Secreted expression of G145R mutant HBsAg preS2+S gene in {\sl Pichia pastoris} expression system and the study of its antigenicity and immunogenicity
In order to study the antigenicity and immunogenicity of G145R mutant HBsAg, G145R mutant hepatitis B surface antigen (HBsAg) preS2+S gene was secretively expressed in Pichia pastoris system. The expressed middle protein of G145R mutant HBsAg was used to immunize mice. The antigenicity and immunogenicity (preS2+S) of G145R mutant HBsAg were studied with ELISA and Western blot tests. The highest-level expression clone MC23 was selected from 150 positive clones. Specific HBsAg middle protein band could be detected with Western blot test. The molecular weight of the monomer was about 34kD and 37kD. The expression level was about 200g/L culture medium when estimated with the AUZYME MONOCLONAL KIT. The antigenicity was tested with 7 commercial HBsAg detection kits and mice polyclonal antisera immunized with HBsAg standard. The results indicated that the detection level of the G145R mutant HBsAg could only be about 50% or less of the wild-type HBsAg in most of the HBsAg detection kits when they were in the same concentration. Only with the AUZYME MONOCLONAL KIT, we could equally detect the G145R mutant HBsAg and that of wild-type. After immunized with G145R mutant HBsAg protein, specific anti-HBs antibody could be detected in the mouse sera, the average titer was about 1:1 600. This anti-sera were also reactive with synthetic peptide P2-145R, which is specific to the "a" determinant of G145R mutant HBsAg. But the anti-HBs did not react with wild-type HBsAg, or with synthetic peptide P1-wt which is specific to "a" determinant of wild-type HBsAg. Further studies with Western blot supported the result too. All the results above suggested that the preS2+S gene of G145R mutant HBsAg could be expressed in the Pichia pastoris system successfully. The G145R mutant HBsAg still had a good immunogenicity. But the antigenicity and immunogenicity when compared with wild-type HBsAg had changed significantly after one amino acid mutation from Glycine to Arginine at amino acid site 145.