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Publication Open Access Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response(2016-04) Don Changsom; Hatairat Lerdsamran; Witthawat Wiriyarat; Warunya Chakritbudsabong; Bunpote Siridechadilok; Jarunee Prasertsopon; Pirom Noisumdaeng; Wanibtisam Masamae; Pilaipan Puthavathana; Mahidol University. Faculty of Medical Technology. Center for Research and Innovation. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showedPublication Open Access COVID-19 Vaccines Help with Herd Immunity(2022) Usa Lek-Uthai; Mahidol University. Faculty of Public Health. Department of Parasitology and EntomologyPublication Open Access Identification of T cell epitopes on the 33-kDA fragment of plasmodium yoelii merozoite surface protein 1 and their antibody-independent protective role in immunity to blood stage malaria(2002) Jiraprapa Wipasa; Chakrit Hirunpetcharat; Yuvadee Mahakunkijcharoen; Huji Xu; Salenna Elliott; Michael F Good; Mahidol University. Faculty of Public Health. Department of Microbiology.; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology.Merozoite surface protein 1 (MSP1) of malaria parasites undergoes proteolytic processing at least twice before invasion into a new RBC. The 42-kDa fragment, a product of primary processing, is cleaved by proteolytic enzymes giving rise to MSP1(33), which is shed from the merozoite surface, and MSP1(19), which is the only fragment carried into a new RBC. In this study, we have identified T cell epitopes on MSPl (33) of Plasmodium yoelii and have examined their function in immunity to blood stage malaria. Peptides 20 aa in length, spanning the length of MSP1(33) and overlapping each other by 10 aa, were analyzed for their ability to induce T cell proliferation in immunized BALB/c and C57BL/6 mice. Multiple epitopes were recognized by these two strains of mice. Effector functions of the dominant epitopes were then investigated. Peptides Cm 15 and Cm21 were of particular interest as they were able to induce effector T cells capable of delaying growth of lethal P. yoelii YM following adoptive transfer into immunodeficient mice without inducing detectable Ab responses. Homologs of these epitopes could be candidates for inclusion in a subunit vaccine.Publication Open Access Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity(1997-12) Tantimavanich S; Pantuwatana S; Bhumiratana A; Panbangred W; Faculty of Science. Department of BiotechnologyChitinase from a high producing strain (TP-1) of Bacillus licheniformis was used with B. thuringiensis subsp. aizawai (B.t.a.) in a combined larvicidal assay against the pest, Spodoptera exigua. With 10 mU of this chitinase, the LD(50) of B.t.a. was reduced by 7.6, 13.8 and 15 times on days 3, 5 and 7, respectively when compared to use of B.t.a. alone. In addition, a combination of chitinase (10 mU) and B.t.a. at a sub-lethal dose retarded growth and development of S. exigua. In preparation for transformation of B.t.a., the TP-1 chitinase gene was cloned in E. coli DH5alpha and sequenced to reveal a single open reading frame of 1,815 bp. This open reading frame encoded for a protein of 604 amino acids and a characteristic signal peptide sequence of 35 amino acids. The gene was subsequently introduced into B.t.a. where it was expressed constitutively. The transformed strain showed slightly improved activity against S. exigua when compared to the non-transformed strain. This was probably due to the low chitinase activity (15 mU/ml) of the transformant, which might be improved by further gene manipulation to overexpress enzyme production.Publication Open Access การแสดงออกของรีคอมบิแนนท์โปรตีนเปลือกนอกของเชื้อไวรัสโรคขนและจงอยปากผิดปกติโดยใช้ระบบเชื้อยิสต์ Pichiapastoris(2557) ลดาวัลย์ สาริยา; ปรุศก์ สุกใส; อดิศักดิ์ ส่งแจ้ง; ภิรมย์ พรมพิราม; มหาวิทยาลัยมหิดล. คณะสัตวแพทยศาสตร์. ศูนย์เฝ้าระวังและติดตามโรคจากสัตว์ป่า สัตว์ต่างถิ่นและสัตว์อพยพPsittacine beak and feather disease virus (BFDV) is a haemagglutination circo virus causing psittacine beak and feather disease (PBFD) in psittacine birds. The infected birds showed feather dystrophy and occasionally associated with beak deformities. Development of vaccine and detection of the disease is difficult and ineffective due to this virus could not be cultured in tissue or cell lines and embryonated eggs. The aim of this study was to express recombinant capsid protein of BFDV using Pichia pastoris system. The recombinant protein was purified by affinity binding with Ni-NTA resin. From the results, the time point of protein expression was 48 hours after inducing with methanol. The recombinant capsid protein was expressed in inclusion bodies form. From SDS-PAGE analysis, the protein had molecular weight of 38 kDa. Moreover, Western blot analysis demonstrated that this recombinant protein could react with convalescent serum from BFDV-infected bird. Based