A new method of producing NS5A antigen of hepatitis C virus.

A task of creating a universal platform for engineering affordable recombinant producers of viral proteins conserving immunogenicity has not been solved yet. High toxicity of the viral proteins for the host cells, low yield and abnormal folding of the products often present severe obstacles to obtaining producers of the viral proteins. In this work, we report a new method of engineering and screening of deletion libraries from the viral antigen genes. This method allows selection of artificial derivatives of these genes adapted for expression in microbial producer cells. The method involves PCR amplification of the gene fragments using a system of randomized and adapter primers, which allows the spontaneous formation of duplexes from the random primers in the absence of the template DNA to be prevented. For selecting variants capable of in vivo expression, the obtained PCR products are cloned to a special vector of a direct phenotypical selection pQL30. It contains E. coli β-galactosidase gene with an inserted polylinker producing a frame-shift mutation. Using this screening method, an artificial variant of hepatitis C (HCV) NS5a gene with optimal biotechnological properties was established. 27 clinical specimens of 1670 bp long HCV1b NS5a fragments were used as a source gene. A PCR bank of the deletion derivatives was produced. 40 LacZ-positive clones based on pQL30 vector with a 50-700 bp long insertion were selected. The LacZ activity of the cell lysates and the immunogenicity of the products were tested. As a result, a single clone encoding a soluble protein with Mr = 114 kDa was selected. Its yield reached 0.3% of the total cell protein. It was highly reactive with sera of HCV 1b infected patients but not with sera of the healthy donors.