[DNA immunization with a plasmid containing gene of hepatitis C virus protein 5A (NS5A) induces the effective cellular immune response].

In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti-NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against abroad spectrum of NSSA epitopes as assessed by T-cell proliferation andsecretion of antiviral cytokines IFN-gamma and IL-2. In in vitro T-cell stimulation experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.

[Structural and functional specificity of the Dichelobacter nodosus pili. Synthesis of the protein pilin of D. nodosus in various bacterial recombinant strains].

The ovine foot rot is a severe infectious disease of sheep. Dichelobacter nodosus is an essential pathogen of this disease. An obligatory anaerobic gram-negative rod-shaped microorganism has slow rate of accumulating bacterial density and fastidious growth requirements. This causes obstacles to vaccine production and makes it difficult to diagnose the disease. The diagnosis in this case is more expensive. Fimbriae (or pili) are one of the major factors of virulence of D. nodosus. Their antigenic and immunogenic properties make them good vaccine components for elevated immunogenicity. Since the nucleotide sequence of the fimA gene encoding fimbrial subunit was determined, attempts to produce recombinant pili were undertaken. The production of the genetic-engineering fimbriae would allow the price of the vaccines to be reduced and their manufacture to be simplified. The vaccine immunogenicity is increased in this case. At first, E. coli was selected as an expression system, but morphogenetic expression of the pili was not achieved on its surface because of some differences in the biogenesis and structure of fimbriae from D. nodosus. Successful morphogenesis of the pili was achieved in Pseudomonas aeruoginosa, which had closest similarity in the structure of pili. The level of the immunity obtained after immunization of the sheep with recombinant pili was similar to the level of the immunity after native pili or whole cells of D. nodosus had been used. This review contains information regarding the recombinant strains of Pseudomonas aeruoginosa obtained using fimbriae of D. nodosus and expression of pilin genes in different bacterial systems.

[Enzyme-linked immunosorbent assay for detection of antibodies to porcine reproductive and respiratory syndrome virus, by using recombinant nucleocapsid protein N].

Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific.

[Genetic variability of the nucleocapsid protein of the virus of the porcine reproductive and respiratory syndrome]. / Variabel'nost' gena belka nukleokapsida virusa reproduktivnogo i respiratornogo sindroma svinei (RRSS).

The porcine reproductive and respiratory syndrome (PRRS) is a contagious viral pathology caused by PRRS virus. There are 2 types of the above virus--the European and American ones. Distribution patterns of the PRRS virus were studied for Russia and Byelorussia. Above 700 porcine sera obtained from 32 households of 21 Russia's administrative regions and from 19 households of 6 Byelorussia's administrative regions were tested for presence of antibodies to the PRRS virus. Simultaneously, the samples were tested for virus presence by polymerase chain reaction (PCR). It was proven serologically that the PRRS virus is widespread in the territories of Russia and Byelorussia. Noteworthily, all field isolates found in Russia and Byelorussia belong to the European type. Not a single viral isolate of the American PRRS type was found. The nucleocapsid (N) recombinant protein was obtained on the basis of the Russian field isolate of the PRRS virus by using the E. coli. expression system. Finally, it was shown as possible to use the recombinant protein in indirect immune enzyme assay for the sake of detecting the antibodies to the PRRS virus.

[The demonstration of the genes controlling enterotoxigenicity in Staphylococcus aureus strains by using the polymerase chain reaction]. / Vyiavlenie genov, kontroliruiushchikh énterotoksigennost' u shtammov Staphylococcus aureus, s pomoshch'iu polimeraznoi tsepnoi reaktsii.

The selection and subsequent synthesis, according to the nucleotide sequences of S. aureus genes responsible for the expression of enterotoxins A and B, of highly specific primers for polymerase chain reaction Pcr were carried out with the use of the program "Primer". The optimum temperature conditions of polymerase chain reaction for all pairs of primers were selected. The method for the rapid determination of the enterotoxigenic properties of S. aureus strains by means of Pcr was proposed. The enterotoxigenic properties of several S. aureus strains were determined, which revealed that 3 clinical isolates had the gene of enterotoxin A in their genome, while laboratory strain FRI-722(H) carried the genes of enterotoxins A and B.

[Expression of Staphylococcus aureus enterotoxin A gene in heterologous systems]. / Ekspressiia gena énterotoksiba A Staphylococcus aureus v geterologichnykh sistemakh.

The genomic library of Staphylococcus aureus genes on the plasmid vector pSL5 has been constructed. The library contains a 2.5 kb HindIII DNA fragment including the gene for enterotoxin A. The entA gene on the high copy number plasmids in the Escherichia coli cells deficient in proteolysis determines the synthesis of enterotoxin A in the amounts comparable to the ones in the parent strain Staphylococcus aureus FRI 722(H).

[The cloning and expression of the Staphylococcus aureus enterotoxin A gene in Escherichia coli cells]. / Klonirovanie i ékspressiia gena énterotoksina A Staphylococcus aureus v kletkakh Escherichia coli.

Gene ent-A has been cloned on phage vector pSL5 with the use of the gene library of S. aureus FR1722(H). It is located within DNA fragment Hind III having 2,500 nucleotide pairs.

[Thermo-regulated expression of the htpR gene under the control of the PR-promotor of bacteriophage lambda induces supersynthesis of heat shock proteins]. / Termoreguliruemaia ékspressiia gena htpR pod kontrolem PR-promotora bakteriofaga lambda indutsiruet sverkhsintez belkov teplovogo shoka.

The mechanisms of induction of heat shock protein synthesis in E. coli have been studied. For this purpose plasmids in which htpR gene expression is controlled by the PR-promoter of bacteriophage lambda and by the Trp-promoter have been constructed. An effective induction of heat shock proteins requires both an increased content of htpR protein and additional cofactors formed in the cell under heat shock conditions.