3/2015

Author

page: 4-10 pp
DOI: 10.11134/btp.3.2015.1
Zhumatov К.Кh., KydyrmanovА.I.
Institute of microbiology and virology
103, Bogenbai batyr str., Almaty, 050010, Kazakhstan 
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Abstract

Viral infections account for more than 60% of all known infectious diseases in human, animals and plants. They are one of the leading causes of death among people living in both developed and developing countries with weak health systems. During the process of constant renewal and emergence of new pathogens, “emerging infections" (emerging-reemerging) are generated regularly in nature.

This review article focuses on a new coronavirus infection found in humans and animals, the Middle East respiratory syndrome (MERS). A brief characterization of members of the family Coronaviridae and their carriers is described, including their natural hosts and a clinical picture of the diseases caused by them. Here, we summarize the data on the occurrence, dynamics of the spread, clinical cases of MERS infection in the Middle East and other regions of the world. The results of the study on the phylogeny of the causative agent (MERS-CoV) are included. We have included a brief description of the Kazakhstani populations that are susceptible to mammalian coronavirus such as those found in bats and camels. We have highlighted the potential risk of the spread of MERS-CoV in Kazakhstan and suggested the importance of a timely study of the natural reservoirs of the virus in the territory of the Republic of Kazakhstan.

Keywords: Middle East respiratory syndrome, coronavirus, genome, phylogenesis, cluster, camel, bat

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Author

page: 11-19 pp
DOI: 10.11134/btp.3.2015.2
Kairzhanova A.D.1, Abisheva G.D.1, Popova O.A.2, Kamalova D.K.1, Shevtsova E.S.1, Shevtsov A.B.1
1National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2 Astana Ecolife
22, Alash road, Astana, 010000, Kazakhstan
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Abstract

Microdeletions in the long arm of the Y chromosome are common causes of male spermatogenesis disorders. The frequency of the AZF locus microdeletions is approximately1 in 1000–1500 males. Such deletions of the Y chromosome are identified in 11% of males with azoospermia and in 8% of males with severe oligospermia. The aim of this study was to develop a multiplex polymerase chain reaction (PCR) protocol for the detection of microdeletions in the AZF locus of the Y chromosome. Based on the literature, the following sequence tagged site (STS) markers were used for development of the PCR protocol for screening the AZF locus microdeletions: AZFa-sY86 иsY84, AZFb-sY127 and sY134, AZFс-sY254 and sY255. As a control, the presence of Y chromosome short arm fragments in the genomic DNA were assessed using the SRY gene sequence and ZFY/Х as an internal control of the PCR. Confirmation of the developed protocol was performed with 40 DNA samples. The protocol recommended by The European Academy of Andrology was used as a complementary method to confirm our results. The results from both methods indicate the specificity and high demand of the developed PCR protocol.

Keywords: male infertility, PCR, microdeletion, AZF locus, Y chromosome, STS markers

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Author

page: 20-32 pp
DOI: 10.11134/btp.3.2015.3
Zhambakin K.Zh., Zatybekov A.K., Volkov D.V., Shamekova M. Kh.
Institute of Plant Biology and Biotechnology
45 Timiryazev str., Almaty, 050040, Kazakhstan
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Abstract

Alphaviruses are enveloped viruses with positive sense RNA genomes, which replicate in the cytoplasm and have no DNA stage in their life cycle. They are natural infection agents of wild and domestic animals, and may also cause epidemics in humans who are dead-end hosts. Alphaviruses have small genomes and show active replication accompanied by the synthesis of large amounts of viral proteins. Representatives of this genus are able to infect many vertebrate and invertebrate cells. Since the genomic RNA of alphaviruses is infectious, viral progeny can be obtained by transfecting in vitro synthesized RNAs into cell culture, thus facilitating genetic engineering. Alphaviruses are attractive vectors for the production of recombinant proteins in cultured cells of mammals, birds, and invertebrates due to high levels of protein expression. Model viruses used for the development of alphavirus expression systems include the Sindbis virus, Venezuelan equine encephalomyelitis virus, and Semliki Forest virus. Use of wild-type alphaviruses as vectors is avoided because of their strong cytopathic effect in cell culture. Hence, numerous mutant alphaviral genomes with reduced cytopathic effect have been developed.

