2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1)
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| collectionurl | https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 |
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| country | Malaysia |
| date | 2022-07-14 |
| format | General Document |
| id | 15853 |
| institution | UniSZA |
| internalnotes | Sila masukkan subject wajib Dissertations, Academic. Terima kasih... |
| originalfilename | RECOMBINANT EXPRESSION AND ANTIGENICITY CHARACTERIZATION OF THE Plasmodium knowlesi APICAL MEMBRANE ANTIGEN 1 (PkAMA1) (MASTER_2022).pdf |
| person | Aina Binti Azazi |
| recordtype | oai_dc |
| resourceurl | https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15853 |
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| spelling | 15853 https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15853 https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 General Document Malaysia Library Staff (Top Management) Library Staff (Management) Library Staff (Support) Terengganu Faculty of Health Sciences English application/pdf 1.5 195 Server storage Scanned document Universiti Sultan Zainal Abidin UniSZA Private Access UNIVERSITI SULTAN ZAINAL ABIDIN SAMBox 2.3.4; modified using iTextSharp™ 5.5.10 ©2000-2016 iText Group NV (AGPL-version) Copyright©PWB2025 2022-07-14 RECOMBINANT EXPRESSION AND ANTIGENICITY CHARACTERIZATION OF THE Plasmodium knowlesi APICAL MEMBRANE ANTIGEN 1 (PkAMA1) (MASTER_2022).pdf 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) Plasmodium knowlesi—Genetics Plasmodium knowlesi is the fifth known human malaria parasite, which previously only causes infection in macaque (simian malaria). Human malaria infection is widespread across the forested areas of Southeast Asia countries, especially in Malaysia. Vaccines are an option for controlling and preventing malaria infection. Generally, Plasmodium is an obligate intracellular parasite and required a complex invasion mechanism. Apical membrane antigen 1 (AMA1) is a surface protein that plays a pivotal role in parasite host cell invasion and potent malaria vaccine candidate. Ectodomain of AMA1 is divided into three immunogenic domains, domain I (DI), domain II (DII) and domain III (DIII). Soluble and functionally active AMA1 immunization conferred immunogenicity that gives protection against malaria infection and thus emphasized the protein solubility is critical for antigenicity assays. Pichia pastoris expression system is an option for the production of soluble recombinant protein for therapeutic and industrial purposes. This study aimed to optimize and compare the expression conditions for recombinant PkAMA1 (rPkAMA1) using P. pastoris system. The antigenicity of rPkAMA1 protein was further validated using serological test and in silico epitope predictions. Multiple expression clones of pPICZαA-PkAMA1 domains (DⅠ, DⅡ, DⅢ, DⅠ-Ⅱ and DⅡ-Ⅲ) were constructed and then cloned into KM71H and X 33 hosts, which allow secretory protein expression. Small-scale protein expression in KM71H and X-33 were induced at optical density (OD600) 3 with four methanol concentrations [0.5%, 1%, 2% and 3% (v/v)] up to six days and the best clone and condition was selected for large-scale expression. The expression conditions for rPkAMA1 (DI-II-III) in KM71H were also optimized for the antigenicity study. The rPkAMA1 (DI-II) and rPkAMA1 (DI-II-III) proteins were tested against immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in 15 malaria serum samples collected from indigenous communities by Western blot assay. Potential B cell epitopes of the ectodomain of rPkAMA1 were predicted using BepiPred and ABCpred bioinformatics servers whereas NetMHCpan-4.1 and NetMHCIIpan-4.0 servers were used for T-cell epitope prediction, followed by antigenicity prediction using the VaxiJen server. Small-scale protein expression in KM71H and X-33 indicated that both strains are capable of producing soluble forms of rPkAMA1 (DⅠ-Ⅱ) at ~43 kDa. Expression in KM71H was higher with lower endogenous proteins compared to X-33 and thus, subsequent large-scale expression was carried out in KM71H (1% methanol, 72 hours) and rPkAMA1 (DI-II-IIII) (2% methanol, 48 hours, ~55 kDa). Pilot serological testing against rPkAMA1 proteins only showed positive reactions against IgG antibody in all infected serum samples and these preliminary findings indicated the possibility of a long exposure of those indigenous communities to Plasmodium infection. In silico epitopes mapping revealed that B- and T-cell epitopes were scattered along the rPkAMA1 domains. Antigenicity characterization of PkAMA1 protein confirmed the presence of protective epitopes that could serve as a target in vaccine development. The soluble and functionally active recombinant PkAMA1 proteins that were produced in this study have the potential to be further developed into an effective vaccine that sustain for long-term protection. Aina Binti Azazi Dissertations, Academic Sila masukkan subject wajib Dissertations, Academic. Terima kasih... Recombinant PkAMA1 Expression Plasmodium Knowlesi Antigenicity AMA1-Based Malaria Vaccine Thesis |
