DEVELOPMENT OF THE ALGORITHM FOR IDENTIFICATION OF THE LEVEL OF VIBRIO CHOLERAE CTXA AND TOXR GENE EXPRESSION USING RT-PCR WITH REAL-TIME HYBRIDIZATION-FLUORESCENT REGISTRATION OF RESULTS

Objective of the study is to design the algorithm for assessment of expression of the structural and regulatory virulence Vibrio cholerae genes by the model of ctxA and toxR genes encoding and controlling biosynthesis of cholera toxin.Materials and methods. Utilized were 10 strains of Vibrio cholerae, classical and El Tor biovars. Cloning of gene fragments was carried out through transformation and ligation. RT-PCR was done in “BIS M112” and “Rotor-GeneQ” amplifiers. Processing of the results was performed by means of the software package in set with Rotor-GeneQ (Software 1.8.17.5).Results and conclusions. Developed has been the algorithm for evaluation of the level of expression of V. cholerae ctxA and toxR genes applying RT-PCR with real-time hybridization-fluorescent registration of results. The stated algorithm allows for rapid and effective statistically significant specification of the expression of structural and regulatory virulence V. cholerae genes and can be used for evaluation of newly discovered cholera vibrio strains

Construction of Recombinant Escherichia coli Strain – Producer of Basic Subunit of Toxin-Coregulated Pilus of Adhesion (TCPA) of Vibrio cholerae Biovar El Tor

Objective of the study is to construct recombinant E. coli strain – producer of TcpA protein of cholera agent El Tor, carrying tcpACIRS gene in its genome, and use the strains for antigen production. Materials and methods. Utilized was non-toxigenic genovariant strain of Vibrio cholerae biovar El Tor from the “State Collection of Pathogenic Bacteria” at the premises of RusRAPI “Microbe”, as well as commercial E. coli strains and plasmids for cloning (Invitrogen, USA). Chromosomal DNA from V. cholerae cells was extracted using Charge Smitch gDNA Mini Bacteria Kit applying nucleo-sorption. To extract plasmid DNA from E. coli cells PureLink Quick Plasmid DNA MiniprepKits were used. The presence of tcpACIRS gene was assayed by PCR, using designed through our own efforts primers. DNA fragments were isolated from agarose gel with the help of PCR Clear-Up-System panel. SDS-PAGE was performed according to U.K.Laemmli method. Protein content of samples was measured by M.M.Bradford method. The panel for affinity chromatography was applied for recombinant TcpA protein purification. Results and conclusions. Constructed safe strain of E. coli is the producer of recombinant TcpA protein, basic subunit of toxin-coregulated pilus of adhesion of cholera agent biovar El Tor. The region of tcpA gene of Vibrio cholerae biovar El Tor was cloned as part of vector plasmid pET302 by the restriction sites XhoI-BamHI in E. coli strain BL21(DE3)Star. In the stated design protein biosynthesis is under transcriptional control of phage promoter T7 and induced by isopropyl-ß-thiogalactoside (IPTG). Tested were the conditions for optimum TcpA protein production and the layout of its purification using affinity chromatography. It was demonstrated that TcpA is present in cells of intestinal bacterium, both in native form and as inclusion bodies. Overall TcpA protein production amounted to 60 mcg/ml. Obtained purified TcpA protein can be used for studies of its immunogenic and physical-chemical properties, as well as development of immune-diagnostic preparations to evaluate the level of TcpA production in various V. cholerae strains, and identification of antigen composition of cholera vaccine preparations

Nucleotide Sequence and Phylogenetic Analysis of Glycoprotein-G of the Russian Fixed Rabies Virus Strain “Moscow 3253”

Fully sequenced have been glycoprotein-G, sha-psi region, as well as H-end site of the L-gene in the rabies virus strain “Moscow 3253”. Compared are amino acid sequences of proteins of “Moscow 3253” strain and other fixed strains of the virus. Established is 98 % DNA homology with RV-97, and 91% homology with PV (Pasteur virus) strain. Constructed has been phylogenetic tree of the strain under study alongside with various groups of fixed rabies virus. It is revealed that “Moscow 3253” strain has closer genetic relations with Japanese group of strains, than with PV strain. Put forward is an assumption that PV strain does not derive from the virus isolated by Pasteur, but relates to the American group of strains