Alphavirus vectors induce stronger cellular and humoral immune responses than other viral vectors, and therefore are used for the construction of live vaccines against infectious and non-infectious diseases (i.e. anti-cancer therapeutic vaccines). The wide cell and tissue tropism allows utilization of alphaviruses as agents for gene delivery under in vivo conditions and gene therapy.

Keywords: alphavirus, replication, vector, protein expression, vaccines, cytopathic effect

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Author

page: 33-43 pp
DOI: 10.11134/btp.3.2015.4
Urazaliyev K.R.
Kazakh Institute of Agriculture and Plant Growing
1, Erlepesov str., Almalybalyk, Karasai district, Almatinskaya oblast, 040909, Kazakhstan
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Abstract

Regeneration of gametic cells to produce haploid or doubled haploid plants is an excellent example of totipotency of plant cells, called gametogenesis. Producing doubled haploid plants through androgenesis or gynogenesis facilitates the possibility of obtaining homozygous plants in a single step (one year). This approach is useful in plant breeding, genetic manipulation, and in many areas of basic research related to the study of plant biology. Achievement of homozygosity in one generation helps reduce the number of inbreeding cross cycles that are required to obtain purebred lines. As an effective system of plant regeneration, gametic cells also are preferred for breeding, genetic transformation, transgenic plant research, and other regeneration efforts.

Doubled haploid plants and the obtained homozygous lines are used in several areas of basic research such as classical plant genetics and cytogenetics, modern molecular genetics, including induced mutagenesis, site-directed mutagenesis, genome mapping and the evaluation of the relative remoteness of genomes, gene dosage effects, and the analysis of the relationships mechanisms of genetic control of chromosome pairs. One of the most important areas of practical application of this technology is plant breeding.

Many plant species have the ability to regenerate from microspores. However, in most cases, it is necessary to perform one or more pre-processing steps, in the form of physical, physiological and/or chemical treatments. Pre-processing must be used to switch microspores from a gametophytic development pathway to a sporophytic development path. It is possible to increase the efficiency of this process by the direct, artificial manipulation of individual microspores, which has enabled the successful production of regenerated plants in more than 300 species.

Keywords: doubled haploids, haploid technology, anther, microspore, homozygote, breeding

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Author

page: 44-52 pp
DOI: 10.11134/btp.3.2015.5
Tursunov K., Begaliyeva A., Inirbay B., Mukanov K.К., Ramanculov Е.М., Shustov А.В., Mukantayev К.Н.
National Center for Biotechnology
13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
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Abstract

Rabies is an important concern in the Republic of Kazakhstan due to its widespread incidence in the country. The main sources of the disease in the Republic are natural, such as viral transmission through populations of wild carnivores and rodents, and anthropurgic, such as viral transmission through populations of domesticated animals. The ability of rabies virus to spread rapidly underscores the importance of developing diagnostic measures during both prevention and quarantine efforts as these could be used to quell emerging out breaks. With the increase in the rate of diagnostic method development, problems have arisen with the inconsistency of antigens and antibodies used to develop diagnostic test systems. The rabies virus nucleoprotein (N protein) is an important target for the immunochemical diagnosis of rabies. Thus, we created a genetic construct based on plasmid pET32 that expresses the С-terminal fragment of the rabies virus nucleoprotein. Our genetic construct was transformed into E. coli competent cells and the parameters of isolation and purification of the recombinant rabies virus nucleoprotein were optimized. We found that the fragment with the most immunogenic site on the nucleoprotein was located at position 360–389 amino acids. The molecular weight of the recombinant antigen was 42 kDa. The serum from mice immunized with fixed rabies virus reacted specifically with this recombinant C-terminal fragment of the nucleoprotein virus. The serum from mice immunized with recombinant antigen and fixed virus showed cross-reactivity at high dilution titers.

