construction and characterization of transgenic strains of the protozoan Leishmania tarentolae for heterologous expression of red fluorescent protein mCherry and nucleobase transporter rSNBT1 from Rattus norvegicus

Postgraduate Thesis uoadl:1318079 527 Read counter

Unit:
ΠΜΣ Μικροβιακή Βιοτεχνολογία
Library of the School of Science
Deposit date:
2015-02-12
Year:
2015
Author:
Δούκας Ανάργυρος
Supervisors info:
Γεώργιος Διαλλινάς Καθηγητής (Επιβλέπων), Χαραλαμπία Μπολέτη Ερευνήτρια Β'
Original Title:
Κατασκευή και χαρακτηρισμός διαγονιδιακών στελεχών παρασίτων Leishmania tarentolae για ετερόλογη έκφραση της κόκκινης φθορίζουσας πρωτεΐνης mCherry και του μεταφορέα νουκλεοβάσεων rSNBT1 (από τον Rattus norvegicus)
Languages:
Greek
Translated title:
construction and characterization of transgenic strains of the protozoan Leishmania tarentolae for heterologous expression of red fluorescent protein mCherry and nucleobase transporter rSNBT1 from Rattus norvegicus
Summary:
We used the L. tarentolae organism to express, the red fluorescent protein
mCherry in order to study its utility as a biosensor in studies for pathogenic
Leishmania species, also use the transgenic parasites L. tarentolae-mCherry for
antileismanial drug screening and the nucleobase transmembrane transporter
rSNBT1 which belongs to the NAT / NCS2 transporters’ family in order to study
its functional characteristics in an eukaryotic organism dividing faster and
growing in less expensive medium than mammalian cell lines with the aim to
isolate from these cells a sufficient amount of the transporter for
crystallization and structural studies.
Initially, a PCR reaction, strengthening mcherry and rsnbt1 genes, followed by
cloning the genes into plasmid vectors. Generation of transgenic strains
performed by electroporation and selection of transgenic populations by culture
on appropriate antibiotic. The expression of mCherry and rSNBT1-mRFP1 confirmed
by fluorescence microscopy.
The correct size of the hybrid protein rSNBT1-mRFP1 confirmed by Western Blot
in parasitic protein extract, enriched in membrane proteins.
The function of rSNBT1-mRFP1 in the transgenic L. tarentolae-rSNBT1-mRFP1 cells
was confirmed by an uptake assay and in competition experiments with other
nucleobases.
Transgenic populations of L. tarentolae-mCherry and L. tarentolae-rSNBT1-mRFP1
were constructed and characterized, expressing the functional protein mCherry
and rSNBT1
Keywords:
Leishmania tarentolae LEXSY, recombinant protein expression, mCherry, Nucleobase transporter rSNBT1, Transmembrane proteins
Index:
Yes
Number of index pages:
13
Contains images:
Yes
Number of references:
91
Number of pages:
121
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