Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method

Centella asiatica is a locally important medicinal plant. It is non-toxic, high in medicinal values, and can serve as a good candidate for genetic manipulation. However, to date no transformation protocol has been developed to fully utilize the potential of this plant. Therefore, this research is to...

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Main Author:	Lai, Kok Song
Format:	Thesis
Language:	English English
Published:	2008
Subjects:	Newcastle disease virus - Case studies
Online Access:	http://psasir.upm.edu.my/id/eprint/7567/1/ABS_---__FBSB_2008_18.pdf
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spelling	my-upm-ir.75672013-05-27T07:35:39Z Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method 2008 Lai, Kok Song Centella asiatica is a locally important medicinal plant. It is non-toxic, high in medicinal values, and can serve as a good candidate for genetic manipulation. However, to date no transformation protocol has been developed to fully utilize the potential of this plant. Therefore, this research is to establish an efficient particle bombardment transformation protocol for C. asiatica embryogenic calli. In addition, an attempt to express the haemagglutinin-neuraminidase (HN) protein from Newcastle disease virus (NDV) strain AF2240 in C. asiatica embryogenic calli were carried out using the developed transformation system. The HN protein can serve as a potential vaccine candidate for Newcastle disease (ND) in poultry. The induced embryogenic calli revealed the presence of extracellular matrix layer (ECM) during the microscopy studies. Particle bombardment transformation protocol was developed using the green fluorescent protein (GFP) as reporter. A total of eight parameters mainly different target distance, helium pressure, gold particles size, chamber vacuum pressure, number of bombardment, precipitation agents, post-bombardment incubation time, and plasmid DNA concentration were identified and successfully optimized. Based on the established protocol, transformations of C. asiatica embryogenic calli were performed using the constructed recombinant pMDC32’HN and HBT95:sGFP(S65T)-NOS’HN plasmids. Genomic PCR analysis revealed the presence of HN transgene in the transformed lines. Unfortunately no protein bands were detected during SDS-PAGE and western blotting, indicating low or no HN protein expression. Transformation using recombinant HBT95:sGFP(S65T)-NOS’HN plasmid resulted in very low GFP expression as compared to the positive control. Nonetheless, the mRNA transcripts were detected in the RT-PCR analysis. Positive signal from the dot blot assay further confirmed the presence of the HN protein expression in the transformed lines. Newcastle disease virus - Case studies 2008 Thesis http://psasir.upm.edu.my/id/eprint/7567/ http://psasir.upm.edu.my/id/eprint/7567/1/ABS_---__FBSB_2008_18.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 Lai, Kok Song Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method
description	Centella asiatica is a locally important medicinal plant. It is non-toxic, high in medicinal values, and can serve as a good candidate for genetic manipulation. However, to date no transformation protocol has been developed to fully utilize the potential of this plant. Therefore, this research is to establish an efficient particle bombardment transformation protocol for C. asiatica embryogenic calli. In addition, an attempt to express the haemagglutinin-neuraminidase (HN) protein from Newcastle disease virus (NDV) strain AF2240 in C. asiatica embryogenic calli were carried out using the developed transformation system. The HN protein can serve as a potential vaccine candidate for Newcastle disease (ND) in poultry. The induced embryogenic calli revealed the presence of extracellular matrix layer (ECM) during the microscopy studies. Particle bombardment transformation protocol was developed using the green fluorescent protein (GFP) as reporter. A total of eight parameters mainly different target distance, helium pressure, gold particles size, chamber vacuum pressure, number of bombardment, precipitation agents, post-bombardment incubation time, and plasmid DNA concentration were identified and successfully optimized. Based on the established protocol, transformations of C. asiatica embryogenic calli were performed using the constructed recombinant pMDC32’HN and HBT95:sGFP(S65T)-NOS’HN plasmids. Genomic PCR analysis revealed the presence of HN transgene in the transformed lines. Unfortunately no protein bands were detected during SDS-PAGE and western blotting, indicating low or no HN protein expression. Transformation using recombinant HBT95:sGFP(S65T)-NOS’HN plasmid resulted in very low GFP expression as compared to the positive control. Nonetheless, the mRNA transcripts were detected in the RT-PCR analysis. Positive signal from the dot blot assay further confirmed the presence of the HN protein expression in the transformed lines.
format	Thesis
qualification_level	Master's degree
author	Lai, Kok Song
author_facet	Lai, Kok Song
author_sort	Lai, Kok Song
title	Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method
title_short	Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method
title_full	Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method
title_fullStr	Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method
title_full_unstemmed	Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method
title_sort	expression of haemagglutinin-neuraminidase envelope protein of newcastle disease virus strain af2240 in centella asiatica (pegaga) embryogenic calli through optimized particle bombardment method
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular sciences
publishDate	2008
url	http://psasir.upm.edu.my/id/eprint/7567/1/ABS_---__FBSB_2008_18.pdf
_version_	1747810701594853376

Expression of Haemagglutinin-Neuraminidase Envelope Protein of Newcastle Disease Virus Strain Af2240 in Centella Asiatica (Pegaga) Embryogenic Calli Through Optimized Particle Bombardment Method

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