Prokaryotic expression and antibody preparation of truncated glycoprotein of Crimean-Congo hemorrhagic fever virus

Abstract

Objective To express and purify truncated glycoprotein(Gn, Gc1, Gc2) of Crimean-Congo hemorrhagic fever virus(CCHFV) strain 79121 from E. coli cells, and to prepare their polyclonal antibodies respectively. Methods The genes encoding Gn, Gc1 and Gc2 from CCHFV strain 79121 were amplified by RT-PCR, and cloned into prokaryotic expression vector pET-28a (+ ) to construct recombinant plasmids. The three recombinant plasmids were then transformed into E. coli BL21(DE3) to obtain the Gn-His, Gc1-His and Gc2-His fusion proteins through IPTG induction. The fusion proteins were purified by Ni-NTA purification system, and then analyzed by using SDS-PAGE. To prepare antisera, the purified Gn-His, Gc1-His and Gc2-His proteins were employed to immunize New Zealand White Rabbit (NZW), respectively. The titer and specificity of the antisera to each truncated glycoprotein were analyzed by using ELISA and Western blot, respectively. Results The recombinant expression vectors of pET-28a-Gn, pET-28a-Gc1 and pET-28a-Gc2 were constructed successfully by restriction enzyme analysis and DNA sequencing. The relative molecular mass(Mr) of Gn-His, Gc1-His and Gc2-His were approximately 25 000, 33 000 and 34 000, and the titers of the corresponding polyclonal antibodies were above 1∶25 600, 1∶6400 and 1∶25 600, respectively. The antisera were able to bind to the corresponding fusion protein specifically. Conclusion The recombinant expression of truncated glycoprotein (Gn, Gc1, Gc2) of CCHFV strain 79121 and the preparation of their high titer polyclonal antibodies laid foundation for further studying the biological function of glycoprotein and the rapid detection of CCHFV. Key words: Crimean-Congo hemorrhagic fever virus; Glycoprotein; Prokaryotic expression; Polyclonal antibody