Preparation of polyclonal antibody to nucleoprotein from Xinjiang hemorrhagic fever virus and its immunological evaluation

Objective: To express and purify the nucleoprotein (NP) from Xinjiang hemorrhagic fever virus(XHFV) strain BA88166 in E.coli, and prepare and identify its polyclonal antibody.

Methods: The cDNA of S gene segment of BA88166 strain was amplified by RT-PCR and cloned into prokaryotic expression vector pET-32a to generate a recombinant plasmid named pET-88166S. The pET-88166S was transformed into E.coli BL21 (DE3). The NP-His fusion protein was induced by IPTG, purified by Ni-NTA purification system, and analyzed by SDS-PAGE. To prepare the antiserum, New Zealand white rabbits were immunized with the purified NP-His protein. The titer and specificity of the antiserum to NP were analyzed by ELISA and Western blotting, respectively.

Results: Restriction endonuclease analysis and DNA sequencing showed that the prokaryotic xpression vector of pET-88166S was constructed successfully. NP-His fusion protein was expressed in E.coli BL21 (DE3) after IPTG induction and its relative molecular mass (Mr;) was about 66 000. ELISA and Western blotting showed that the titers of the antisera were above 1:25 600, and that the antisera can specifically bind with the entire and truncated NP protein of XHFV strain YL04057.

Conclusions: NP-His fusion protein can be successfully expressed in E.coli and the specific anti-NP rabbit polyclonal antibody has been obtained, which will provide the basic information for the studies on the diagnosis, treatment and prevention of Xinjiang hemorrhagic fever.