Priprava biološko aktivnega konjugata dejavnika tumorske nekroze alfa

diplomsko delo univerzitetnega študijskega programa

Marjeta Simončič (Author), Uroš Potočnik (Mentor), Mateja Novak Štagoj (Mentor)

Abstract

Dejavnik tumorske nekroze alfa (TNF-α) je proinflamatorni citokin, saj vzpodbuja sistemsko vnetje, z izrazitim pleiotropnim delovanjem. Opravlja torej več različnih vlog v človeškem telesu, lahko tudi hkrati. Osnovna vloga TNF-α je sodelovanje in uravnavanje imunskega odziva. Prva opisana funkcija TNF-α je bila razgradnja določenih vrst celic, predvsem tumorskih celic. Danes je znano, da TNF-α povzroča nekrozo ali apoptozo celic, povzroča tudi vnetje in zavira tumorogenezo ter razmnoževanje virusov. Prav zaradi delovanja proti malignim celicam in večjim tvorbam ter močno izraženi afiniteti do folne kisline le teh, je bil namen naloge pripraviti biološko aktiven analog dejavnika tumorske nekroze alfa (LK-801) in njegov konjugat s folno kislino ter nadalje smo želeli analog in konjugat delno okarakterizirati z analitskimi metodami in določiti njegovo biološko aktivnost. V prvi fazi smo izvedli izolacijo biološko aktivnega proteina dejavnika tumorske nekroze α iz bakterije Escherichia coli. Gojenje biomase je potekalo v bioreaktorju za razmnoževanje kultur, čiščenje izbranega proteinskega analoga LK-801 smo opravili v kromatografskih kolonah. Izolirani analog smo v nadaljevanju pretvorili v pripadajoč ester s pomočjo estrifikacije z aktivirano folno kislino. Reakcije smo izvajali z različnimi prebitki aktivirane folne kisline, s kontrolnim eksperimentom neaktivirane folne kisline ter s starim (že predhodno pripravljenim) in sveže izoliranim - novim analogom LK-801. Večji izkoristek reakcije konjugacije smo dosegli z novim analogom LK-801, ki je v nižjih prebitkih folne kisline imel tudi zelo dobro bološko aktivnost. Konjugacija s starim analogom je bila precej slabša, kar nas je presenetilo, saj sta imela tako predhodno pripravljeni kot sveže izolirani analog TNF-α zadovoljivo biološko aktivnost. Vsekakor smo uspeli pokazati pot sinteze proteina na folno kislino. Sintezna pot na folno kislino se lahko uporabi za različne proteine, ki so morda boljša alternativa analogu TNF-α.

Keywords

dejavnik tumorska nekroze alfa;folna kislina;konjugacija;biološka aktivnost;

Data

Language:	Slovenian
Year of publishing:	2011
Source:	Maribor
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[M. Simončič]
UDC:	579.25:577.21(043.2)
COBISS:	15653142
Views:	2738
Downloads:	206
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Preparation of biologically active tumor necrosis factor alpha conjugate
Secondary abstract:	Tumor Necrosis Factor Alpha (TNF-α) is an anti-inflammatory cytokine, for it enhances a systematic inflammation with an expressive pleiotropic activity. It thus serves various (potentially simultaneous) functions in human body. Its basic function is cooperation and balancing of the immune response. Decomposition of certain cell-types (esp. tumor cells) was the first function of the TNF-α to be discovered. Today, however, the TNF-α is known for causing necrosis or apoptosis of cells, inflammation, as well as hinders tumor-genesis and propagation of viruses. And for the reason of its activities against malign cells and larger formations as well as its strong affinity to folic acid was our aim to prepare a biologically active LK801 analogue of the TNF-α and its folic acid conjugate. Or aim was also to partly characterize the conjugate and determine its biological activity by using some analytical methods. Firstly, we isolated biologically active TNF-α from Escherichia coli bacteria. This was done in a bioreactor and the LK-801 protein was cleaned by chromatographic columns. We then turned an isolated analogue into a corresponding ester by esterification with an active folic acid. Reactions were conducted based on various folic acid redundancies, by controlled inactive folic acid experiment, and by old (previously prepared) and just-isolated new LK-801 analogue. A conjugation process achieved higher yield by the fresh LK801 analogue that also showed a very good biological activity at lower parts of the folic acid redundancies. Conjugation by the old analogue was substantially weaker, which was something contrary to our expectations, as both old and new TNF-α analogue both showed very good biological activity. We thus demonstrated a synthesizing path on folic acid of the protein. The path can well be used for various other proteins that may prove a better alternative to the TNF-α analogue.
Secondary keywords:	tumor necrosis factor alfa;folic acid;connjugation;biological activity;
URN:	URN:SI:UM:
Type (COBISS):	Undergraduate thesis
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	XI, 57 f.
Keywords (UDC):	mathematics;natural sciences;naravoslovne vede;matematika;biological sciences in general;biologija;microbiology;mikrobiologija;general microbiology;mathematics;natural sciences;naravoslovne vede;matematika;biological sciences in general;biologija;material bases of life;biochemistry;molecular biology;biophysics;biokemija;molekularna biologija;biofizika;
ID:	1015813