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All Journal HAYATI Journal of Biosciences
Patricia Gita Naully
School of Life Science and Technology, Institut Teknologi Bandung, Bandung
Agriculture, Biological Sciences & Forestry Earth & Planetary Sciences

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Integration Stability of sHBsAg-Multi Expression Cassettes in Pichia pastoris GS115 during Methanol Induction Patricia Gita Naully; Neni Nurainy; Elvi Restiawaty; Dessy Natalia; Debbie Soefie Retnoningrum; Wardono Niloperbowo; Ernawati Arifin Giri-Rachman
HAYATI Journal of Biosciences Vol. 27 No. 4 (2020): October 2020
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.4308/hjb.27.4.283

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.
Co-Authors DEBBIE SOEFIE RETNONINGRUM Dessy Natalia Elvi Restiawaty Ernawati Arifin Giri-Rachman Neni Nurainy Wardono Niloperbowo