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Your search keyword '"Phan, Tuan-Nghia"' showing total 11 results
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11 results on '"Phan, Tuan-Nghia"'

5. Cloning and Expression of 3 Chymotrypsin-like Protease of SARS-CoV-2 in E. coli using pET28a Vector

Author

Nguyen Thi Hong Loan, Phan Tuan Nghia, Dinh Nho Thai, Ngo Thu Trang, Tran Thi Xuan Giang, and Vu Thi Duy Ly

Subjects
General Medicine
Abstract

The 3 chymotrypsin-like protease (3CLpro) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is one of the primary targets for the development of antiviral drug therapies as it plays a critical role in viral replication. In this study, the gene encoding for SARS-CoV-2 3CLpro (918 bp) was amplified from the cDNA of the virus by polymerase chain reaction (PCR) and cloned into the pGEM-T vector. 3CLpro was then inserted into the expression vector pET28a at the end of the 6 histidine residue encoding sequence to form a fusion protein (6xHis-3CLpro). The 6xHis-3Clpro construct was successfully expressed in E. coli. The expression of 3CLpro was highest when E. coli BL21(DE3) RIL harboring pET28a-3CLpro vector was cultured in LB medium at 20 oC, induced by 1.0 mM Isopropyl thiogalactopyranosie (IPTG) when cell density measured by optical density at 600 nm (OD600) reached 0.7-0.8 and harvested after 24 hours of induction. The recombinant 3CLpro was purified by Ni-sepharose affinity chromatography under denaturation conditions. The purified 3CLpro showed to have a 41 kDa band on sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using polyclonal anti-3CLpro antibody and hydrolyzed a fluorescent specific substrate of 3CLpro after renaturation.

Published
2023

8. Creation of Standard DNA for Detection and Quantification of White Spot Syndrome Virus in Shrimps by Real-time PCR (qPCR)

Author

Nguyen Thi Hong Loan, Phan Tuan Nghia, and Tran Viet Cuong

Subjects
General Medicine
Abstract

White spot syndrome virus (WSSV) is a double stranded DNA virus that causes WSS diseases for many crustaceans including the Penaeidae family shrimp. In this study, standard DNA for detection and quantification of WSSV by real-time polymerase chain reaction (qPCR) method was created. A specific gene fragment of 126 bp from WSSV genome was successfully amplified by PCR and cloned into pGEM-T vector. qPCR calibration curves using the created recombinant pGEM vector harboring 126 bp gene fragment as the standard DNA at concentrations of 3x102 to 3x108 copies/mL showed to have good linear regression with an efficiency of 99.1%, correlation coefficient R2 of 0.998 and the slope of -3.344. The recombinant vector was also used as a positive standard for detection and quantification of WSSV in a number of WSSV-infected shrimp samples and the values ranging from 3.69x103 copies/mL to 1.25x108 copies/mL WSSV were found in the collected samples.

Published
2022

11. Proteases from Penaeus monodon Fabricius, 1798 (Decapoda, Penaeidae): partial characterization and localization.

Author

Nguyen, Hong-Loan T., Quach, Hong-Thai, Trinh, Dinh-Quynh, Nguyen, Huong-Quynh, Pham, Bao-Yen, Dinh, Thai-Nho, Ngo, Thi-Trang, and Phan, Tuan-Nghia

Subjects
PENAEUS monodon, PENAEIDAE, DECAPODA, PROTEOLYTIC enzymes, SODIUM dodecyl sulfate, MOLECULAR weights, POLYACRYLAMIDE gel electrophoresis, CASEINS
Abstract

This study aimed to characterize and determine the distribution of proteases in various anatomical parts of Penaeus monodon using mainly electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels impregnated with casein (casein-SDS-PAGE) and specific protease inhibitors. Twelve caseinolytic bands designated A-L, with estimated molecular masses of 11.4-49.6 kDa were detected by SDS-PAGE, and were most abundant in the hepatopancreas. Among the 12 bands, E, J, K and L were predominant, A, G and H were identified as chymotrypsin-like proteases, D, E, I, J, K and L were identified as trypsin-like proteases, and B, C and F were identified as serine proteases. The proteases were most active at 50°C and pH 7.0. The proteolytic and trypsin-like activities of Penaeus monodon were most abundant in hepatopancreas extracts and least abundant in extracts from the haemolymph. Trypsin-like proteases in the hepatopancreas extract were found to activate prophenoloxidase (proPO) to form phenoloxidase (PO). Résumé: Cette étude vise à caractériser et à déterminer la distribution des protéases dans diverses parties anatomiques de Penaeus monodon , en utilisant principalement la technique de l'électrophorèse en gel de polyacrylamide contenant du dodécylsulfate de sodium (SDS) imprégné de caséine (casein SDS-PAGE) et des inhibiteurs spécifiques de protéase. La technique de l'électrophorèse SDS-PAGE révèle l'existence de douze bandes d'activité caséinolytique, désignées A-L et de poids moléculaire variant de 11,4 à 49,6 KDa. Ces bandes sont les plus abondantes dans l'hépatopancréas. Sur les douze bandes révélées, quatre ont été trouvées prédominantes à savoir E, J, K et L. Les bandes A, G et H ont été identifiées comme des protéases de type chymotrypsine, les bandes D, E, I, J, K et L comme des protéases de type trypsine et les bandes B, C et F des comme des protéases à sérine. La meilleure activité protéasique a été observée à 50°C et à pH 7,0. Les activités protéolytiques et celles de type trypsine de Penaeus monodon ont été trouvées plus abondantes dans l'hépatopancréas et moins abondantes dans l'hémolymphe. La formation de la phénoloxydase (PO), dans l'hépatopancréas, est due à l'activation de la prophénoloxydase (proPO) par des protéases de type trypsine. [ABSTRACT FROM AUTHOR]

Published
2022
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