Keywords: rabies virus, nucleoprotein, recombinant antigen, genetic construct, immunogenic domain, producing strains

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Author

page: 53-60 pp
DOI: 10.11134/btp.3.2015.6
Tursunov K., Raiymbek G., Shustov A.V., Begalieva A., Іnіrbay B., Sadiknabi I., Mukanov K.K., Ramanculov E.M., Mukantayev K.N.
National center of biotechnology
13/5, Korgalzhyn rd., Astana, 010000, Kazakhstan
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Abstract

Bovine leukemia is an important infectious diseasethat affects farm animals. The viral etiology of the disease is well recognized. The disease causes significant economic damage to agricultural enterprises, including losses associated with the death and premature culling of high producing cows, reduced productivity, reduced milk quality, the cost of anti-leukemic measures. All these factors endanger the preservation of breeding herds, and threaten the management and breeding efforts to improve productive traits in dairy cattle.

In the present study, we determined that the optimal concentration of recombinant gp51 antigen to immobilize on a nitrocellulose membrane is 500 µg/ml. For the preparation of a colloidal gold conjugate, the optimal concentration of protein G is 8 µg/ml. The amount of colloidal gold conjugate for the membrane was 7 µl per 1 strip.

We found that the diagnostic characteristics of the immunochromatographic assay clearly differentiate the positive control sera of infected animals from sera of healthy animals, and also differentiates between serum samples with brucellosis and FMD. When determining the sensitivity of the test system, specific antibodies in the positive control sera were detected at a dilution of 1:1600.

Comparison of 145 positive serum samples in a lateral flow assay with immunoenzyme analysis and reaction immunodiffusion showed 97% agreement between the results.

Keywords: virus, leukemia, gp51, recombinant antigen, immunochromatographic analysis, reaction diffusion precipitation

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Author

page: 61-69 pp
DOI: 10.11134/btp.3.2015.7
Umarov B.R. 1, Sagdiev N.G.2, Kim A.V.2, Inagamov U.K.2
Institute of Microbiology, Academy of Sciences of Uzbekistan
7b, A. Kadyri str., Tashkent, 100128, Uzbekistan
2 A.S. Sadykov Institute of Bioorganic Chemistry, Academy of  Sciences of the Republic of Uzbekistan

83, M. Ulugbek str., Tashkent, 100125, Uzbekistan
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Abstract

The local strain of Trichoderma harzianum В1 produces cellulolytic enzymes when cultured submerged in a medium containing 2% wheat bran as the sole carbon source. Chromatography systems using different carriers and different matrices, including Sephadex G-75, P-60 Akrileks, and ion exchange chromatography on DEAE 650M gel Toyperl, were used to isolate three isoforms of endo-1,4-β-glucanase (EC 3.2.1.4), EG 1, EG 2, and EG 3 and determined a molecular weight (MW) of 35±1 kDa. The isolates exhibited a cellulolytic activity of 90.4, 77.52, and 78.92 U/mg protein. In addition, we isolated four isoforms of cellobiase (1.3-β-glucosidase, EC 3.2.1.21) CBH 1, CBH 2, CBH 3, and CBH 4 with aMW 24±1 kDa, and a cellulolytic activity of 2.60, 3.80, 4.3, and 3.0 U/mg protein. The influence of the temperature, pH, and metal ions on the activity was determined for the cellulolytic enzymes. The optimal pH and temperature of endo-1,4-β-glucanase and cellobiase (1,3-β-glucosidase) was observed to bepH 4.8 and 50°C. The effect of the monovalent and divalent metal ions K, Na, Fe, Mg, and Mn in a buffered medium increased the enzymatic activity of 1,4-β-glucanase by10%, and of 1,3-β-glucosidase by 15%.

Keywords: Trichoderma harzianum В1, cellulolytic enzymes, endo-1,4-β-glucanase, 1,3-β-glucosidase

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