[Cloning and expression of the nucleoprotein gene of Puumala-like virus]

In order to detect Hokkaido virus (HOKV), a recombinant baculovirus containing the nucleoprotein (NP) gene of HOKV was constructed, and then the NP was expressed in insect cell.

The NP gene was cloned into plasmid PCR 2.1TA vector and then was ligated into baculovirus donor plasmid pFastBac after cutting by the restriction enzyme Kpn I and Not I. pFastBac 1 was subsequently transferred into the One Short TOP10 competent cells and then into DH1OBac E. coli competent cells, which contained the baculovirus shuttle vector (Bacmid) and the helper plasmid to generate a recombinant bacmid.

The NP gene was successfully expressed in Sf9 insect cell. The expressed recombinant nucleoprotein had been identified in the Sf9 insect cell by indirect immunofluorescence assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The results showed that the recombinant nucleoprotein appeared a molecular weight of 50 x 10(3) Mr, and could reacted with anti-recombinant Puumala virus (PUUV) nucleocapsid monoclonal antibodies and polyclonal antibodies against hantavirus.

Our results indicated that the recombinant nucleoprotein was successfully expressed and having the immunogenicity and reactivity of natural nucleoprotein of HOKV.