STABILITY TEST AND DETERMINATION OF INTEGRATED sHBsAg GENE COPY NUMBER ON CHROMOSOME OF Pichia pastoris GS115

• Main Author: Gita Naully, Patricia
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/35016
• Institution: Institut Teknologi Bandung

Hepatitis B virus is one of the causes of liver inflammation in hepatitis infected patients. This infection can be prevented by vaccination which contains HBV surface protein (sHBsAg). Recently, Indonesia still imports sHBsAg protein to meet the nation’s need. Therefore, it is crucial to produce sHBsAg protein that match with HBV subtype in Indonesia. The production of sHBsAg recombinant protein can be done by integrating multiple copies of sHBsAg genes at one locus, resulting in sequential repetition of the sHBsAg gene in the chromosome of Pichia pastoris. The integration process can cause loss of sHBsAg genes from the chromosome that is caused by excisional recombination (looping out). The purpose of this study was to determine the stability and the number of sHBsAg gene in the chromosome of P. pastoris GS115. In this study, P. pastoris GS115 with integrated sHBsAg gene was cultivated for 105 generations in MGY media and induced by 2% methanol on FM22 media. Genomic DNA from the whole culture was isolated and the concentration was determined using fluorometer. Determination of stability and integrated sHBsAg gene copy number in P. pastoris GS115 chromosome were done qualitatively using PCR and quantitatively using absolute quantification qPCR. 208 bp-sized DNA bands were formed with the same intensity from every PCR samples. In qPCR analysis, all the samples produced the same Ct value, 22, which equal with four copies sHBsAg gene per genome. Based on qualitative and quantitative analysis, integration of four copies HBsAg gene in the chromosome of P. pastoris GS115 was stable for 105 generations in MGY medium and during cultivation and induction of 2% methanol on FM22 medium.