Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris

• Published in: Protein expression and purification 2006-10, Vol.49 (2), p.168-175
• Main Authors: Han, Xue, Ye, Lin-Bai, Li, Bao-zong, Bo, Gao, Cai, Wei-jia, Hong, Zheng, She, Ying-Long, Li, Ye, Kong, Ling-Bao, Wu, Zheng-Hui
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Viral - immunology; Antibody Formation - immunology; Dose-Response Relationship, Immunologic; Fermentation; Hepatitis B - genetics; Hepatitis B - immunology; Hepatitis B - prevention & control; Hepatitis B Antigens - biosynthesis; Hepatitis B Antigens - genetics; Hepatitis B Antigens - immunology; Hepatitis B Antigens - isolation & purification; Hepatitis B Antigens - pharmacology; Hepatitis B Vaccines - biosynthesis; Hepatitis B Vaccines - genetics; Hepatitis B Vaccines - immunology; Hepatitis B Vaccines - isolation & purification; Hepatitis B Vaccines - pharmacology; Hepatitis B virus; Hepatitis B virus (HBV); Hepatitis B virus - genetics; Hepatitis B virus - immunology; Humans; Immunization; Large protein; Pichia - genetics; Pichia pastoris; Purification; Rabbits; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - immunology; Recombinant Proteins - isolation & purification; Recombinant Proteins - pharmacology; Time Factors; Vaccine; Viral Envelope Proteins - biosynthesis; Viral Envelope Proteins - genetics; Viral Envelope Proteins - immunology; Viral Envelope Proteins - isolation & purification; Viral Envelope Proteins - pharmacology
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2006.05.002

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10 μg, was detected on day 14, whereas rabbits vaccinated with 10 and 2 μg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2 μg L protein induced a significant and rapid anti-HBsAg antibody response than 10 μg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.