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.