Characterization of Hepatitis B surface Antigen gene containing recombinant Pichia pastoris

Abstract

Hepatitis B is a potentially life-threatening liver infection caused by the Hepatitis B virus. It is a major health problem worldwide. So, the development of safe and effective vaccines against this disease can be a huge triumph for mankind. Pichia. pastoris is a suitable host for inducing the production of the Hepatitis B surface antigen (HBsAg) inside it’s body as well as it can be used for commercially large scale production. This can be done by inserting the HBsAg gene into the plasmid vector of P. pastoris yeast under the alcohol oxidase (AOX) promoter and let them grow in suitable condition. Our study was conducted to ensure the insertion of the HBsAg gene under the AOX promoter into the yeast P. pastoris after allowing them to be stored as Master Cell Bank (MCB) and Working Cell Bank (WCB). Thus, the first aim of this study was to characterize the Master Cell Bank and Working Cell Bank by Phenotypic tests; like microbial purity, identity and viability. Secondly, HBsAg gene in P. pastoris was analyzed by polymerase chain reaction (PCR) using two sets of primers named F1R1 and F2R2, followed by performing gel electrophoresis and Sanger sequencing. Characterization of the cell banks as well as ensuring the recombinant HBsAg gene indicates that visually all the P. pastoris contain the HBsAg gene. Finally, the main objective of this study was to ensure the expression, purification, and characterization of HBsAg in yeast P. pastoris. The study was carried out successfully.