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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my-upm-ir.115922024-06-24T02:29:36Z 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.pdf text en public masters Universiti Putra Malaysia Viral antigens Escherichia coli infections in swine Faculty of Environmental studies Tan, Wen Siang English |
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Universiti Putra Malaysia |
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PSAS Institutional Repository |
language |
English English |
advisor |
Tan, Wen Siang |
topic |
Viral antigens Escherichia coli infections in swine |
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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.pdf |
_version_ |
1804888641923710976 |