Enhancement of humoral immune responses to HBsAg by heat shock protein gp96 and its N-terminal fragment in mice

Figure 1 Gp96 expressed in E. coli and purification from normal murine livers. A and B: Elutions of gp96 from POROS 20QE column with 300-800 mmol/L NaCl were detected by SDS-PAGE. The gp96 expressed in E.coli. A: or purified from mice livers; B: were collected as 1 mL fractions during the purification on POROS 20QE column and a total of six individual fractions (lanes 1-6) were run on the SDS-PAGE gel with Coomassie blue staining afterwards; C: Gel-filtration analysis of gp96 purified from mice livers. The protein samples are run on Superdex G200 column; D: Proteins collected from the peak of gel filtration were run on 10% SDS-PAGE and Western blot are shown on the right.

Figure 2 Expression and purification of the N-terminal fragment of gp96 in the GST fusion expression system. A: The resulting protein N-terminal fragment of gp96 (named after NTF) was separated from the digestion product on a glutathione±Sepharose 4B column to remove GST and GST-3C protease. Lane 1, 10% SDS±PAGE of GST-NTF eluted by reduced glutathione; lane 2, NTF after GST-3C protease digestion (16 h, 5 °C); lane 3, NTF after removing GST and GST-3C protease; lane 4, purified GST as control; M, protein molecular weight markers as indicated in kilo Daltons; B: Gel-filtration analysis of NTF protein. NTF protein subjected to Superdex G75 gel-filtration; C: The proteins from the peak run on 10% SDS-PAGE and Western blot. The standard proteins are superimposed in both A and C.

Figure 3 Induced humoral immune response in Balb/c mice. A: Groups (eight mice per group) of Balb/c mice were immunized with different gradient: group 1, PBS only; group 2, 10 μg gp96 only; group 3, 10 μg NTF only; group 4, 10 μg HBsAg only; group 5, 10 μg HBsAg+10 μg NTF; group 6, 10 μg HBsAg+10 μg gp96; group 7, 10 μg HBsAg+IFA; group 8, 10 μg HBsAg+10 μg NTF (95 °C heated for 30 min) at d 0 (day of first vaccination). At d 21, and 42 mice were re-immunized with the same material. At d 63 anti-HBsAg antibodies from different groups were compared using the value at 450 nm absorbance. The values of antibody were expressed as geometry mean±SD; B: Sera from the mice immunized with HBsAg plus heated NTF, HBsAg plus NTF or gp96 (groups 5, 6 and 8) were collected on d 14, 35 and 64 from first vaccination and stored at -20 °C and titers at different dilution were tested at the same time.

Figure 4 T-cell response of splenocytes of immunized Balb/c mice. Mice immunized with PBS, HBsAg plus heated NTF, HBsAg plus NTF or gp96 (the same groups as groups 5, 6, and 8 respectively in Figure 3) were killed on d 63 and the splenocytes were collected for ELISPOT assay with 10⁵ cells per well. The number of SFCs was calculated. The photograph of the representative wells from different groups is shown below the statistics.