on this information, it would be a potential to develop in house BFDV capsid protein-based diagnostic test and vaccine in the future.Publication Open Access การแสดงออกของรีคอมบิแนนท์โปรตีนเปลือกนอกของเชื้อไวรัสโรคขนและจงอยปากผิดปกติโดยใช้ระบบเชื้อยิสต์ Pichiapastoris(2557) ลดาวัลย์ สาริยา; ปรุศก์ สุกใส; อดิศักดิ์ ส่งแจ้ง; ภิรมย์ พรมพิราม; มหาวิทยาลัยมหิดล. คณะสัตวแพทยศาสตร์. ศูนย์เฝ้าระวังและติดตามโรคจากสัตว์ป่่า สัตว์ต่างถิ่นและสัตว์อพยพ; มหาวิทยาลัยมหิดล. คณะแพทยศาสตร์ศิริราชพยาบาล. หน่วยวิจัยโรคไข้เลือดออกเชื้อไวรัสโรคขนและจงอยปากผิดปกติเป็น haemagglutination circo virus และเป็นสาเหตุของโรคขนและจงอยปากผิดปกติที่พบในกลุ่มนกตระกูลนกปากขอ นกที่เป็นโรคจะมีอาการขนร่วงและอาจมีจงอยปากยื่นยาวไม่สวยงาม เนื่องจากเชื้อนี้ไม่สามารถเพาะเลี้ยงในเนื้อเยื่อหรือเซลล์เพาะเลี้ยงและไข่ไก่ฟักได้ เป็นผลให้การพัฒนาวิธีการตรวจและการพัฒนาวัคซีนต่อโรคทำได้ค่อนข้างยาก ดังนั้น ในการศึกษาครั้งนี้จะทำการผลิตรีคอมบิแนนท์โปรตีนเปลือกนอกของเชื้อไวรัสโรคขนและจงอยปากผิดปกติ โดยให้ยีนมีการแสดงออกโดยอาศัยระบบเชื้อยีสต์ Pichia pastoris จากนั้นทำให้รีคอมบิแนนท์โปรตีนบริสุทธิ์ โดยอาศัยหลักการ Affinity binding กับโมเลกุลของโลหะนิเกิล จากผลการแสดงออกของรีคอมบิแนนท์โปรตีนเปลือกนอกพบว่า มีการแสดงออกได้ดีที่สุดหลังจากกระตุ้นด้วยเมทานอล 48 ชั่วโมง และพบว่าส่วนใหญ่เป็น inclusion bodies เมื่อนำโปรตีนมาทำการแยกขนาดด้วยกระแสไฟฟ้า พบว่าโปรตีนมีขนาดประมาณ 38 kDa นอกจากนี้ยังพบว่ารีคอมบิแนนท์โปรตีนสามารถจับกับซีรัมของนกที่ติดเชื้อไวรัสโรคขนและจงอยปากผิดปกติได้ เมื่อตรวจด้วยวิธี Western blot analysis ซึ่งข้อมูลที่ได้นี้จะช่วยให้มีการพัฒนาวิธีการตรวจหาเชื้อและผลิตวัคซีนต่อเชื้อไวรัสโรคขนและจงอยปากผิดปกติ โดยใช้รีคอมบิแนนท์โปรตีนเปลือกนอกในอนาคตPublication Open Access Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.(2015-03) Usa Boonyuen; อุษา บุญยืน; Kamoltip Promnares; กมลทิพย์ พรหมณเรศ; Suwapat Junkree; Day, Nichloas P.J.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Mallika Imwong; Mahidol University. Faculty of Tropical Medicine. Mahidol Oxford Research Unit.; Mahidol University. Faculty of Tropical Medicine. Central Equipment Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.Human liver carboxylesterase 1 (CES1) plays a critical role in the hydrolysis of various ester- and amide-containing molecules, including active metabolites, drugs and prodrugs. However, it has been problematic to express recombinant CES1 in bacterial expression systems due to low solubility, with the CES1 protein being mainly expressed in inclusion bodies, accompanied by insufficient purity issues. In this study, we report an efficient in vitro method for refolding recombinant CES1 from inclusion bodies. A one-step purification with an immobilized-metal affinity column was utilized to purify His-tagged recombinant CES1. Conveniently, both denaturant and imidazole can be removed while the enzyme is refolded via buffer exchange, a dilution method. We show that the refolding of recombinant CES1 was successful in Tris-HCl at pH 7.5 containing a combination of 1% glycerol and 2mM β-mercaptoethanol, whereas a mixture of other additives (trehalose, sorbitol and sucrose) and β-mercaptoethanol failed to recover a functional protein. His-tagged recombinant CES1 retains its biological activity after refolding and can be used directly without removing the fusion tag. Altogether, our results provide an alternative method for obtaining a substantial amount of functionally active protein, which is advantageous for further investigations such as structural and functional studies.Publication Open Access Homology modeling of mosquito cytochrome P450 enzymes involved in pyrethroid metabolism: insights into differences in substrate selectivity(2011) Panida Lertkiatmongkol; Ekachai Jenwitheesuk; Pornpimol Rongnoparut; Mahidol University. Faculty of Science. Department of BiochemistryBackground: Cytochrome P450 enzymes (P450s) have been implicated in insecticide resistance. Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds. Findings: Homology models of the three An. minimus P450 enzymes were constructed using the multiple template alignment method. The predicted enzyme model structures were compared and used for molecular docking with insecticides and compared with results of in vitro enzymatic assays. The three model structures comprise common P450 folds but differences in geometry of their active-site cavities and substrate access channels are prominent. The CYP6AA3 model has a large active site allowing it to accommodate multiple conformations of pyrethroids. The predicted CYP6P7 active site is more constrained and less accessible to binding of pyrethroids. Moreover the predicted hydrophobic interface in the active-site cavities of CYP6AA3 and CYP6P7 may contribute to their substrate selectivity. The absence of CYP6P8 activity toward pyrethroids appears to be due to its small substrate access channel and the presence of R114 and R216 that may prevent access of pyrethroids to the enzyme heme