Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System

Hepatitis B virus (HBV) infection is a worldwide health problem, which can lead to severe liver disease mainly hepatocellular carcinoma and cirrhosis. Hepatitis B surface antigen (HBsAg) is a glycoprotein found on the surface of the virus and its presence in the blood is an indicator of positive hep...

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Main Author:	Mohamad Sinang, Fazia
Format:	Thesis
Language:	English English
Published:	2010
Subjects:	Antigens Yeast Liver - Diseases
Online Access:	http://psasir.upm.edu.my/id/eprint/21265/1/FBSB_2010_3_R.pdf
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id	my-upm-ir.21265
record_format	uketd_dc
spelling	my-upm-ir.212652013-05-27T08:16:01Z Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System 2010-08 Mohamad Sinang, Fazia Hepatitis B virus (HBV) infection is a worldwide health problem, which can lead to severe liver disease mainly hepatocellular carcinoma and cirrhosis. Hepatitis B surface antigen (HBsAg) is a glycoprotein found on the surface of the virus and its presence in the blood is an indicator of positive hepatitis B infection. The existing commercially available vaccine is based on the produced small HBsAg (S-HBsAg) via recombinant DNA technology. Although it is effective, about 5-10% of normal individuals demonstrates no or low response to the standard vaccination schedule. The efficacy of the large HBsAg (L-HBsAg) derived vaccine may differ from S-HBsAg, but such study needs to be conducted to evaluate the potential of L-HBsAg. To date, there is no infonnation on the expression of L-HBsAg intracellularly. Therefore, this study was carried out to express the L-HBsAg protein in yeast Pichia pastoris (P. pastoris) intracellularly. The L-HBsAg gene is inserted into the pPICZ C plasmid and introduced into P. pastoris. The positive clones were confinned by DNA sequencing. Small scale expression of the recombinant L-HBsAg was analyzed by using Western blotting. The glycosylated L-HBsAg of about 42 kDa was detected by Western blotting and its concentration increase from 24 hours to 72 hours sample. The production of L-HBsAg was then scaled up and purified by using sucrose density gradient centrifugation. ELISA showed that the purified L-HBsAg was antigenic. Electron microscopic analysis revealed that the L-HBsAg assembled into spherical particles about 25 nm. The LHBsAg produced in P. pastoris provides an alternative to the vaccines available in market Antigens Yeast Liver - Diseases 2010-08 Thesis http://psasir.upm.edu.my/id/eprint/21265/ http://psasir.upm.edu.my/id/eprint/21265/1/FBSB_2010_3_R.pdf application/pdf en public phd doctoral Universiti Putra Malaysia Antigens Yeast Liver - Diseases Faculty of Biotechnology and Biomolecular Sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Antigens Yeast Liver - Diseases
spellingShingle	Antigens Yeast Liver - Diseases Mohamad Sinang, Fazia Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System
description	Hepatitis B virus (HBV) infection is a worldwide health problem, which can lead to severe liver disease mainly hepatocellular carcinoma and cirrhosis. Hepatitis B surface antigen (HBsAg) is a glycoprotein found on the surface of the virus and its presence in the blood is an indicator of positive hepatitis B infection. The existing commercially available vaccine is based on the produced small HBsAg (S-HBsAg) via recombinant DNA technology. Although it is effective, about 5-10% of normal individuals demonstrates no or low response to the standard vaccination schedule. The efficacy of the large HBsAg (L-HBsAg) derived vaccine may differ from S-HBsAg, but such study needs to be conducted to evaluate the potential of L-HBsAg. To date, there is no infonnation on the expression of L-HBsAg intracellularly. Therefore, this study was carried out to express the L-HBsAg protein in yeast Pichia pastoris (P. pastoris) intracellularly. The L-HBsAg gene is inserted into the pPICZ C plasmid and introduced into P. pastoris. The positive clones were confinned by DNA sequencing. Small scale expression of the recombinant L-HBsAg was analyzed by using Western blotting. The glycosylated L-HBsAg of about 42 kDa was detected by Western blotting and its concentration increase from 24 hours to 72 hours sample. The production of L-HBsAg was then scaled up and purified by using sucrose density gradient centrifugation. ELISA showed that the purified L-HBsAg was antigenic. Electron microscopic analysis revealed that the L-HBsAg assembled into spherical particles about 25 nm. The LHBsAg produced in P. pastoris provides an alternative to the vaccines available in market
format	Thesis
qualification_name	Doctor of Philosophy (PhD.)
qualification_level	Doctorate
author	Mohamad Sinang, Fazia
author_facet	Mohamad Sinang, Fazia
author_sort	Mohamad Sinang, Fazia
title	Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System
title_short	Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System
title_full	Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System
title_fullStr	Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System
title_full_unstemmed	Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System
title_sort	cloning, expression and characterization of hepatitis b large surface antigen in yeast system
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Biotechnology and Biomolecular Sciences
publishDate	2010
url	http://psasir.upm.edu.my/id/eprint/21265/1/FBSB_2010_3_R.pdf
_version_	1747811484722790400

Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System

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