Analysis of Genetic Determination of <I>Vibrio cholerae</I> Tweenase Activity

Studied are Vibrio cholerae of different serogroups on the presence of cef (CHO cell elongating factor) gene and activity against tweens and tributyrin using HDS-agar, prepared on the basis of bakery yeast pancreatic digest. Determined is the fact that all cef-positive strains hydrolyze tweens 20, 40, 60, 80, 85 and all but toxigenic V.cholerae O139, hydrolyze tributyrin. In contrast, non-toxigenic cef-negative strains of O139 serogroup, are active only against the latter. Apparently, the tweenase activity of Vibrio cholerae is provided, partly, by Cef, and the ability to hydrolyze tributyrin is the result of combined activity of Cef and other ferments. Shown is the efficiency of HDS-agar application to determine these characteristics

Recombinant Escherichia coli Strain with Enhanced Production of Vibrio cholerae Neuraminidase

Objective of this work was cloning of the Vibrio cholerae nanH gene as part of a plasmid vector, providing expression of foreign genes under the control of T5 promoter, and construction of a E. coli strain – producer of V. cholerae recombinant neuraminidase.Materials and methods. V. cholerae о1 strain served as a DNA donor, pQE30 – as a vector plasmid. The gene was PCR-amplifi ed, the cloning was carried out by means of conventional methods, performance of recombinants and localization of the required protein was determined based on the results of electrophoresis of cell lysates. Neuraminidase activity was identifi ed by fl uorescence in ultraviolet light after incubation with specifi c substrate (4-methylumbelliferyl-N-acetylneuraminic acid).Results and discussion. Recombinant plasmid pNanH, containing the cloned gene nanH V. cholerae, has been constructed. The gene is inserted into BamHI-PstI sites of the polylinker of pQE30. Expression of the cloned gene in the producer strain E. coli JM103pNanH occurs under the control of T5 promoter after isopropyl-1-thio-β-D-galactopyranoside (IPTG) induction. The strain shows neuraminidase activity. The recombinant NanH protein is accumulated in the producer’s cells in two forms. The fi rst form, with molecular mass (MM) of 89.5 kDa, is an unprocessed protein with the hexahistidine block (6His-tag) at its N-terminus, it is located in inclusion bodies. Its percentage is 5.6–6.6 % of the total cell proteins. The second one, with MM of 83 kDa, is found in the periplasmic space and corresponds to the mature NanH, its percentage being 3.4–3.8 %. The total percentage of both forms is 9–10 % of total cell proteins, which allows to consider the strain E. coli JM103pNanH to be a super-producer of the required protein. The strain may be used for purifi cation of NanH preparations for construction of specifi c diagnostic, therapeutic and pharmaceutical preparations as well as for investigation of the protein as a virulence/persistence factor of the pathogen

Cholera Vibrio Membrane Protein <I>OmpT</I> as an Omptin Belonging to <I>Vibrionaceae</I> Family

Concerned are the issues related to enterobacteria omptins, their structure and functionality, as well as alternative role in pathogenesis of the infections induced by them. Isolated from cholera vibrio, and later purified using differential centrifugation and column chromatography has been porin protein of the OmpT outer membranes, with the molar mass of approximately 40 kDa. Synthesis of porin is under control of the complex regulatory system. It does not contain cysteine, but possesses proteolytic activity with broad substrate specificity: it hydrolyzes fibrin, protamin, gelatine; transduces human plasminogen into plasmin, which provides for the well-known advantages for the vibrios in the intestine of a susceptible host. Comparative computer-assisted analysis of amino acid sequence has revealed that cholera vibrio OmpT protein relates to the omptins of enterobacteria as a far-remotely one, and has 13 % identity and similarity to it. OmpT protein is probably affiliated to a new class of porins of the family Vibrionaceae

Crimean Hemorrhagic Fever: Ecologic and Epizootic Role of the <i>Corvidae</i> Family

The role of birds in the circulation of the virus of Congo-Crimean hemorrhagic fever (CCHF) is described in the paper. Investigation of 758 samples of birds revealed the virus CCHF antigen to be present in 19 (2.51 % ± 0.57) rooks' samples. Corvidae were shown to be a full-fledged co-member of the Crimean hemorrhagic fever (CHL) natural foci in the territory of Rostov Region. Epidemiologic significance of this bird family consists in dissemination of the CCHL virus, expansion of the existing natural CHL foci, and thereby supporting the formation of new ones. Ecologic and epizootiologic role of Corvidae should be taken into consideration while conducting epidemiologic surveillance in HCL natural foci