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 ß-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.

[Study of the Accumulation of Rec A from Bacillus subtilis in the Mitochondria of a Recombinant Strain of the Yeast Yarrowia lipolytica].

No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic systembased on the pQ-SRUS construct that allows the expression of the RecA recombinase from Bacillus subtilis and its transportation to mitochondria of Yarrowia lipolytica. The targeting of recombinant RecA to mitochondria is provided by leader sequences (5'-UTR and 3-UTR) derived from the SOD2 gene mRNA, which exhibit affinity to the outer mitochondrial membrane and provides cotranslational import of RecA to the inner space of mitochondria. The accumulation of RecA in mitochondria of the Y. lipolytica recombinant strain bearing the pQ-SRUS construct has been shown by immunoblotting of purified mitochondrial preparations.

[Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

[The regulation of peroxisomal matrix enzymes (alcohol oxidase and catalase) formation by the product of the gene Mth1 in methylotrophic yeast Pichia methanolica].

Two independent mutant strains of methylotrophic yeast Pichia methanolica (mth1 arg1 and mth2 arg4) from the initial line 616 (ade1 ade5) were investigated. The mutant strains possessed defects in genes MTH1 and MTH2 which resulted in the inability to assimilate methanol as a sole carbon source and the increased activity of alcohol oxidase (AO). The function of the AUG2 gene encoding one of the subunits of AO and CTA1, a probable homolog of peroxisomal catalase of Saccharomyces cereviseae, was investigated by analyses of the molecular forms of isoenzymes. It was shown that optimal conditions for the expression of the AUG2 gene on a medium supplemented with 3% of methanol leads to an increasing synthesis of peroxisomal catalase. The mutant mth1 possessed a dominant formation of AO isoform with electrophoretic mobility which is typical for isogenic form 9, the product of the AUG2 gene, and a decreased level of peroxisomal catalase. The restoration of growth of four spontaneous revertants of the mutant mth1 (Rmth1) on the methanol containing medium was accompanied by an increase in activity of AO isogenic form 9 and peroxisomal catalase. The obtained results confirmed the functional continuity of the structural gene AUG2 in mutant mth1. The correlation of activity of peroxisomal catalase and AO isogenic form 1 in different conditions evidenced the existence of common regulatory elements for genes AUG2 and CTA1 in methilotrophic yeast Pichia methanolica.

[A comparative study of the components of the antioxidant defense system during growth of the mycelium of a wild-type Neurospora crassa strain and mutants, white collar-1 and white collar-2].

A comparative study of the changes in the components of the antioxidant defense system (ADS), the activity of superoxide dismutase (SOD) and catalase and the level of extractable SH-groups, during the growth of wild-type and mutant (white collar-1 and white colar-2) Neurospora crassa strains was performed. Oxidative stress developing during spore germination and upon the transition to a stationary growth phase was accompanied in all strains by an increase in the level of extractable SH-groups and SOD activity, whereas the total catalase activity decreased during growth. However, in contrast to the wild-type strain, the activity of the catalase in the mutant strains wc-1 and wc-2 slightly increased upon the transition to the stationary phase. In the wc-2 mutant, SOD activity and the level of extractable SH-groups in the exponential growth phase were always lower than in the wild-type and wc-2 strains. The role of wc-1 and wc-2 genes in the level regulation of reactive oxygen species is discussed.