Pilot-scale purification of rF1-V fusion protein of Yersinia pestis and characterization of its immunogenicity.
- Author:
Ting FANG
;
Jun REN
;
Jinlong ZHANG
;
Kexin YIN
;
Xiuxu YANG
;
Rui YU
;
Xiaopeng ZHANG
;
Changming YU
- Publication Type:Journal Article
- MeSH:
Adjuvants, Immunologic;
Animals;
Antibodies, Bacterial;
blood;
Antibody Formation;
Antigens, Bacterial;
immunology;
Blotting, Western;
Chromatography, Ion Exchange;
Enzyme-Linked Immunosorbent Assay;
Mice;
Plague;
prevention & control;
Plague Vaccine;
immunology;
Recombinant Fusion Proteins;
immunology;
Vaccines, Subunit;
immunology;
Yersinia pestis
- From:
Chinese Journal of Biotechnology
2016;32(1):95-104
- CountryChina
- Language:Chinese
-
Abstract:
Recombinant Fl-V (rFl-V) fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of rF1-V in Escherichia coli BL21 (DE3) that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters: the culture was induced at the middle exponential phase with 50 µmol/L of IPTG at 25 °C for 5 h. Soluble rFl-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 µg rFl, rV and rFl-V and 10 µg rFl+10 µg rV, were assayed by ELISA after 2 doses. The antibody titers of anti-Fl with 20 µg rFl-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that rFl-V would be the active pharmaceutical ingredient of the plague subunit vaccine.
