[Immunogenic properties of recombinant vaccinia virus containing the human IL-2 gene].

A genetic construct of the human interleukin-2 (IL-2) gene within vaccinia virus (L-IVP strain) has been designed. The authors show the capacity of CV-1 cells infected with the recombinant vaccinia virus VV-SIL2 to secrete human IL-2 into the culture medium. Human IL-2 has been detected by immunoblotting. The sera from the animals immunized with the recombinant virus VV-SIL2 exhibited both human IL-2 and its antibodies throughout the observation period. This recombinant virus immunization induced both humoral and cell-mediated immune responses to human IL-2; the observed changes in the concentrations of cytokines are likely to suggest that the response predominantly followed a Th1 pathway. The study construct was nontoxic at the used concentrations and administration routes. The findings point that it is promising to investigate the adjuvant properties of the recombinant VV-SIL2 vaccine-based preparation for immunization in combination with various vaccines and to study this construct in therapy for cancer diseases.

[Comparative study of variola virus and monkeypox virus interferon-gamma-binding].

DNA fragments containing genes for coding IFN-gamma-binding proteins (IFNgammaBPs) of variola virus (VARV) and monkeypox virus (MPXV) were obtained from viral genomes using PCR. Isolated genes coding desired proteins were expressed in the insect Sf21 cells using baculovirus expression system. Secreted recombinant IFNgammaBPs were isolated from culture medium of infected Sf21 cells through affinity chromatography procedure. SDS-PAAG and Western blot analysis of culture medium of infected insect cells and preparations of purified recombinant IFNgammaBPs indicated that recombinant viral proteins were dimerized even in the absence of ligand (hIFNgamma) unlike their cell (eucaryotic) analogs. Biological activity of the recombinant IFNgammaBPs were studied in the test of protective effect inhibition of hIFNgamma on L68 cells infected with murine encephalomyocarditis virus. It was shown that recombinant IFNgammaBPs had dose-dependent IFNgamma-inhibiting activity. A possibility of the elaboration of new therapeutics for anti-hIFNgamma therapy on the base of IFNgammaBPs is discussed.

[Expression of genes for orthopoxviral TNF-binding proteins and study resulted recombinant proteins].

Genes for TNF-binding proteins (CrmBs) of variola virus (VARV), monkeypox virus (MPXV) or cowpox (CPXV) were isolated with PCR from viral genomes and expressed within baculovirus DNAs in Sf21 insect cell line. Properties of resulted recombinant proteins were studied with physical-chemical and immunological methods. It was shown with solid phase enzyme-linked immunoassay that viral proteins inhibited hTNF binding with polyclonal hTNF-antibodies. The strongest inhibitor was VARV-CrmB, the less one was MPXV-CrmB. Biological activity of recombinant protein preparations was studied in the test of neutralization of TNF cytotoxicity for L929 murine fibroblast cells. It was shown that recombinant CrmBs neutralized cytotoxicity of hTNF, mTNF or rTNF in species-specific manner. It was shown also that effectiveness of hTNF cytotoxicity inhibition in vitro with VARV-CrmB exceeded the same effect of polyclonal hTNF-antibody. A possibility of the elaboration of new therapeutics for anti-TNF therapy on the base of CrmB-like proteins is discussed.

[Presentation of HIV epitopes by HBcAg].

The hepatitis B core antigen (HBcAg) was used to present the HIV epitopes and mimics selected by phage display. The HIV epitopes were inserted into the el loop of HBcAg. The influence of insertions on the ability of chimeric HBcAg to assemble itself was studied. Special soft was made use of to detect the regularities between certain physical-and-chemical properties of amine-acid residua (belonging to an inserted alien peptide) and the presence or loss of the ability of HBcAg to assemble itself. Recommendations are provided of how to overcome difficulties related with the presentation of alien epitopes.

[Expression of the gene for vaccinia virus 36K nonstructural protein in Escherichia coli and study of the protective properties of expression products]. / Ekspressiia gena nestrukturnogo belka 36K virusa ospovaktsiny v Escherichia coli i izuchenie protektivnykh svoistv produktov ékspressii.

The nonstructural 36K protein of vaccinia virus (VV) mapped in HindIII-P and HindIII-J fragments of VV strain L-IVP has been expressed in E. coli as a fusion protein. The products of 36K gene preserved the antigen homology with the native protein 36K and the capacity to bind specific immunoglobulins of rabbit antiVV serum. The protective properties of 36K gene products and their joint effect with the immunodominant protein 35K were investigated. The non-structural 36K gene products showed no protective activity, but increased the production of specific antibodies in mice immunized with a mixture of both protein preparations, this increase being compatible with that observed after immunization with the inactivated virus preparations.

