EKSPRESI EKSTRASELULER PROTEIN PERMUKAAN VIRUS HEPATITIS B (sHBsAg) PADA Pichia pastoris

• Main Author: Nurfitriani
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/32803
• Institution: Institut Teknologi Bandung

Recently, hepatitis B virus infects 2 million people worldwide. 350 thousand of them are in the chronic condition with the death rate about 600.000 people per year. Therefore, there is a need to discover a method that can inhibit its proliferation. During the infection step, hepatitis B virus is secreting a surface protein, namely sHBsAg. In the blood, sHBsAg is able to form virus like particle (VLP) which induce the formation of antibody (anti-sHBsAg) that can be used as a vaccine to fight the disease and primary reagent for diagnostic kit. In the large scale, sHBsAg can be produced by recombinant technology using Pichia pastoris expression system. The recombinant sHBsAg is expressed in intracellular and purified in several purification steps. This purification is the limiting step of the sHBsAg production because in the final step only small amount of the protein can be recovered (approximately 85 ?g/g of the dry cells). Therefore, extracellular expression of sHBsAg in Pichia pastoris is one of the choices to overcome this problem. The aim of the research was to do extracellular surface protein expression of hepatitis B virus in P. pastoris. In the previous research, the recombinant pPICZ?-A-sHBsAg was successfully generated. In present research, sHBsAg gene was successfully integrated into the chromosom of P. pastoris strain GS115 and KM71 by homolog recombination single cross over event. P. pastoris transformant was screened to determine the phenotype and the transformants which had highest zeocin resistance (2mg/mL). Phenotypic test was done by growing the transformant in MMH (minimal methanol + Histidin) medium and MDH (minimal Dextrose + Histidine) medium. Zeocin resistance test was done in rich medium YPDS with the zeocin concentration range from 0.1-2 mg/mL. The transformant with Muts phenotype and the highest gene copy number was chosen for HBsAg production. The SDS-PAGE analysis showed that HBsAg protein was successfully expressed with the molecular weight about 29 kDa and ELISA analysis revealed that sHBsAg was secreted as VLPs form at 8 days after induction.