Title:
Investigation Of The Genetic Polymorphism And Erythrocyte Binding Activity Of The Plasmodium knowlesi Duffy Binding Protein Alpha Region II (PkDBPαII) From Isolates In Malaysia
Created by Khor Siang Hooi
Supervised by Dr. Cheong Fei Wen, Prof. Fong Mun Yik
Background
This research investigates the genetic diversity and erythrocyte binding activity of Plasmodium knowlesi Duffy Binding Protein Alpha Region II (PkDBPαII), particularly focusing on samples collected from various regions of Malaysia between 2019 and 2021.
Plasmodium knowlesi, a monkey malaria parasite, is currently the most significant cause of human malaria infections in Malaysia, with Malaysian Borneo reporting the highest case numbers. This study focuses on understanding the genetic polymorphisms of PkDBPαII and its ability to bind to human red blood cells, which is crucial for the malaria parasite to invade human hosts.
The study has two main objectives:
1. To evaluate the genetic polymorphisms of PkDBPαII in P. knowlesi isolates:
The study aims to assess genetic variations in PkDBPαII among recent P. knowlesi isolates collected from Malaysia between 2019 and 2021. Ten isolates are randomly selected from both Peninsular Malaysia (n=5) and Malaysian Borneo (n=5), representing a mix of high and low parasitemia blood samples.
To achieve this objective, the employed techniques involve PCR to amplify PkDBPαII, followed by DNA cloning into the pGEM-T vector. Subsequently, two positive recombinant clones were picked from each sample and conducted sequencing. The collected data were then utilised to construct a phylogenetic tree using MEGA X. Comparing these sequences to earlier ones from Fong et al.'s studies (2014; 2015), the analysis presented all 20 sequences fell into two allele groups, consistent with previous research.
2. To investigate the erythrocyte binding activity of PkDBPαII in isolates with different parasitemia & geographical regions:
It involves studying how PkDBPαII from these isolates binds to human erythrocytes. For that, four isolates were selected. I first digested pGEM-T-PkDBPαII recombinant plasmids using the BglII restriction enzyme and cloned them into the pDisplay-AcGFP1 vector. The resulting constructs were sent for sequencing.
The next step involves transfecting these recombinant plasmids into COS-7 cells before conducting an erythrocyte binding assay (EBA). During EBA, human erythrocytes were added to the transfected COS-7 cells, stained with Hoechst 33342 dye, and observed under a fluorescence microscope. Rosettes, defined as aggregates of erythrocytes adhering to more than 50% of COS-7 cells, were counted, and their sizes were determined using imaging software. The statistical analysis is planned using IBM SPSS version 22.
Methodology:
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Cloning of PkDBPαII (PCR, colony PCR, plasmid extraction)
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Cloning into the pDisplay-AcGFP1 vector (RE BglII digestion, directional PCR)
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Maintenance of COS-7 cells (thawing, subculture, cryopreservation)
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Erythrocyte Binding Assay (plating, transfection, staining, rosette analysis)
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Gene sequencing & analysis; Phylogenetic study (UGENE, MEGA X)
Conclusion:
From what has been presented in the phylogenetic trees of the current study, there has been no novel genetic clustering of P. knowlesi-infected PkDBPαII isolates in recent years, particularly from 2019 to 2021. These findings highlight the importance of further investigation into how these genetic variations might influence malaria transmission and severity.
The study also sets the groundwork for future research that can explore potential vaccine targets or therapeutic interventions by analyzing more samples and additional factors, such as environmental influences on parasite transmission.