| spellingShingle | 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) |
| state | Terengganu |
| subject | Plasmodium knowlesi—Genetics Dissertations, Academic |
| summary | Plasmodium knowlesi is the fifth known human malaria parasite, which previously only causes infection in macaque (simian malaria). Human malaria infection is widespread across the forested areas of Southeast Asia countries, especially in Malaysia. Vaccines are an option for controlling and preventing malaria infection. Generally, Plasmodium is an obligate intracellular parasite and required a complex invasion mechanism. Apical membrane antigen 1 (AMA1) is a surface protein that plays a pivotal role in parasite host cell invasion and potent malaria vaccine candidate. Ectodomain of AMA1 is divided into three immunogenic domains, domain I (DI), domain II (DII) and domain III (DIII). Soluble and functionally active AMA1 immunization conferred immunogenicity that gives protection against malaria infection and thus emphasized the protein solubility is critical for antigenicity assays. Pichia pastoris expression system is an option for the production of soluble recombinant protein for therapeutic and industrial purposes. This study aimed to optimize and compare the expression conditions for recombinant PkAMA1 (rPkAMA1) using P. pastoris system. The antigenicity of rPkAMA1 protein was further validated using serological test and in silico epitope predictions. Multiple expression clones of pPICZαA-PkAMA1 domains (DⅠ, DⅡ, DⅢ, DⅠ-Ⅱ and DⅡ-Ⅲ) were constructed and then cloned into KM71H and X 33 hosts, which allow secretory protein expression. Small-scale protein expression in KM71H and X-33 were induced at optical density (OD600) 3 with four methanol concentrations [0.5%, 1%, 2% and 3% (v/v)] up to six days and the best clone and condition was selected for large-scale expression. The expression conditions for rPkAMA1 (DI-II-III) in KM71H were also optimized for the antigenicity study. The rPkAMA1 (DI-II) and rPkAMA1 (DI-II-III) proteins were tested against immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies in 15 malaria serum samples collected from indigenous communities by Western blot assay. Potential B cell epitopes of the ectodomain of rPkAMA1 were predicted using BepiPred and ABCpred bioinformatics servers whereas NetMHCpan-4.1 and NetMHCIIpan-4.0 servers were used for T-cell epitope prediction, followed by antigenicity prediction using the VaxiJen server. Small-scale protein expression in KM71H and X-33 indicated that both strains are capable of producing soluble forms of rPkAMA1 (DⅠ-Ⅱ) at ~43 kDa. Expression in KM71H was higher with lower endogenous proteins compared to X-33 and thus, subsequent large-scale expression was carried out in KM71H (1% methanol, 72 hours) and rPkAMA1 (DI-II-IIII) (2% methanol, 48 hours, ~55 kDa). Pilot serological testing against rPkAMA1 proteins only showed positive reactions against IgG antibody in all infected serum samples and these preliminary findings indicated the possibility of a long exposure of those indigenous communities to Plasmodium infection. In silico epitopes mapping revealed that B- and T-cell epitopes were scattered along the rPkAMA1 domains. Antigenicity characterization of PkAMA1 protein confirmed the presence of protective epitopes that could serve as a target in vaccine development. The soluble and functionally active recombinant PkAMA1 proteins that were produced in this study have the potential to be further developed into an effective vaccine that sustain for long-term protection. |
| title | 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) |
| title_full | 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) |
| title_fullStr | 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) |
| title_full_unstemmed | 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) |
| title_short | 2022_Recombinant Expression and Antigenicity Characterization of the Plasmodium knowlesi Apical Membrane Antigen 1 (PkAMA1) |
| title_sort | 2022_recombinant expression and antigenicity characterization of the plasmodium knowlesi apical membrane antigen 1 (pkama1) |