Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli

Nipah virus (NiV) possesses a nonsegmented, single-stranded negative sense RNA genome that contains six structural genes arranged in the order of 3' N-P-M-F-G-L 5'. The nucleocapsid (N) gene of Nipah virus isolated from swine was amplified from the viral RNA by reverse transcription pol...

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Main Author:	Ong, Swee Tin
Format:	Thesis
Language:	English English
Published:	2002
Subjects:	Viral antigens Escherichia coli infections in swine
Online Access:	http://psasir.upm.edu.my/id/eprint/11592/1/FSAS_2002_57_A.pdf
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spelling	my-upm-ir.115922012-05-18T03:16:42Z Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli 2002-12 Ong, Swee Tin Nipah virus (NiV) possesses a nonsegmented, single-stranded negative sense RNA genome that contains six structural genes arranged in the order of 3' N-P-M-F-G-L 5'. The nucleocapsid (N) gene of Nipah virus isolated from swine was amplified from the viral RNA by reverse transcription polymerase chain reaction (RT-PCR). The nucleocapsid (N) gene of Nipah virus was cloned into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a 63 kDa fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. The N gene sequence shared 99% homology with that of Nipah virus isolated from human. The coding region of N protein of NiV was cloned into different vectors and subsequently introduced into different E. coli strains. The yield of the N protein produced in different vectors and different hosts was compared. It was found that the amount of N protein expressed by the pTrcHis2 vector containing the trc promoter in E. coli SG 935 was four-fold higher than that of vector pRSETB and pGEX-4T-l in E. coli strain BL 21 series. Deletion of the N- or/and C-terminal region of the N protein revealed that the N-terminal region plays a role in N protein solubility, but a mutant (MN50fus) containing the second half of the N protein showed the highest expression level in all the three E. coli strains. Lowering the growth temperature of E. coli cell cultures to 25°C improved the solubility of the full-length and truncated Nfus protein from 50% to 80%. This study addresses the fundamental problems encountered in production of Nipah viral N protein in E. coli which may be useful as an alternative antigen for the detection of anti-NiV in swine. Viral antigens Escherichia coli infections in swine 2002-12 Thesis http://psasir.upm.edu.my/id/eprint/11592/ http://psasir.upm.edu.my/id/eprint/11592/1/FSAS_2002_57_A.pdf application/pdf en public masters Universiti Putra Malaysia Viral antigens Escherichia coli infections in swine Faculty of Environmental studies English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Viral antigens Escherichia coli infections in swine
spellingShingle	Viral antigens Escherichia coli infections in swine Ong, Swee Tin Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli
description	Nipah virus (NiV) possesses a nonsegmented, single-stranded negative sense RNA genome that contains six structural genes arranged in the order of 3' N-P-M-F-G-L 5'. The nucleocapsid (N) gene of Nipah virus isolated from swine was amplified from the viral RNA by reverse transcription polymerase chain reaction (RT-PCR). The nucleocapsid (N) gene of Nipah virus was cloned into the bacterial expression vector, pTrcHis2, for intracellular expression in three Escherichia coli strains: TOP 10, BL 21 and SG 935. The N protein was expressed as a 63 kDa fusion protein containing the myc epitope and His-tag at its C-terminal end. The amount of the fusion protein expressed in strain SG 935 was significantly higher than the other two strains, and was detected by the anti-myc antibody, anti-His and swine anti-NiV serum. The N gene sequence shared 99% homology with that of Nipah virus isolated from human. The coding region of N protein of NiV was cloned into different vectors and subsequently introduced into different E. coli strains. The yield of the N protein produced in different vectors and different hosts was compared. It was found that the amount of N protein expressed by the pTrcHis2 vector containing the trc promoter in E. coli SG 935 was four-fold higher than that of vector pRSETB and pGEX-4T-l in E. coli strain BL 21 series. Deletion of the N- or/and C-terminal region of the N protein revealed that the N-terminal region plays a role in N protein solubility, but a mutant (MN50fus) containing the second half of the N protein showed the highest expression level in all the three E. coli strains. Lowering the growth temperature of E. coli cell cultures to 25°C improved the solubility of the full-length and truncated Nfus protein from 50% to 80%. This study addresses the fundamental problems encountered in production of Nipah viral N protein in E. coli which may be useful as an alternative antigen for the detection of anti-NiV in swine.
format	Thesis
qualification_level	Master's degree
author	Ong, Swee Tin
author_facet	Ong, Swee Tin
author_sort	Ong, Swee Tin
title	Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli
title_short	Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli
title_full	Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli
title_fullStr	Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli
title_full_unstemmed	Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli
title_sort	production of the nucleocapsid protein of a swine nipah virus isolate in escherichia coli
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Environmental studies
publishDate	2002
url	http://psasir.upm.edu.my/id/eprint/11592/1/FSAS_2002_57_A.pdf
_version_	1747811245315063808

Production Of The Nucleocapsid Protein Of A Swine Nipah Virus Isolate In Escherichia Coli

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