2/2016

Author

page: - pp
DOI: 10.11134/btp.2.2016.1
Urazaliyev К.R.
Kazakh Institute of Agriculture and Plant Growing
1 Erlepesov Str., Almalybak, Karasajskiy raiyon, Almatinskaya obl.,040909, Kazakhstan
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Abstract

A major feature distinguishing plants from bacteria and animals is the variety of synthetic processes in plants, which result in a broad range and variety of compounds, many of which are biologically active. Thus far, more than 30 000 such compounds have been identified across various chemical classes, and every year this number is increasing.

In recent years, plant cell and tissue culture has advanced to the extent that sufficient plant matter can be produced and economically important secondary compounds can be produced in amounts greatly exceeding that of their naturally occurring content in plants. The way in which these valuable chemical compounds are obtained, including plant cell and tissue culture methods, significantly impact the economic viability of the production of such compounds. To develop an economical production method, different techniques were compared: the use of immobilised cells and enzymes, semi-continuous and continuous systems, higher plant cells for biotransformation, and two-stage cultivation methods, and increased growth rates of the cultured cells and yields of secondary compounds from cultured tissue and cells. This comparative study was conducted by applying a variety of techniques, including chemical and physical mutagenesis, cloning of individual lines, the use of differentiated cultures, and optimisation of growth conditions, where the primary aim was to develop cheaper technologies for the production of secondary compounds from cultured cells and tissues of higher plants.

Keywords: secondary metabolites, cell and tissue culture, biologically active compounds.

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Author

page: - pp
DOI: 10.11134/btp.2.2016.2
Kozhamkulov U., Kairov U., Yerezhepov D., Akhmetova A., Molkenov A., Akilzhanova A.
Nazarbayev University, National Laboratory Astana
53, Kabanbai Batyr Avenue, 010000, Astana, Kazakhstan
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Abstract

Tuberculosis (TB) remains a major cause of morbidity and mortality in many countries and thus, represents a major public health problem in Kazakhstan and globally. An alternative and more promising option in the fight against this disease is the application of genetic diagnostic methods, for example, the detection of point mutations or other genetic determinants of TB drug resistance and genotyping of M. tuberculosis. Whole-genome sequencing and next-generation sequencing (NGS) technologies have developed rapidly in recent decades and are now applied in many branches of biomedical research. New NGS technologies allow us to study whole-genome sequences of pathogens associated with various infectious diseases, to study their molecular and genetic features, and thus, to understand these pathogens on a fundamental and practical basis in terms of microbiology, virology, and epidemiology.

This paper describes the application of high-performance “shotgun” next-generation sequencing, using a Roche 454 GS FLX + Titanium platform to determine the complete genome sequences of twenty clinical isolates of M. tuberculosis with different drug sensitivity profiles. The paper furthermore defines the quality indicators of genome sequencing, describes the de novo assembly of the complete genomes of M. tuberculosis isolates, and details the mapping and alignment to the reference genome of M. tuberculosis (H37Rv strain; GenBank database). The assembled genomes of two M. tuberculosis isolates were deposited in the NCBI GenBank database and are available for public access under the numbers AZBA00000000 and AZAZ00000000. The other genomes are available on request.

Keywords: whole-genome sequencing, tuberculosis, Mycobacterium tuberculosis, DNA, pyrosequencing, genome

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Author

page: - pp
DOI: 10.11134/btp.2.2016.3
IskakovaA.N.,1 Zholdybayeva E.V.2
1Al-Farabi Kazakh National University
71, al-Farabi ave., Almaty 050040, Kazakhstan
2National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

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Abstract

Introduction. Different drug classes in addition to lifestyle adjustments are used to reduce glucose level. Class of dipeptidyl peptidase-4(DPP4) inhibitors is one of them.

The aim of our study was to compare the glucose-lowering efficacy of DPP4 inhibitors among different populations.

Methods. MEDLINE database has been searched. Randomized controlled trials were included in the analysis if there were comparison of DPP4 inhibitors vs. placebo in a form of monotherapy or combination therapy with treatment duration of at least 12 weeks. Other criteria were information on ethnicity, baseline glycated hemoglobin (HbA1c) levels, fasting plasma glucose (FPG) levels and its levels after therapy, or HbA1c and FPG data compared to the baseline.

