Cloning and expression of the nucleoprotein gene of Puumala-like virus / 中华流行病学杂志

ABSTRACT

Objective 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.Methods The NP gene was cloned into plasmid PCR[R]2.1TA vector and then Was ligated into baculovims donor plasmid pFastBacTM1 after cutting by the restriction enzylne Kpn I and Not I.pFastBacTM1 was subsequently transferred into the One ShortTMTOP10 competent cells and then into DH10BacTM E.coli competent cells.which contained the baculovirus shuttle vector(Bacmid)and the helper plasmid to generate a recombinant bacmid.Results The NP gene was successfully expressed jn St9 insect cell.The expressed recombinant nucleoprotein had been identified in the S19 insect cell by indirect immunofluorescence assay,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Westem blot.The results showed that the recombinant nucleoprotein appeared a molecular weight of 50×103M.and could reacmd with anti-recombinant Puumala virus(PUUV)nucleocapsid monoclonal antibodies and polyclonal antibodies against hantavirus.Conclusion Our results indicated that the recombinant nucleoprotein was SUCCESSfully expressed and having the immunogenicity and reactivity of natural nucleoprotein of HOKV.