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


RESULTS
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.


CONCLUSIONS
Our results indicated that the recombinant nucleoprotein was successfully expressed and having the immunogenicity and reactivity of natural nucleoprotein of HOKV.
Yao
Wen-rong
WR
National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Zou
Yang
Y
Li
Ming-hui
MH
Zhang
Yong-zhen
YZ
D000818:Animals; D016367:Baculoviridae / Q000145:classification / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D007313:Insecta / Q000166:cytology; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000276:immunology*; D010957:Plasmids; D029262:Puumala virus / Q000235:genetics* / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D019590:Nucleocapsid Proteins; C100886:nucleocapsid protein, Puumala virus