Results. Of the 527 publications identified from the databases, 22 studies were included in the analysis. The difference between "Japan", "China", "Korea" and "Europe" subgroups was -0.84% ​​(95% CI -1.07, -0.62; I2 = 65.2%) which was statistically significant (p<0.05). The FPG value difference between the subgroups was -1.07 mmol/L (95% CI -1.26, -0.87; I2 = 83.6%) with a statistically significant probability (p <0.05).

Conclusion. HbA1c-lowering efficacy of DPP4 inhibitors is higher in the Japanese population than in Korean, while FPG-lowering efficacy higher in the Korean population when compared to the Japanese and Chinese populations.

Keywords: meta-analysis, DPP4 inhibitors, gliptins, HbA1c, FPG, type 2 diabetes.

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Author

page: - pp
DOI: 10.11134/btp.2.2016.4
Omasheva M.Y., Pozharskiy A.S., Maulenbay A.D., Ryabushkina N.A., Galiakparov N.N.
Institute of Plant Biology and Biotechnology
45, Timiryazev str., 050040, Almaty, Kazakhstan
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Abstract

The Kazakhstan territory belongs to the Middle Asian centre of origin and diversity of apple species. Despite having favourable agro climatic conditions, Kazakhstan represents only 20% of the internal apple market. Modern international requirements for selection and zoning include both phenotypic and genetic characterisation of varieties, which depends on genetic analysis resources. In the present study, molecular-genetic passports for 31 Kazakhstani apple varieties were made for the first time. Samples were collected from five different gardens and analysed using 16 simple sequence repeat (SSR) markers. Established genotypic properties of the varieties (passports) verified information gathered by selectors regarding parentage of the majority of the varieties. The genotyping furthermore allowed for individual discrepancies of samples from certain varieties to be identified. Three samples of the Alexander Aport apple were, for example, genetically identified as hybrids of the Aport variety and an unknown variety. The analysed varieties were tested for the presence of alleles associated with resistance to various aggressive pathogens: apple scab, powdery mildew, and fire blight. Nine markers were used for apple scab, two for fire blight, and five for powdery mildew. The Maksat, Saltanat, and Maximus varieties, which bear the genes of resistance to scab and fire blight, were identified as the most promising varieties for further selection. The Kazakhstani varieties Rashid’s Aport, Bes Zhuldyz, Voshod, Zhana Tan, and Maximus were found to harbour valuable genes for long-term storage, which also makes them an appealing choice for selection.

Keywords: Malus domestica, apple varieties, genotyping, SSR markers, PCR

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Author

page: - pp
DOI: 10.11134/btp.2.2016.5
Baltabekova A.Zh., Shagyrova Zh.S., Kim Y.G., Voikov M.S.,Zhiyenbay E., Shustov A.V.
National Center for Biotechnology
13/5, Korgalzhyn Road, Astana, 010000, Kazakhstan
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Abstract

Venezuelan equine encephalomyelitis virus (VEE) is a well-studied member of the genus Alphavirus in the family Togaviridae. The VEE virus is a popular vector for gene delivery in cell culture as well as for heterologous expression of recombinant proteins in cultures of vertebrate and insect cells. All VEE RNA synthesis occurs in the cytoplasm and during the course of recombinant protein expression from a gene cloned into the VEE genome, a number of molecular processes are excluded, which may otherwise represent a bottleneck in the expression system. Complementary DNA (cDNA) representing the full-length VEE genome was assembled and cloned into an Escherichia coli plasmid under the control of the SP6 RNA polymerase promoter. The gene encoding GFP was engineered into the VEE genome under the control of a synthetic copy of a viral 26S subgenomic RNA promoter. The RNA transcript of the recombinant virus was transfected into BHK-21 cells and by 36 h post-transfection, almost all cells in the culture exhibited bright GFP fluorescence. Titres of the virus produced in the transfected culture were measured for 5 days and found to be 8.4*105 TCID50 24 h post-transfection and 2.9*108 TCID50 by 3 days post-transfection.

This article illustrates the basic steps of constructing a VEE-based vector and furthermore, demonstrates the rescue of the recombinant VEE variant engineered to produce model recombinant protein GFP. The VEE vector may be applicable in biopharmaceutical production in Kazakhstan.

