Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1

Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are the most important pa...

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Main Authors: Abubakar, Mustapha Bala, Ideris, Aini, Omar, Abdul Rahman, Bejo, Mohd Hair
Format: Conference or Workshop Item
Language:English
Published: 2009
Online Access:http://psasir.upm.edu.my/id/eprint/65127/1/PGM-1-7.pdf
http://psasir.upm.edu.my/id/eprint/65127/
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Institution: Universiti Putra Malaysia
Language: English
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spelling my.upm.eprints.651272018-09-04T04:04:09Z http://psasir.upm.edu.my/id/eprint/65127/ Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1 Abubakar, Mustapha Bala Ideris, Aini Omar, Abdul Rahman Bejo, Mohd Hair Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are the most important pathogens for the poultry industry in recent times. The haemagglutinin protein and neuraminidase, serves as the target for the immune response of the host. Due to recurrent genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not withstand pressure by the novel hybrid virus. The emergence of genetic engineering technology provided the industry with new methods of manufacturing diagnostics tools and vaccines. After extraction of RNA from the cell culture of strain influenza A/Chicken/Malaysia/2004(H5N1) of AIV, the viral RNA was converted to cDNA by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed by agarose gel electrophoresis. The intact PCR product of full length haemagglutinin gene was cloned in TOPOTM TA Cloning vector. The full-length HA-encoding gene of H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed plasmid was transformed into E.coli.Top10, Plasmid DNA from transformed bacteria was extracted in white colony and positive clones were confirmed by restriction digestion with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing. Construction of a recombinant pPICZA/H5HA plasmid containing the full length haemagglutinin gene was achieved as a first step towards the expression in Pichia pastoris. 2009 Conference or Workshop Item PeerReviewed text en http://psasir.upm.edu.my/id/eprint/65127/1/PGM-1-7.pdf Abubakar, Mustapha Bala and Ideris, Aini and Omar, Abdul Rahman and Bejo, Mohd Hair (2009) Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1. In: 8th Malaysia Congress on Genetics, 4-6 Aug. 2009, Genting Highlands, Malaysia. (pp. 180-184).
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description Avian Influenza viruses belonging to the Orthomyxoviridae family are enveloped viruses with segmented negative sense RNA genome surrounded by a helical symmetry capsid. Influenza viruses, especially the highly pathogenic avian influenza virus (HPAI) such as H5 or H7 subtype are the most important pathogens for the poultry industry in recent times. The haemagglutinin protein and neuraminidase, serves as the target for the immune response of the host. Due to recurrent genetic reassortments between avian and human influenza viruses, global pandemics may emerge and the naive human immunity could not withstand pressure by the novel hybrid virus. The emergence of genetic engineering technology provided the industry with new methods of manufacturing diagnostics tools and vaccines. After extraction of RNA from the cell culture of strain influenza A/Chicken/Malaysia/2004(H5N1) of AIV, the viral RNA was converted to cDNA by a specific primer. The cDNA was amplified by the polymerase chain reaction (PCR) and analyzed by agarose gel electrophoresis. The intact PCR product of full length haemagglutinin gene was cloned in TOPOTM TA Cloning vector. The full-length HA-encoding gene of H5N1 AIV was subcloned into a pPICZA vector. After successful ligation, the constructed plasmid was transformed into E.coli.Top10, Plasmid DNA from transformed bacteria was extracted in white colony and positive clones were confirmed by restriction digestion with Sacl and Not1 restriction enzymes, colony PCR screening and nucleotide sequencing. Construction of a recombinant pPICZA/H5HA plasmid containing the full length haemagglutinin gene was achieved as a first step towards the expression in Pichia pastoris.
format Conference or Workshop Item
author Abubakar, Mustapha Bala
Ideris, Aini
Omar, Abdul Rahman
Bejo, Mohd Hair
spellingShingle Abubakar, Mustapha Bala
Ideris, Aini
Omar, Abdul Rahman
Bejo, Mohd Hair
Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1
author_facet Abubakar, Mustapha Bala
Ideris, Aini
Omar, Abdul Rahman
Bejo, Mohd Hair
author_sort Abubakar, Mustapha Bala
title Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1
title_short Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1
title_full Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1
title_fullStr Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1
title_full_unstemmed Molecular cloning of full-length haemagglutinin (H5HA) gene of highly pathogenic avian influenza virus (HPAI) H5N1
title_sort molecular cloning of full-length haemagglutinin (h5ha) gene of highly pathogenic avian influenza virus (hpai) h5n1
publishDate 2009
url http://psasir.upm.edu.my/id/eprint/65127/1/PGM-1-7.pdf
http://psasir.upm.edu.my/id/eprint/65127/
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