Mycoplasma contamination of continuous cell cultures and collection viral strains remains a serious problem in the biotechnology industry and experimental research. The frequency of mycoplasma contamination of cultured cell lines and viruses is 15–35

Hantaviruses are highly pathogenic causative agents of hantaviral fevers, including hemorrhagic fever with renal syndrome, registered among people in countries of the Eurasian continent and a disease called «hantavirus pulmonary syndrome» – in the countries of North and South America. More recently, the spread of hantaviral diseases has been detected in Africa. There are still no drugs for specific antiviral therapy. The most promising method of dealing with hantaviral fevers is specific prophylaxis, that is, vaccination of the population against hantaviruses, which determine the endemicity of different territories. This review summarizes current data on existing and developed vaccines against hantaviral fevers.

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.

No eukaryotic species has a system for homologous DNA recombination of the mitochondrial genome. We report on an integrative genetic system based 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.

Существующие методы исследования гликозилирования белков остаются громоздкими. В своем развитии они отстают от высокопроизводительных протеомных технологий. Существует реальная потребность в разработке простого, недорогого и специфичного метода выявления сайтов гликозилирования в белках, совместимого с общепринятыми процедурами подготовки биологического материала к протеомному и фосфопротеомному анализу. В статье описан новый метод дегликозилирования гликопротеинов с помощью бета-элиминирования с последующим введением ковалентной метки и визуализацией паттерна первично негликозилированных и дегликозилированных белков в предобработанном клеточном гомогенате с использованием J-агрегатов, образующихся в геле in situ. Новый метод подготовки материала к протеомному и фосфопротеомному анализу может быть использован при проведении фосфопротеомного анализа в формате RPMA для регистрации изменения фосфорилирования белкового состава клеток, в том числе в условиях развития вирусной инфекции.

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 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.

This review is dedicated to structural and functional properties of IgA1-proteases in pathogenic microorganisms. The interest to structural and functional studies of IgA-proteases is caused essentially by their putatively great impact on set up and progression of infectious diseases. IgA degradation at mucosal surfaces is commonly suggested to be the main function of IgA1-proteases. In turn, this effect may lead to abatement of the immune response efficiency at the epithelial barrier. In our review, structural comparison of “classic” IgA1-proteases with their functional analogues and structural homologues allows to hypothesize IgA-protease dependent effector mechanisms mediated by serum IgA.

In this study, we used two mutants of the methylotrophic Pichia methanolica which have lost the ability to assimilate methanol and with low level of alcohol oxidase activity (AOD) and their revertants to investigate the function of two genes: AUG2, which encodes one of AOD subunits and CTAI, a putative homolog of Saccharomyces cereviseae peroxisomal catalase (PCAT). Growing the mutants in conditions favorable for AUG2 expression (3% methanol-containing growth medium) resulted in increased expression of PCAT, whereas either carbon source free or 1% glycerol-containing growth media resulted in decreased expression of PCAT. The AOD isozyme pattern of the mthl mutant gave a dominant isozyme band with electrophorectic mobility typical to isogenic form 9, the product of gene AUG2. Four spontaneous revertants of the mthl mutant (Rmthl) were able to use methanol as the sole carbon source, and had increased activity of both AOD isozyme species 9 (the product of AUG2), and PCAT. These results confirmed the existence of regulatory elements in common that are likely to affect an expression of both AUG2 and CTAI genes in the yeast P. 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 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-1 strains. The role of wc-1 and wc-2 genes in the level regulation of reactive oxygen species is discussed.