Expression of HPV Subtype 52 L1 Protein of Indonesian Isolate in Pichia pastoris

• Main Author: Aprilia Helena, Grace
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/39503
• Institution: Institut Teknologi Bandung

HPV (Human Papillomavirus) is member of DNA virus Papillomaviridae which infects basal cell of human epithelial tissue. According to the previous data, HPV16, 18 and 52 have the highest prevalence for causing cervical cancer in Indonesia. The prevention of cervical cancer can be done through administration of HPV vaccine in VLP (Virus-Like Particle) form. VLP is a particle consisted of proteins without viral DNA that is capable of initiating adaptive immune response. However, up until this time, there is no local vaccine developed to prevent the spread of HPV infection in Indonesia. Hence, this research is aimed to express L1 protein of HPV subtype 52 of Indonesian isolate with pPICZ A in Pichia pastoris for HPV vaccine candidate. DNA isolate of HPV52 L1 in pPICZ A comes from the previous research. The plasmid is then confirmed with PCR (Polymerase Chain Reaction) and DNA sequencing. The confirmed plasmid is transformed into Escherichia coli for cloning purpose. The construct of pPICZ A-HPV52 L1 is then transformed into Pichia pastoris GS115 electrocompetent cells by means of electroporation. Selection of the transformants is done by growing the randomly picked colonies in a master plate with medium contained Zeocin in the following concentration: 100 ?g/ml, 200 ?g/ml, 500 ?g/ml dan 1000 ?g/ml. The transformants that are able to grow in medium with Zeocin 1000 ?g/ml are then confirmed with PCR. Induction of transformant culture is done with the addition of methanol until 0.5% concentration every 24 hour. The isolation of protein is done on samples from the following time points. The analysis of protein profile is done with SDS-PAGE in 12.5% polyacrylamide gel. The result shows the confirmed fragment (1.7 kb) in pPICZ A plasmid. Sequencing analysis shows that the L1 fragment has the appropriate ORF with in-silico design. The result of protein expression shows a protein band around 58.8 kDa size on 48 hour time point which expected to be the L1 protein. Hence, it is concluded that L1 protein of HPV subtype 52 of Indonesian isolate is presumed to be successfully expressed in Pichia pastoris.