Cloning and Expressing Hepatitis B Virus Surface Protein (HBsAg) In Hansenula polymorpha

• Main Author: HERYAKUSUMA (NIM : 10512018), CHRISTIAN
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28501
• Institution: Institut Teknologi Bandung

<p align="justify">Indonesia has the second-highest endemicity of hepatitis B following Myanmar in Southeast Asia. In 2013, there were about 28 million people infected by hepatitis B in Indonesia. The number is higher than that of 2007 and predicted to increase in the future. Therefore, preventive action with vaccination should be applied. Hepatitis B surface protein (HBsAg) has been used as a hepatitis B vaccine in which the recombinant HBsAg is produced in yeasts Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha. The purposes of this research were to construct pHIPX4-HBsAg recombinant plasmid and to express HBsAg under the control of MOX promoter in H. polymorpha NCYC495. A 681 base pair gene fragment encoding HBsAg was amplified by PCR technique using pPICZ&#945;-A-HBsAg as a DNA template. The DNA fragment encoding HBsAg was recombined into H. polymorpha expression vector pHIPX4 on the restriction site of HindIII and SalI to obtain a 7.7 kb recombinant plasmid pHIPX4-HBsAg. H. polymorpha NCYC495 was transformed by pHIPX4-HBsAg using electroporation method and was selected on YND media in the absence of leucine. H. polymorpha NCYC495-pHIPX4- HBsAg transformant was grown in mineral media and methanol 0,5% (v/v) was then added to induce the expression of HBsAg protein. Analysis of SDS-polyacrylamide gel electrophoresis suggested that HBsAg protein is produced with molecular mass of ~38 kDa. Crude extract of HBsAg protein samples also gave a positive result on HBsAg diagnostic kit test and enzyme- linked immunosorbent assay.<p align="justify">