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2. Master Thesis / Tesis Master
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Title:	Functional analysis of the L protein of nipah virus using serial deletion mutagenesis
Authors:	Shahandeh Ali. (P41140)
Supervisor:	Dr. Amir Bin Rabu
Keywords:	Dissertations, Academic -- Malaysia Nipah virus (NiV) Mutated genes
Issue Date:	8-Dec-2011
Description:	Nipah virus (NiV) is a paramyxovirus that has been identified as the etiologic agent of severe febrile encephalitis. NiV L gene encodes RNA-dependent RNA polymerase (RdRp) which is an enzyme required for replication. This research is an effort to recognize the characteristics of the N and the C terminal of NiV L gene. The objectives of this research are to construct the mutated L gene and to examine these mutated genes in a replication model of virus. In order to fulfill the objectives firstly, high fidelity PCR was applied to make serial deletions on the L gene of NiV. Five serial mutations with 20 amino acids residues distance between them were engineered at both ends of the L gene. Subsequently the truncated genes were cloned into the plasmid vector and formed five new recombinant plasmids at the carboxyl-terminus and five new recombinant plasmids at the amino-terminus of the L gene. Sequence analysis indicated the originality of the new recombinant plasmids and also confirmed that deletions were executed as they designed. The ability of new recombinants plasmids in viral replication were tested by using minigenome system which is based on an intracellular and plasmid-based replication assay. Analysis of intracellular transcription-replication process of new recombinant plasmids required introducing them into cell culture. Replication activity of new recombinant plasmids was assessed by measuring the reporter protein (CAT), extracted from the cell culture. CAT-ELISA which is a colorimetric enzyme immunoassay test was used to determine the quantity of the CAT protein. Furthermore, in order to visualize the CAT protein in the samples extracted from the cell cultures, Western blot was performed. The results of the study suggest that there is a difference in functionality between the C and the N terminal of NiV L during viral replication. Five serial mutants at the N terminal were completely defective and they failed to express the reporter gene. The mutants at the C terminal part of L protein showed their enzymatic activity reduced.,Master
Pages:	89
Call Number:	QR404.2.N55 S535 2011
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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