Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris.
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 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg 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, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.
Han
Xue
X
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, PR China.
Ye
Lin-Bai
LB
Li
Bao-Zong
BZ
Bo
Gao
G
Cai
Wei-Jia
WJ
Hong
Zheng
Z
She
Ying-Long
YL
Li
Ye
Y
Kong
Ling-Bao
LB
Wu
Zheng-Hui
ZH
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000917:Antibody Formation / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D006509:Hepatitis B / Q000235:genetics / Q000276:immunology / Q000517:prevention & control; D006511:Hepatitis B Antigens / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D017325:Hepatitis B Vaccines / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007114:Immunization; D010843:Pichia / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D013997:Time Factors; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology
D000914:Antibodies, Viral; D006511:Hepatitis B Antigens; D017325:Hepatitis B Vaccines; C096362:L protein, hepatitis B virus; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins