Prokaryotic expression of a multi-epitope gene of Xinjiang hemorrhagic fever virus and development of an indirect ELISA for virus detection

Abstract

Objective To express and purify the multi-epitope peptide of Xinjiang hemorrhagic fever virus (XHFV) in a prokaryotic expression system and to further use it as diagnostic antigen to establish an indirect ELISA for the detection of XHFV in animal sera. Methods A double copy multi-epitope gene (MEPX2) segment with repeated tandem including six highly conservative and dominant B cell epitopes was designed based on the analysis of hydrophilicity and antigenic determinant sites in amino acid sequences of nucleoprotein and glycoprotein from XHFV strain YL04057. The multi-epitope gene MEPX2 was synthesized by chemical method and then inserted into the prokaryotic expression vector pET-32a (+ ) to construct the prokaryotic expression plasmid pET-32a (+ )-MEPX2. The recombinant expression plasmid was transformed into E.coli BL21 (DE3) to induce the expression of rMEPX2 by IPTG. The expressed products were purified by Ni-NTA purification system. The antigenicity of rMEPX2 was identified by Western blot assay. The purified rMEPX2 was used as coating antigen to establish an indirect ELISA by checkerboard for the detection of XHFV in animal serum samples. The results of virus detection were compared with those by using commercial ELISA kit. Results The recombinant expression plasmid pET-32a (+ )-MEPX2 was constructed successfully as confirmed by restriction enzyme digestion and DNA sequencing analysis. Results of the SDS-PAGE and Western blot assay confirmed that the rMEPX2 was expressed correctly with a relative molecular mass (Mr) of about 49×103. The rMEPX2 was recognized specifically by His-tag mouse monoclonal antibody and by positive serum samples of sheep naturally infected with XHFV. The indirect ELISA for the detection of XHFV IgG antibody was established by using the purified rMEPX2 as antigen. Compared with the commercial ELISA kit, the established indirect ELISA showed good specificity for the detection of XHFV in 108 sheep serum samples. Conclusion The recombinant multi-epitope protein rMEPX2 with good antigenicity was obtained successfully. The established indirect ELISA based on the rMEPX2 could be used for the detection of specific antibodies against XHFV. Key words: Xinjiang hemorrhagic fever virus; Multi-epitope peptide; Prokaryotic expression; Indirect ELISA