An efficient method of constructing recombinant adenoviruses (Ads)

SUMMARY:Recently, the adenovirus expression vector attracts much attention for the application to gene therapy and the method to purify and concentrate adenovirus without loss of infectivity has become very important, especially for animal experiments and gene therapy of humans.In this report, we show a simple and efficient method for purifying infectious adenovirus. The method consists of sequential centrifugation in CsCl step gradients without loss of infectivity and can be completed in one day. The method maintained the viral infectivity after 10-fold concentration and seemed to remove more than 99.9% of carried-over proteins. We showed also that the buffers for dialyzing the purified virions influenced the stability of infectivity.The buffers of 10 mM HEPES-1 mM EDTA -10% glycerol and PBS (-)-10% glycerol resulted in higher stability than did 10 mM HEPES-1 mM MgC12-10% glycerol. The method is may be useful in many applications of recombinant adenovirus.

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Hepatitis C virus (HCV) is a main causative agent for transfusion-associated and sporadic cases of non-A, non-B hepatitis throughout the world. HCV has a positive-strand RNA of about 9,400 nucleotides as its genome, whose organization is similar to those of animal pestiviruses or human fiaviviruses. In spite of the lack of an effective replication system in tissue culture cells, genes coding for viral proteins of HCV have been identified. The putative nucleocapsid (p22) and envelope (gp35 and gp60) proteins have been expressed in cells by different vectors under various foreign promoters. Furthermore, a truncated core protein and association of envelope proteins with nonstructural proteins have also been observed. These synthesized viral proteins have been shown to be useful for diagnostic assays.