Expression And Immunogenicity Of Hepatitis B Virus Middle Surface Protein

The hepatitis B virus (HBV) envelope (env) protein is composed of three regions: the 108- or 119-residue preS1 region involved in the direct interaction with hepatocytes, the 55-residue preS2 region associated with the polymerized albumin-mediated interaction, and the major 226-residue S protein region. Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preSl and preS2 sequences could be an ideal candidate for a new HBV vaccine with higher efficacy. The methylotrophic yeast Pichia pastoris has been developed as production systems for recombinant proteins recently. The favourable and most advantageous characteristics of the species have resulted in an increasing number of biotechnological applications.To determine the activity of HBV env protein M, polypeptide preS2 was expressed in prokaryotic system. A gene coding for preS2 was artificially synthesized. The gene was subcloned into Escherichia coli expression vector pThioHisA and expressed at high level as a fusion protein of thioredoxin. The thioredoxin-preS2 fusion protein was initial purified by the treatment of osmotic pressure, displayed preS2 antigenicity.To express HBV env protein M at a high level in P. pastoris, the same segments of pPIC3.5k and pAO815 digested with restriction enzyme SacI and SalI were exchanged to obtain the new vector pAO818. Genes of HBV env protein S was placed in this vector and expressed at sable and high level. After success in reconstruction of this high level expression vector, gene of HBV env protein M was placed under the control of AOX1 promoter in this integrative Pichia expression vector carrying eight copies of the expression cassette. Through electroporation transformation of SMD1168 and G418 selection, a strain of Pichia pastoris capable of secreting protein M into the...