This invention relates to a method for producing virus RNA polymerases of RNA
viruses, more specifically, virus RNA polymerases of RNA viruses free of virus
genomic RNA.
The methods described in this invention includes the procedures for preparation
of cDNAs for the genes for the component proteins of RNA polymerase of an RNA virus,
incorporation of the cDNA into baculovirus genome to construct recombinant virus,
and the infection of insect cells with the recombinant virus to express RNA polymerase.
In this method, it is recommended that all species of the recombinant viruses,
each of which is designed for expressing each of the above-mentioned component
protein genes of RNA polymerase, are coinfected into insect cells. Thus, cDNA is
prepared for each of the component proteins of RNA polymerase and incorporated
into baculovirus genome to construct recombinant virus for independently expressing
the corresponding protein. In addition, the RNA viruses described above include
influenza virus especially. In this method, RNA polymerase may be tagged to facilitate
the subsequent purification. Furthermore, the tagged RNA polymerase described above
may be purified using an adsorbent to trap the RNA polymerase at the tag. Another
subject matter of this invention is to artificially prepare a complex of the component
proteins of RNA polymerase of an RNA virus, or, in other words, to prepare RNA
polymerase free from RNA virus genomic RNA. The RNA viruses described here include
influenza virus, and the component proteins of RNA polymerase include PA, PB1 and PB2.