Stability Assay of sHBsAg Gene Integration in Hansenula polymorpha RB11 Genome in Hepatitis B Vaccine Production

• Main Author: Syari Intan, Novia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/39891
• Institution: Institut Teknologi Bandung

Hepatitis B infection is one of the major public health problem both in Indonesia and in the world. This infection can be prevented by administering a vaccine containing the Hepatitis B virus surface antigen namely SHBsAg. Recombinant SHBsAg can be produced using Hansenula polymorpha RB11 which genome has been integrated by multicopy sHBsAg genes. Although it is potential to produce a large amount of proteins using this system, the integrated multicopy gene may undergo gene excision through loop out mechanism. This may cause a problem because the culture used in vaccine production must have a stable expression system throughout the whole production process. This research aims to examine the stability of sHBSAg gene integration in Hansenula polymorpha RB11 genome. The sample tested in this research was taken from the working seed and some other production processes. Repetitive subculture until the 44th generation of recombinant Hansenula polymorpha RB11 was also done for eight days to examine the stability of sHBSAg gene integration during repetitive subculture. Each sample’s genome was isolated and the presence of sHBSAg gene was then evaluated using PCR and qPCR. sHBSAg gene sequence resemblance to the parent seed was also evaluated by sequencing. The visualization of PCR result using agarose gel electrophoresis shows the sHBsAg gene amplicon as a band about 681bp long corresponding to the reference. The sequencing result also shows a resemblance in sHBSAg gene sequence between all samples and parent seed. The Cq value results from qPCR analysis for all samples with the same DNA concentration shows a close value ranging from 22.1-23.4. Through this research, it was concluded that the integration of sHBsAg gene in Hansenula polymorpha RB11 genome is stable throughout the repetitive subculture until the 44th generation and throughout the production process.