Protein Expression of Two Cassettes L-HBsAg gene in Methylotrophic Yeast Pichia pastoris

• Main Author: STEPHANIE (NIM : 21115019), NADYA
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29503
• Institution: Institut Teknologi Bandung

<p align="justify"> Indonesia has a high prevalence of hepatitis B infection. Hence since 1997, the administration of hepatitis B vaccine containing surface protein S (product of the S gene) HBV (S-HBsAg) in infants was mandatory. Unfortunately, about 5% of children and young adults who were vaccinated did not give the positive response. Therefore, hepatitis B vaccine containing HBV surface protein L (product of preS1 + preS2 + S gene/ L-HBsAg) is developed to overcome this problem. This research objective is to obtain recombinant P.pastoris that integrated with 2 copies of L-HBsAg protein genes in its genome. P.pastoris transformation was performed using electroporation. The recombinant colony was selected using polymerase chain reaction (PCR) method to determine transformants that produce a PCR fragment of 462 bp using specific primers. The addition of methanol (1%, 1.5%, 2%, 3%) for 144 hours was conduct to induce the protein expression. The presence of L-HBsAg protein was confirmed using Western Blot analysis. Total protein content for 1%, 1.5% , 2%, and 3% methanol induction were 89.45 mg / mL, 87.7 mg / mL, 94.9 mg / mL and 106.68 mg /L, respectively. Western blot analysis showed the presence of HBsAg band (about 45 kDa), which is similar to a commercial L-HBsAg protein. Based on the data, we obtained the P.pastoris recombinant that integrated with 2 copies of L-HBsAg gene in its genome and could express the L-HBsAg protein. Therefore this clon can be used for the development of the latest HBV vaccine. <p align="justify">