Identification of potential receptors for surface proteins of Toxoplasma gondii in humans / Lai Meng Yee
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that invades any nucleated cells in humans and other warm-blooded animals, with a great infection rate. Approximately 25% to 30% of the world’s human population is infected by T. gondii. Many proteins involved in T. gon...
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Format: | Thesis |
Published: |
2018
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Online Access: | http://studentsrepo.um.edu.my/9204/7/meng_yee.pdf http://studentsrepo.um.edu.my/9204/ |
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Summary: | Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that invades
any nucleated cells in humans and other warm-blooded animals, with a great infection
rate. Approximately 25% to 30% of the world’s human population is infected by T.
gondii. Many proteins involved in T. gondii invasion have been characterized, and the
contribution for parasite entry has been proposed. The identification of receptors or
binding host proteins for surface antigens is an important activity throughout the study.
A better understanding of the interplay between T. gondii and its hosts may provide a
starting point for the discovery of novel therapeutics. The surface antigens (SAGs) of T.
gondii play a major role during the host cell invasion process. In this study, the yeast twohybrid
system was used to analyze the interaction of T. gondii SAG1 and SAG2 with the
human host cell. Protein interaction was performed using commercial human cDNA in
pGADT7-RecAB. A total of 39 and 25 clones which interacted with the respective SAG1
and SAG2 were detected based on a series of the selection procedures. Twenty-nine and
18 clones for SAG1 and SAG2 were sent for sequencing after colony PCR. Following
analysis of sequencing results, Y187 cells transformed with each of these potential prey
plasmids (22 and 13 prey clones of each SAG1 and SAG2) was mated with the respective
Y2HGold containing pGBKT7-SAG2 or pGBKT7-SAG1 and Y2HGold (pGBKT7).
Homo sapiens lysine rich coil-coiled (abbreviated as HLY) and Homo sapiens zinc finger
(abbreviated as HZF) proteins were identified as potential candidate interacting with
SAG1 and SAG2, respectively. The interaction were further examined by betagalactosidase
assay and the enzyme activity between SAG1 and SAG2 with their host
proteins were 449.4 U and 437.7 U, respectively. In comparison to positive control (424.3
U), both interaction between prey and SAG1 and SAG2 were demonstrated. The
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interaction between prey and bait proteins was further determined using coimmunoprecipitation
assay. The result indicated the binding between these prey and
SAG1 and SAG2 proteins were significant. Finally, with the aid of isothermal titration
calorimetry (ITC) method, binding strength for recombinant pRSET-A-SAG1/pRSET-AHLY
proteins and recombinant pRSET-A-SAG2/pRSET-A-HZF proteins were 0.0075
µM and 18.75 µM, respectively. Both thermodynamic curves revealed that there were
exothermic and endothermic reaction for recombinant pRSET-A-SAG1/pRSET-A-HLY
proteins and recombinant pRSET-A-SAG2/pRSET-A-HZF proteins, respectively. These
prey proteins may serve as the potential drug candidates during the vaccination study in
future |
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