Figure legends
Fig. 1. Preparation scheme of ORF8-CRM and ORF8-PEG.
Fig. 2. Purification and characterization of ORF8. The cell
cultures were analyzed by SDS-PAGE (A). Lane 1, marker; Lane 2, the cell
culture without IPTG induction; Lane 3, the cell culture with IPTG
induction; Lane 4, the inclusion bodies; Lane 5, the supernatant. ORF8
was purified from the cell culture by a Ni Sepharose HP column (B). The
two conjugates were analyzed by an analytical Superdex 200 column (C)
and SDS-PAGE (D). Lane 1, marker; Lane 2, ORF8; Lane 3,
CRM197; Lane 4, ORF8-CRM; Lane 5, ORF8-PEG.
Fig. 3. Antibody response after immunization of the samples.
The measurements of ORF8-specific IgG (A), IgG1 (B), IgG2a (C) and IgM
(D) were carried out using ELISA. The lymphocyte proliferation of mice
spleen suspensions was restimulated by ORF8 (E). Blood samples after
immunization on days 7, 14 and 21 were obtained for antibody
measurement. Antibody avidity of ORF8-specific IgG (F) was analyzed
using the blood samples on day 21. Values represent mean ± S.D. from six
mice per group.
Fig. 4. Determination of the cytokine secretions in the
immunized BALB/c mice. IFN-γ (A), IFN-β (B), TNF-α (C) and IL-5 (D) in
the culture supernatant were analyzed using the ELISA kits. Values
represented the mean value± S.D. from 6 mice per group.
Table 1. Toxicity study on the cardiac, hepatic and renal
functions of the mice
Sample LDH a CK b ALTc ALB d TP eUAf
(U/L) (U/L) (U/L) (g/L) (g/L) (μmol/L)
PBS 214.6 205.4 13.6 15.7 26.3
95.4
ORF8 215.2 201.1 16.5 21.6 25.4 103.1
ORF8-CRM 187.4 128.6 12.2 14.1 22.4 93.4
ORF8-PEG 174.7 197.6 14.4 16.1 28.0 90.3
ORF8/AL 157.8 110.7 12.6 13.6 21.4 82.1
a Lactate dehydrogenase. b Creatine
kinase. c Alanine aminotransferase.d Albumin. e Total protein.f Uric acid.