[Construction of recombinant strain Brevibacillus choshinensis for chimeric borrelia Dbpa antigen production.]

The aim of this work was creation of recombinant chimeric protein using TAKARA expression system Brevibacillus choshinensis with fused gene dbpAAG, which include the parts of dbpAA and dbpAG genes coding the major antigenic determinants of decorinbinding proteins А (DbpA) from two species of borreliosis agents - Borrelia afzelii and Borrelia gаrinii. Such plasmid should be able to support the synthesis of recombinant chimeric polypeptide consisting immunogenic domains of DbpA Borrelia afzelii and Borrelia gаrinii in the stable and soluble forms, that important for effective using in Lyme diseases serodiagnosis. We chose the TAKARA expression system based on the strain Brevibacillus choshinensis and plasmid pNCMO2. It give us possibilities to obtain the scale quantity of the secreted soluble target proteins with native conformation in particular with conserve antigenic determinants. As results, the plasmid pNCMO2 with a fusion gene dbpAAG was constructed. Recombinante plasmide DNA pNCMO2/dbpAAG was used for Brevibacillus choshinensis trasformation. We were able to show that during cultivation in a liquid medium recombinant cells of В. choshinensis/pNCMO2/dbpAAG produced secreted chimeric 30кD protein with high immunoreactivity to Lyme borreliosis patient's serum.

[Search for protective antigens in Ixodes persulcatus (ixodidae) salivary gland extracts].

RT-PCR evaluation of the activity of eight Ixodes persulcatus salivary gland genes shows clear distinctions in their expression depending of the stage of tick feeding. Out of them, only Salp 10 and Salp 15 proteins may be regarded as candidates for protective antigens to develop anti-tick and anti-Borrelia vaccines. Firstly they play an important role in feeding a tick and modifying a host's immune response. Secondly, the increasing expression of the salp 10 and salp 10 genes begins at early tick feeding stages. Thirdly, the activity of these genes increases with the beginning of feeding by tens and hundreds times and keeps at this level until the third tick feeding stage is over.

[Changes in the expression of salivary gland genes in Ixodes persulcatus (Ixodidae) depending on the stage of tick feeding].

By using the guanidine-isothiocyanate test, the authors isolated a summary RNA preparation from Ixodes persulcatus salivary gland extracts. Activity products of the genes responsible for the expression of some salivary proteins were first identified using the RT-PCR. It has been shown that, firstly, I. persulcatus synthesizes at least 3 transcripts homologous to the respective salivary components of the related species I. scapularis, the translation product of which is likely to be immunodominant antigens; secondly, the number of each of these transcripts, as in I. scapularis, depends on the stage of tick feeding. The changes in the expression of each transcript are specific: monotonously increasing changes in Salp 17 and cyclic ones in Salp 16, and synthesis, only when the ticks are fully ingested, in Salp 25.

[Study of the transfer of foreign genes into mussel Mytilus Galloprovincialis Lam. eggs by spermatozoa]. / Issledovanie perenosa chuzherodnykh genov spermatozoidami v iaitsekletki midiii Mytilus Galloprovincialis Lam.

The possibility of transferring exogenous DNA into eggs by mussel Mytilus galloprovincialis Lam. sperms both with the use of certain methods of transfection and without them was studied. The efficacy of egg fertilization by sperms treated with foreign DNA and the development of larvae at early stages of embryogenesis were evaluated. Negative effects of the contact between mussel sperms and exogenous DNA on fertilization and subsequent development were noted. The proportion of developing larvae decreased with increasing DNA concentration and sperm exposure. Transfer of plasmids pCMVlacZ and pMTbGH into eggs was observed in group crosses. With the use of PCR, foreign DNA sequences were found in the larvae at the stage of veliger 48 h after fertilization. An intense signal was recorded after sperm electroporation in 10% DMSO.

[The use of molecular biological methods for the individual characterization of Mycobacterium tuberculosis strains]. / Ispol'zovanie molekuliarno-biologicheskikh metodov dlia individual'noi kharakteristiki shtammov Mycobacterium tuberculosis.

The expediency of using molecular biological methods for the evaluation of M. tuberculosis clinical strains by individual genetic certification of circulating M. tuberculosis strains has been substantiated. Considerable genetic heterogeneity of M. tuberculosis strains isolated from patients in different regions of the Russian Federation has been established; this heterogeneity is due to the presence of differences in the number of copies (5-26) of element IS6110 in M. tuberculosis cells.

[Molecular mechanisms of mycobacterium tuberculosis strains resistance to rifampicin and isoniazid]. / Molekuliarnye mekhanizmy ustoichivosti k rifampitsinu i izoniazidu klinicheskikh shtammov mycobacterium tuberculosis.

The levels and spectra of the drug resistance of clinical M. tuberculosis strains were defined. There was a relationship of treatment regimens to the drug resistance of mycobacteria isolated from the strains. The fragments of genes rpoB, inhA, and katG were analyzed by polymerase chain reaction and sequencing. In addition to earlier identified substitutions, new mutations were found in rpoB: A-->T/233, G-->A/395, C-->T/232, G-->T/202, C-->T/221, C-->T/260, GA-->TT/202-203, delta 199-207 ATGGACCAG. Strain 12/7 was found to have 30 point mutations leading to substitution of only 3 amino acids and to have GGG(Gly)354 deletion as well. Most mutations in this strain are "silent". Substitutions at 944 and 463 positions were revealed in katG.

Drug-resistant strains of Mycobacterium tuberculosis isolated in Russia.

SETTING: State Research Center for Applied Microbiology, Russian Research Institute of Phthisiopulmonology (Ministry of Health, Moscow). OBJECTIVE: To analyze drug-resistant clinical isolates of Mycobacterium tuberculosis obtained from patients referred to the institute from different parts of Russia, and to study the mechanisms of their rifampicin resistance. DESIGN: Fifty clinical isolates of M. tuberculosis were analysed. Polymerase chain reaction (PCR) and sequencing were used to study the mechanisms of rifampicin resistance in 25 isolates. RESULTS: Among cultures isolated from 50 patients, drug resistance was detected in 33. Most of the isolates were resistant to rifampicin (25 isolates), isoniazid (14 isolates), and streptomycin (seven isolates). Only 6% of the isolates were resistant to one drug, while 14% were resistant to two, 32% to three, 40% to four, and 8% to five drugs. Susceptible isolates were derived from 17 patients. The following point mutations and deletions in the rpoB locus, responsible for high level rifampicin resistance (more than 50 microg/ml in egg-based medium), were detected: G-->A/395 (Arg-->Gln), C-->T/232 (His-->Tyr), C-->T/221 (Ser-->Leu), G-->T/202 (Asp-->Tyr), GA-->TT/202-203 (Asp-->Phe), deltaATGGACCAG/199-207 (Met, Asp, Gin), A-->T/91 (Met-->Leu), TG-->CC/227-228 (Leu-->Ser), GAG-->AGT/349-350-351 (Gln-->Ser), deltaGGG/354(Gly). CONCLUSION: A number of previously unrecognised genetic modifications in the rpoB region were found in rifampicin-resistant strains isolated from patients from different parts of Russia.