CONSTRUCTION OF RECOMBINANT VECTOR PPICZΑ-A-HBSAG FOR EXPRESSION OF HEPATITIS B SURFACE ANTIGEN (HBSAG) IN PICHIA PASTORIS

• Main Author: (NIM : 10508066); Pembimbing : Dessy Natalia, Ph.D, NURFITRIANI
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/16739
• Institution: Institut Teknologi Bandung

Nowadays, it is estimated that more than 350 million people in the world have chronic hepatitis B disease with a high risk to become liver cirrhosis and hepatocellular carcinoma. Therefore, an early diagnosis method to prevent further progression of the disease from the acute phase to the chronic phase is required. Hepatitis B surface antigen (HBsAg) is a subparticle secreted by hepatitis B virus (HBV) during the HBV infection. The HBsAg presence in the cell can induce production of antibody against HBV (anti-HBs), and hence the course of HBV infection could be determined by the interplay between viral replication via HBV protein production and the host’s immune response. Therefore, the diagnosis of infection in clinical practice could be established by the serological detection of HBV protein product as well as be measuring the level of antibodies produced by the host. The long term goal of this research is to produce recombinant HBsAg in the Pichia pastoris expression system, which will be used for detecting the presence of anti-HB in the blood. This study aimed at construction of a P. pastoris expression vector pPICZα-A harboring the gene encoding for S-HBsAg gene from subtype adw2 genotype B. The research work included amplification of HBsAg gene by Polymerase Chain Reaction (PCR), cloning of the PCR-amplified product of HBsAg gene fragment into the p-GEMT Easy vector and followed by subcloning of the HBsAg gene fragment into the pPICZα-A. The restriction enzyme and nucleotide sequence analysis revealed that the recombinant plasmids pGEMT-Easy-HBsAg and pPICZα-A-HbsAg have been successfully obtained.