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


Citation

Mohamad Sinang, Fazia (2010) Cloning, Expression and Characterization of Hepatitis B Large Surface Antigen In Yeast System. PhD thesis, Universiti Putra Malaysia.

Abstract

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


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Additional Metadata

Item Type: Thesis (PhD)
Subject: Antigens
Subject: Yeast
Subject: Liver - Diseases
Call Number: FBSB 2010 3
Chairman Supervisor: Prof. Dr. Tan Wen Siang
Divisions: Faculty of Biotechnology and Biomolecular Sciences
Notes: Prof. Dr. Tan Wen Siang
Depositing User: Haridan Mohd Jais
Date Deposited: 18 Mar 2013 06:38
Last Modified: 27 May 2013 08:16
URI: http://psasir.upm.edu.my/id/eprint/21265
Statistic Details: View Download Statistic

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