Expression of Hantaan virus A9 M genome segment by using a vaccinia virus recombinant

The plasmid pJSBA9M, containing the M clone segment, was constructed and controlled by the promoter P11, and also was proved by restriction enzyme digestion. This expression plasmid pJSBA9M was transfected using lipofectin reagent into Vero cells, which were superinfected with the vaccinia virus containing the Lac Z gene in the TK region. The reccombinant vaccinia viruses were isolated by plaque assay of transfected-cell lysate on Vero cells with 1% low-melting-point agarose overlay containing 300mug of X-gal per ml. White plaques were removed and subjected to six round plaque purification. From 16 white plaques, only one was identified as the recombinant vaccinia virus by PCR. In addition, immunofluorescent assay (IFA) and Radio-immunoprecipitation (RIP) were used to identify the products of the recombinant vaccinia virus. Furthermore, the neutralizing antibody against hantaan virus with the titer of 1:320 in sera of mice inoculated with the recombinant vaccinia virus was detected.