[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.

CONCLUSION

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.