Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression

The anchoring of proteins to the cell surface of Lactococcus using recombinant DNA techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. Presently available anchoring systems are based on recombinant bacteria displaying pro...

Full description

Saved in:

Bibliographic Details
Main Author:	Yasid, Nur Adeela
Format:	Thesis
Language:	English English
Published:	2009
Subjects:	Newcastle disease virus - Case studies
Online Access:	http://psasir.upm.edu.my/id/eprint/7540/1/ABS_----__FBSB_2009_26.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-upm-ir.7540
record_format	uketd_dc
spelling	my-upm-ir.75402013-05-27T07:35:28Z Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression 2009 Yasid, Nur Adeela The anchoring of proteins to the cell surface of Lactococcus using recombinant DNA techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. Presently available anchoring systems are based on recombinant bacteria displaying proteins or peptides on the cell surface. This research is focused on using lactococcal cell wall binding (cwb) region for display of viral epitopes and antigenic determinants. Newcastle disease virus (NDV) is the aetiological agent of Newcastle disease which can result in 100% morbidity and mortality in chicken. Immune response of haemagglutinin-neuraminidase (HN) protein antigens of NDV plays an important role in the prevention of viral infection. A lactococcal domain coding region, acmA’ (300 bp) was cloned into the mammalian expression vector, pcDNA3.1/His. The 1.7 kb HN gene was then inserted at the 5’-end of the acmA’ coding region. Plasmid extraction and restriction enzyme digestion analyses showed that cloning was carried out successfully. Sequencing results indicated that the inserts (acmA’, HN and acmA’HN) were 99%, 97% and 98% homologous to the published sequence of Lactococcus lactis and NDV strain AF2240. The condition for transfection was optimised by testing various amounts of transfected DNA (1 μg, 2 μg and 3 μg) and different charge ratio of liposome reagent (μl): DNA (μg) in a 6-well plate. Transient expression of the fusion protein in Chinese hamster ovary (CHO) cells was analysed by SDS-PAGE and Western blotting. The results indicated that the best combination of DNA used for transfection was 2 μg in 1:3 charge ratio of the liposome:DNA. This study showed that a recombinant acmA’, HN and acmA’-HN have been successfully constructed, transfected and expressed in CHO cells. This work could contribute towards the development of a recombinant vaccine delivery system for NDV. Newcastle disease virus - Case studies 2009 Thesis http://psasir.upm.edu.my/id/eprint/7540/ http://psasir.upm.edu.my/id/eprint/7540/1/ABS_----__FBSB_2009_26.pdf application/pdf en public masters Universiti Putra Malaysia Newcastle disease virus - Case studies Faculty of Biotechnology and Biomolecular Sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Newcastle disease virus - Case studies
spellingShingle	Newcastle disease virus - Case studies Yasid, Nur Adeela Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression
description	The anchoring of proteins to the cell surface of Lactococcus using recombinant DNA techniques is an exciting and emerging research area that holds great promise for a wide variety of biotechnological applications. Presently available anchoring systems are based on recombinant bacteria displaying proteins or peptides on the cell surface. This research is focused on using lactococcal cell wall binding (cwb) region for display of viral epitopes and antigenic determinants. Newcastle disease virus (NDV) is the aetiological agent of Newcastle disease which can result in 100% morbidity and mortality in chicken. Immune response of haemagglutinin-neuraminidase (HN) protein antigens of NDV plays an important role in the prevention of viral infection. A lactococcal domain coding region, acmA’ (300 bp) was cloned into the mammalian expression vector, pcDNA3.1/His. The 1.7 kb HN gene was then inserted at the 5’-end of the acmA’ coding region. Plasmid extraction and restriction enzyme digestion analyses showed that cloning was carried out successfully. Sequencing results indicated that the inserts (acmA’, HN and acmA’HN) were 99%, 97% and 98% homologous to the published sequence of Lactococcus lactis and NDV strain AF2240. The condition for transfection was optimised by testing various amounts of transfected DNA (1 μg, 2 μg and 3 μg) and different charge ratio of liposome reagent (μl): DNA (μg) in a 6-well plate. Transient expression of the fusion protein in Chinese hamster ovary (CHO) cells was analysed by SDS-PAGE and Western blotting. The results indicated that the best combination of DNA used for transfection was 2 μg in 1:3 charge ratio of the liposome:DNA. This study showed that a recombinant acmA’, HN and acmA’-HN have been successfully constructed, transfected and expressed in CHO cells. This work could contribute towards the development of a recombinant vaccine delivery system for NDV.
format	Thesis
qualification_level	Master's degree
author	Yasid, Nur Adeela
author_facet	Yasid, Nur Adeela
author_sort	Yasid, Nur Adeela
title	Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression
title_short	Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression
title_full	Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression
title_fullStr	Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression
title_full_unstemmed	Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression
title_sort	construction of a recombinant plasmid from newcastle disease virus antigen, haemagglutinin-neuraminidase and lactococcal n-acetylmuramidase and its expression
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2009
url	http://psasir.upm.edu.my/id/eprint/7540/1/ABS_----__FBSB_2009_26.pdf
_version_	1747810696668643328

Construction of a Recombinant Plasmid from Newcastle Disease Virus Antigen, Haemagglutinin-Neuraminidase and Lactococcal N-Acetylmuramidase and its Expression

Similar Items