Diagnosis and correction of hemostasis injury in surgery for cholepathia

Целью исследования было уменьшение частоты геморрагических осложнений при оперативном лечении пациентов по поводу заболеваний желчных протоков. Состояние гемостаза до, сразу после, а также на следующие сутки после операции контролировали по данным низкочастотной пьезоэлектрической тромбоэластографии (НПТЭГ). Установлено, что при использовании транексамовой кислоты частота геморрагических осложнений уменьшается на 38, 65 %.The aim of the study was to reduce the frequency of hemorrhagic complications in the surgical treatment of patients for diseases of bile ducts. Hemostatic state before, immediately after and on the following day after the operation was controlled by the data of frequency piezoelectric thromboelastography. It is established, that the application of tranexamic acid to promote incidence of hemorrhagic complications are reduced by 38.65%

Antirabies DNA Immunization

Cited are literary data related to the development of DNA vaccines against rabies virus. Research results regarding gene vaccination of different models of laboratory animals and different ways of vaccine introduction are presented. Possibility to potentiate immunogenicity of DNA vaccines using adjuvants and cytokines is considered. Ways of improving of polynucleotide vaccines are discussed

Construction of <i>Yersinia pestis</i> Strain Producing Fluorescent Protein GFP and Prospects of Its Usage

Objective of the study was to construct the recombinant Y. pestis strain producing fluorescent protein GFP and analysis of the prospects of its usage for investigation of interaction of the agent with the protozoa, and also macroorganism of mammals (rodents). Materials and methods. To create the strain producing fluorescent protein GFP, we used the natural Y. pestis strain isolated in 2016 in Gorno-Altai high-mountain plague focus. Fluorescent protein gen was inserted into commercial vector pTurboGFP-B via electroporation. Properties of the obtained recombinant Y. pestis strain 367 pTurboGFP-B were studied using microbiological, biological, and molecular-genetic methods. Results and conclusions. The plague agent strain, containing vector plasmid pTurboGFP-B which encodes synthesis of green fluorescent protein GFP, and being resistant to ampicillin was constructed applying electroporation. The designed Y. pestis strain, producing green fluorescent protein, does not differ from the stock strain by its cultural-morphological, biochemical properties, virulence and survivability in mixed culture with Acanthamoeba castellani. On solid nutrient media, recombinant strain and its subcultures, obtained from infected animals, formed colonies of greenish-yellow color, becoming fluorescent under UV-light. In preparations from animals and from co-cultures with amoeba, fluorescent cells of plague agent were observed. The constructed strain can be utilized as a model one for investigation of interactions between plague agent and cells of protozoa (soil amoeba, nematodes) and mammals under laboratory conditions

Thrombohemorrhagic diagnosis and correction of disorders in patients after prostatectomy on surgical treatment stages

У роботі узагальнено результати обстеження системи гемостазу та профілактики тромбогеморагічних ускладнень при хірургічному лікуванні 114 хворих на доброякісну гіперплазію передміхурової залози. Була встановлена форма реагування системи гемостазу, що визначає характер післяопераційних тромбогеморагічних ускладнень. Розроблений комплекс профілактичних заходів дозволяє посилити їх взаємний позитивний ефект, нормалізувати порушені механізми гемостазу, знижуючи інтенсивність внутрішньосудинного мікрозгортання крові і зменшуючи тим самим частоту розвитку ускладнень.Prostatectomy is associated with a high risk of thrombohemorrhagic complications and identifies as the most problematic surgery in urology. Complex use of prolonged epidural anesthesia, bemiparin and doxazosin on the stages of surgical treatment in patients with benign prostatic hyperplasia normalize the functional state of the hemostatic system, eliminates the symptoms DIC - syndrome, decreases the amount of thrombohemorrhagic complications and reduce time of hospitalization.В работе обобщены результаты обследования системы гемостаза и профилактики тромбогеморрагических осложнений при хирургическом лечении 114 больных доброкачественной гиперплазией предстательной железы. Была установлена форма реагирования системы гемостаза, которая определяет характер послеоперационных тромбогеморрагических осложнений. Разработанный комплекс профилактических мер позволяет усилить их взаимный положительный эффект, нормализовать нарушенные системы гемостаза, снижая интенсивность внутрисосудистого микросвертывания крови и уменьшая тем самым частоту развития осложнений