[Localization of the N-terminal epitope of the HBe antigen of the hepatitis B virus]. / Lokalizatsiia N-kontsevogo épitopa HBe-antigena virusa gepatita B.

A new antigenic site was located at the N-terminus of HBeAg. Comparison of antigenic properties of the recombinant proteins HBeAgR and HBeAg delta N truncated at the N-terminus has lead to this conclusion. The proteins have been studied by ELISA and western-blot analysis using monoclonal antibodies E1A7 and polyclonal antiserum to HBeAg. Polyclonal antiserum to HBeAg binds to HBeAg delta N and HBeAgR while monoclonal antibodies E1A7 interact only with HBeAgR. The data obtained supports evidence that the epitope is located at the N-terminus of HBe-polypeptide chain. It is classified as a sequential type one and at least one amino acid from the Met-Asp-Ile sequence participates in its formation.

[The use of fractionation in a polyethylene glycol-dextran two-phase system for the testing and purification of restriction endonucleases]. / Ispol'zovanie fraktsionirovaniia v dvukhfaznoi sisteme poliétilenglikol'-dekstran dlia testirovaniia i ochistki éndonukleaz restriktsii.

A simple procedure was developed for testing and purification of restriction endonucleases Msp I, Pst I, Bam HI, Pvu I, Pvu II that includes biomass destruction, fractionation of cell-free extracts in the aqueous two-phase (polyethylene glycol-dextran) system and chromatography oh phosphocellulose. Optimal conditions for the fractionation of Msp I, Pst I, Bam HI, Pvu II, EcoR I, EcoR II, BspR I, Alu I were chosen. For separation of Pvu I and Pvu II gel filtration through biogel A-0.5 m was additionally introduced.

[Purification and characteristics of an RNA-ligase preparation from bacteriophage T4]. / Ochistka i kharakteristika preparata RNK-ligazy bakteriofaga T4.

A purification techniques was developed to obtain a preparation of the bacteriophage T4 RNA-ligase (EC 6.5.1.3) free of contaminating nuclease activities. It includes fractioning on phosphocellulose and DEAE-cellulose, chromatography on DEAE-cellulose, isoelectric sedimentation, gel filtration through Sephadex G-100 and chromatography on hydroxylapatite and aminohexyl-sepharose. The enzyme yield amounts to 30-37%. By means of biochemical tests, the preparation of RNA-ligase was found to be good for molecular biology and gene engineering, in particular for such areas where oligodeoxyribonucleotides and high molecular-weight RNA are used as substrates. The ligating ability of the enzyme was demonstrated by means of the preparative synthesis of nonaribonucleotide ApUpG(pU)6 from trinucleoside diphosphate ApUpG and hexoribouridylic acid, as well as by insertion of [5'-32P]-labelled cytidine-3',5'-diphosphate in the 3'-end of the bacteriophage MS 2 RNA.

[Influence of the structure of photoreactive ATP analogs on the affinity modification of phenylalanyl-tRNA synsthetase. Modification of the enzyme at two types of nucleotide sites]. / Vliianie struktury fotoaktivnykh analogov ATP na affinnuiu modifikatsiiu fenilalanil-tRNK sintetazy. Modifikatsiia fermenta po dvum tipam nukleotidsviazyvaiushchikh uchastkov.

ATP gamma-(p-azidoanilidate) (1) and ATP gamma-(p-azidobenzyl)-methylanilidate (2) were shown to be competitive inhibitors for ATP and amino acid in tRNA aminoacylation catalyzed by E. coli MRE-600 phenylalanyl-tRNA synthetase (E.C.6.1.1.20). Low concentration (10(-5)--10(-6) M) of either ATP, gamma-anilidate or GMP stimulates the aminoacylation of tRNA suggesting their interaction with some nucleotide binding sites of the enzyme other than catalytic ones. Covalent photobinding of (1) to the enzyme does not inhibit aminoacylation, nor does it prevent nucleotides from activating the enzyme. UV-irradiation of the synthetase in the presence of (2) results in complete inactivation of the enzyme which can be prevented by phenylalanine or phenylalanine-ATP to save 50% of the enzyme activity but not ATP and tRNA. The photobinding of (2) to the enzyme in the presence of phenylalanine and ATP removes the activation of the enzyme by nucleotides suggesting that both the catalytic and effector sites of the synthetase are blocked in the same manner by compound (2).