center. Conclusions: Differences in active site topologies among CYPAA3, CYP6P7, and CYP6P8 enzymes may impact substrate binding and selectivity. Information obtained using homology models has the potential to enhance the understanding of pyrethroid metabolism and detoxification mediated by P450 enzymes.Publication Open Access Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ(2016) Jitlada Vasuvat; Atcha Montree; Sangduen Moonsom; Ubolsree Leartsakulpanich; Songsak Petmitr; Federico Focher; Wright, George E.; Porntip Chavalitshewinkoon‑Petmitr; Mahidol University. Faculty of Tropical Medicine. Department of ProtozoologyBackground: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. Methods: The coding sequences of DNA polymerase δ catalytic subunit (PfPolδ-cat), DNA polymerase δ small subunit (PfPolδS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC–MS/MS. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPolδ-cat activity and on in vitro parasite growth using SYBR Green I assay. Results: The purified recombinant protein PfPolδ-cat, PfPolδS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPolδ-cat and PfPolδS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPolδ-cat possessed both DNA polymerase and 3′–5′ exonuclease activities. It used both Mg2+ and Mn2+ as cofactors and was inhibited by high KCl salt (>200 mM). PfPolδS stimulated PfPolδ-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPolδ-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 μM) and 7-acetoxypentyl-( 3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 μM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 μM). Conclusions: Recombinant PfPolδ-cat, PfPolδS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPolδ-cat. The high sensitivity of PfPolδ to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.Publication Open Access Mud crab susceptibility to disease from white spot syndrome virus is species-dependent(2010) Naraporn Somboonna; Seksan Mangkalanan; Attasit Udompetcharaporn; Chartchai Krittanai; Kallaya Sritunyalucksana; TW Flegel; Mahidol University. Center of Excellence for Shrimp Molecular Biology and Biotechnology; Mahidol University. Institute of Molecular Biosciences; Mahidol University. Faculty of Science. Department of BiochemistryBackground Based on a report for one species (Scylla serrata), it is widely believed that mud crabs are relatively resistant to disease caused by white spot syndrome virus (WSSV). We tested this hypothesis by determining the degree of susceptibility in two species of mud crabs, Scylla olivacea and Scylla paramamosain, both of which were identified by mitochondrial 16 S ribosomal gene analysis. We compared single-dose and serial-dose WSSV challenges on S. olivacea and S. paramamosain. Findings In a preliminary test using S. olivacea alone, a dose of 1 × 106 WSSV copies/g gave 100% mortality within 7 days. In a subsequent test, 17 S. olivacea and 13 S. paramamosain were divided into test and control groups for challenge with WSSV at 5 incremental, biweekly doses starting from 1 × 104 and ending at 5 × 106 copies/g. For 11 S. olivacea challenged, 3 specimens died at doses between 1 × 105 and 5 × 105 copies/g and none died for 2 weeks after the subsequent dose (1 × 106 copies/g) that was lethal within 7 days in the preliminary test. However, after the final challenge on day 56 (5 × 106 copies/g), the remaining 7 of 11 S. olivacea (63.64%) died within 2 weeks. There was no mortality in the buffer-injected control crabs. For 9 S. paramamosain challenged in the same way, 5 (55.56%) died after challenge doses between 1 × 104 and 5 × 105 copies/g, and none died for 2 weeks after the challenge dose of 1 × 106 copies/g. After the final challenge (5 × 106 copies/g) on day 56, no S. paramamosain died during 2 weeks after the challenge, and 2 of 9 WSSV-infected S. paramamosain (22.22%) remained alive together with the control crabs until the end of the test on day 106. Viral loads in these survivors were low when compared to those in the moribund crabs. Conclusions S. olivacea and S. paramamosain show wide variation in response to challenge with WSSV. S. olivacea and S. paramamosain are susceptible to white spot disease, and S. olivacea is more susceptible than S. paramamosain. Based on our single-challenge and serial challenge results, and on previous published work showing that S. serrata is relatively unaffected by WSSV infection, we propose that susceptibility to white spot disease in the genus Scylla is species-dependent and may also be dose-history dependent. In practical terms for shrimp farmers, it means that S. olivacea and S. paramamosain may pose less threat as WSSV carriers than S. serrata. For crab farmers, our results suggest that rearing of S. serrata would be a better choice than S. paramamosain or S. olivacea in terms of avoiding losses from seasonal outbreaks of white spot disease.