Keywords: alphavirus, eukaryotic expression, vector, transient expression, virus rescue, complementary DNA (cDNA)

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Author

page: - pp
DOI: 10.11134/btp.2.2016.6
Assanzhanova N.N., Kydyrbayev Zh.K., Ryskeldinova Sh.Zh., Kassenov M.M., Khairullin B.M., Tabynov K.K.
Research Institute for Biological Safety Problems
Gvardeiskiy, Kordaiskiy rayon, Zhambylskaya oblast, 080409, Republic of Kazakhstan
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Abstract

The development of the technological stages of manufacturing an experimental, inactivated seasonal trivalent influenza split vaccine in the Republic of Kazakhstan is presented here. Master and working seed lots of the A/NIBRG-121хр (H1N1), A/NYMC X-217 (H3N2), and B/NYMC BX-49 (type B) strains were obtained for manufacturing a seasonal influenza vaccine. The purification and concentration of the three influenza virus strains were optimised and achieved by flow-through centrifugation on a linear sucrose gradient (0-60%). The optimal modes of inactivation were achieved using β-propiolactone at a final concentration of 0.1% and the subsequent splitting was carried out using the detergents Triton X-100 and Tween-80. The purified and inactivated virus concentrates were subjected to technological quality control at each production stage, as well as to stability testing. Quality control was carried out in accordance with regulations approved by the biological industry for the production and vaccines control for health care. For the first time in the Republic of Kazakhstan, a pilot batch of seasonal trivalent influenza split vaccine was produced from the NIBRG-121xp, A/NYMC X-217 (H3N2), and B/NYMC BX-49 strains. Quality-control results demonstrated that the prepared batch of vaccine meets National Pharmacopeia of Republic of Kazakhstan and European Pharmacopeia requirements and is suitable for further clinical trials.

Keywords: seasonal influenza virus, cultivation, purification and concentration, inactivation, splitting, stabilization, vaccine

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Author

page: - pp
DOI: 10.11134/btp.2.2016.7
Taylakova E.T., ChervyakovaO.V.
The Research Institute for Biological Safety Problems
Gvardeiskiy, Kordaiskiy rayon, Zhambylskaya oblast, 080409, Kazakhstan
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Abstract

Vaccination is an effective means of preventing the dissemination of infectious diseases and therefore, improvement of existing prophylactic measured is urgent. Recombinant proteins with antigenic and immunogenic properties have been widely used in recently developed prophylactic preparations. The objective of this study was to generate recombinant proteins of the sheep pox virus for the development of a subunit SPV vaccine, since outbreaks of the infection have been more frequently reported in the republic and threaten the progress of animal husbandry. In this study, the conditions for the expression of the SPV genes sppv-95 and sppv-141 in a bacterial system were optimised. It was shown that recombinant protein expression levels were optimal after incubation at 37°С for 4 h, following induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for gene sppv-95, and at 25°С and 0.25 mM IGPT for gene sppv-141. The expression of the target recombinant proteins was confirmed by immunoblotting using serum against polyhistidine. The recombinant proteins were shown to interact in serological tests with antibodies to SPV, thus confirming the retention of their specific antigenic activity following expression in a prokaryotic system. The resulting recombinant proteins will be used in the development of prophylactic preparations.

Keywords: recombinant protein, cloning, expression, induction, sheep pox virus

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Author

page: - pp
DOI: 10.11134/btp.2.2016.1
Mussakhmetov A., Turgimbayeva A., Khassenov B.
National center for biotechnology
13/5,Korgalzhyn road, Astana, Z05K8D5, Kazakhstan
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Abstract

Human interferon-gamma (IFN-γ) is an important immune component that protects against pathogens and tumours. The IFN-γ cytokine, the only member of the type II interferon family, is produced predominantly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and by Th1 CD4 and CD8 cytotoxic T lymphocyte (CTL) effector T cells upon the development of antigen-specific immunity. The recombinant production of IFN-γ has significant practical value. Here, we describe the production of recombinant human IFN-γ, the gene of which (hIFNG) was efficiently expressed in E. coli. The hIFNG gene was assembled from synthetic fragments, amplified by PCR, and subsequently cloned into several plasmid vectors. The target gene was cloned into recombinant vectors both with and without a hexa-histidine tag. The Rosetta2 (DE3) E. coli strain was used as the host strain for recombinant protein expression and high levels of synthesis of the recombinant human IFN-γ were achieved by the optimisation of induction conditions. The optimal concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) was found to be 0.005 mM. The recombinant protein was shown to accumulate in the E. coli cells as inclusion bodies, and highly pure recombinant human IFN-γ was obtained by multi-stage extraction with chaotropic agents. Purification was carried out by a combination of anion and cation exchange chromatography using Q- and SP-sepharose.

Keywords: Interferon-gamma, cytokine, recombinant protein

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