Abstract
Hepatitis B is the major health problem worldwide including in Indonesia. Vaccination is the best prevention strategy for the disease. For the purpose of vaccine development and to decrease drug import, production of Hepatitis B Virus (HBV) small surface antigen (sHBsAg) from Indonesian HBV subtype is needed. The recombinant protein production can be conducted by integrating multi expression cassettes of sHBsAg gene in Pichia pastoris chromosome using gene replacement method. Such integration method turns out to allow loss of foreign gene from chromosome by excisional recombination-mediated looping out. This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium. The methanol induction was conducted twice at 63-h and 75-h. Integration stability determination was conducted qualitatively using PCR and quantitatively using qPCR absolute quantification. A band of 208 bp with similar intensity was observed after amplification of genomic DNA. All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction.
Highlights
Hepatitis B is an infectious disease affecting hepatocytes and is caused by Hepatitis B Virus (HBV)
This research was aimed to determine integration stability of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome inducted with 2% methanol in FM22 medium
All samples generated the same Ct value of around 22 with four copies of sHBsAg gene per genome. The result from this experiment shows that integration of four copies of sHBsAg expression cassette in P. pastoris GS115 chromosome is stable during methanol induction
Summary
Hepatitis B is an infectious disease affecting hepatocytes and is caused by Hepatitis B Virus (HBV). Hepatitis B can be prevented by vaccination which contains HBV’s small surface antigen (sHBsAg). Presence of sHBsAg in the form of Virus-Like Particle (VLP) in blood can induce antibody to eliminate HBV (Lunsdorf et al 2011). Hepatitis B vaccine has been available in Indonesia since 1997, but up until now Indonesia still imports sHBsAg protein to Production of sHBsAg recombinant protein from Indonesian isolate can be conducted by expressing sHBsAg gene in certain organism. Other microorganism than can be utilised as expression platform is P. pastoris (Bo et al 2005). Expression of sHBsAg gene in P. pastoris can be increased in several ways such as using strong and regulated promoter (AOX promoter), inducing promoter with optimum methanol concentration, and increasing number of integrated expression cassette in chromosome
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