLACIJA BAKTERIOFAGNE DNA................................................................................................................ 59 DOLO"ANJE NUKLEOTIDNEGA ZAPOREDJA NA FAGNI DNA.......................................................................... 59 RA"UNALNIKA ANALIZA LASTNOSTI PEPTIDOV........................................................................................... 61 KVANTITATIVNI TEST ELISA ....................................................................................................................... 61 METODA PLAZMONSKE POVRINSKE RESONANCE (SPR) .............................................................................. 62 ENCIMSKI TEST ZA LIPOPROTEIN-LIPAZO ...................................................................................................... 63 REZULTATI ....................................................................................................................................................... 65 BAKTERIOFAGNI PRIKAZ ................................................................................................................................. 65 SELEKCIJA Z NESPECIFI"NO ELUCIJO ............................................................................................................. 66 SELEKCIJA S SPECIFI"NO ELUCIJO ................................................................................................................. 74 IZBRANI BAKTERIOFAGNI KLONI ................................................................................................................... 84 POVRINSKA PLAZMONSKA RESONANCA ........................................................................................................ 86 MERJENJE AFINITETE BAKTERIOFAGNIH KLONOV ......................................................................................... 86

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