1/2014

Author

page: 4-11pp
DOI: 10.11134/btp.1.2014.1
Zhambakin K.J., Shamekova M.H., Volkov D.V., Zatybekov A.K.
Institute of Plant Biology and Biotechnology, 45, Timirjazeva str., 050040, Almaty, Kazakhstan 
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Abstract

Commercial cultivation of safflower appeal lies in its high resistance to drought and high quality of the oil obtained. At the same time, despite the fact that safflower has been known since antiquity, this culture is still poorly understood. Safflower highly varies in morphology, content and composition of the oil, depending on the region of cultivation. The review provides data on the use of safflower as a plant for multipurpose use in food industry, medicine and as a food source for animals and birds. Demand for safflower oil has been increasing over the last period due to the high level content of polyunsaturated fatty acids. For industrial applications, the role of breeding aimed at obtaining varieties with certain combination of oil, safflower oleic, linoleic fatty acids. To improve selection, applications of molecular markers in plant breeding and safflower seeds using culture cells and tissues and methods of genetic engineering are found to be promising. Molecular markers for safflower are used to study the phylogenetic relationships and the origin of the Carthamus tinctorius species, to control the commercial use of varieties - the food or the technical direction, and to identify genes associated with specific selection and valuable traits. Use of male gametophyte culture for production of homozygous lines is also promising. Application of genetic engineering techniques may result into development of new safflower varieties with high level of oleic acid and resistant to non-selective herbicide. The problems and prospects of safflower cultivation in Kazakhstan are under discussion.
Keywords: safflower, breeding, fatty acids, DNA markers, allele, oil, tissue culture, microspores, genetic engineering, and medicine.

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Author

page: 12-16pp
DOI: 10.11134/btp.1.2014.2
S. Abeldenov, B. Khassenov
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
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Abstract

Polymerase chain reaction (PCR) is a method that is used in solving of many molecular biology problems. At the beginning of PCR development the DNA-polymerase from Escherichia coli was used for amplification of DNA segments. After discovery of DNA-polymerase from Thermus aquaticus (Taq-polymerase) this enzyme due to its thermostability is widely used in PCR for DNA amplification. Taq-polymerase is highly thermostable DNA-polymerase. Nowadays this is the most commonly used enzyme in polymerase chain reaction and DNA sequencing. In this article we describe a procedure for obtaining the recombinant Taq-polymerase, including steps of cloning the gene and its expression in a heterologous environment, as well as the purification of recombinant enzyme.

Gene of Taq-polymerase was cloned into expression vector. The identity of cloned gene was confirmed by sequencing. Analysis of nucleotide sequence showed that recombinant Taq-polymerase consists of 852 amino acid residues (including 20 additional amino acids at N-terminus that contain 6xHis-tag) and has a molecular mass of 96 kDa. The recombinant protein has been purified, characterized and showed polymerase activity and thermostability.

Despite availability of a variety of commercial sources of thermostable DNA polymerases from bacteria genus Thermus, the most widely used enzyme is Taq DNA polymerase. However enzymes for molecular biology are not produced in Kazakhstan, and therefore, organization of recombinant enzymes production for use in research and diagnostic practice is an important and urgent task. The expression system and purification method used in this article allow obtaining sufficient amount of the recombinant enzyme - Taq DNA-polymerase.
Keywords: Taq DNA polymerase, polymerase chain reaction, Thermus aquaticus, recombinant protein.

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Author

page: 17-27 pp
DOI: 10.11134/btp.1.2014.3
A.T. Kulyyassov 1, G.S. Zhubanova 1, E.M. Ramankulov 1, V.V. Ogryzko 2
National Center for Biotechnology under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/1 Valikhanov str., Astana, 010000, Kazakhstan
Institut Gustave Roussy, CNRS UMR8126, 94805, Villejuif, France, 39 Rue Camilles Desmoulin
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Abstract

The development and progression of cancer is accompanied by changes in gene expression which are often can be caused by both genetic and epigenetic alterations and therefore proteins НР1α, β и γ are potential oncomarkers.

We have used method, called the Proximity Utilizing Biotinylation (PUB), based on co-expression within a single cell of the recombinant proteins - the protein of interest fused with biotin ligase BirA and his partner with the biotin acceptor peptide BAP, which allows an accurate quantitative assessment of the extent of their interaction in vivo.

The aim of this work is to develop a method for quantifying interactions in vivo of oncomarker proteins HP1α and HP1β.

In experiments on protein expression of BAP and BirA fusions of HP1α, HP1β and Tap54β in HEK293T cells we found elevated levels of biotinylation due to in vivo interaction of homologous proteins BAP-HP1 and BirA-HP1 (BAP-Tap54β and BirA-Tap54β). Signal ratio of heterologous to homologous interaction in all repeated experiments was 0,4 ± 0,14 (for samples containing BAP-HP1α) and 0,32 ± 0,08 (for samples with BAP-HP1β). Qualitative analysis by LC-MS/MS method of the gel fragments corresponding to the immunoblot bands of expressed proteins allowed to identify peptides relevant to BAP, Tap54β,   HP1α and HP1β.

Keywords: heterochromatin, euchromatin, protein-protein interactions, biotinylation, oncomarkers, biotin ligase, biotin acceptor peptide, plasmids, transient transfection, western blot, mass spectrometry.

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Author

page: 28-35pp
DOI: 10.11134/btp.1.2014.4
Danlybaeva G.A., Imanbekova M.K., Sekenova A.E.
National Center for Biotechnology, 13/1 Valikhanova str., Astana, 010000, Kazakhstan
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Abstract

The widespread using of cell cultures for scientific and applied research implicates the long-term cell cultivating process. Wherein this cell culture may have a tendency to change the basic characteristics due to as the cultivating conditions and as contamination by extraneous agents. Normally, when working with mammalian cell cultures researchers concerns about the contamination by various microorganisms (bacteria, yeasts, mycoplasma), while the cross-contamination by other types of cell cultures (both intra- and inter-species) may remain unnoticed. Purity and delicate description of the used model, which in many cases serves continuous cell cultures, are an absolute necessary condition for conducting of biomedical scientific researches, is requires their reliable identification. According to data of the International Cell Line Authentication Committee up to 50% of cell cultures used in research, misidentified or cross-contaminated. This fact is significant justification for authentication of cells and cell lines. In this paper presents the results of determining the species identity 6 new diploid strains obtained in Kazakhstan and 5 continuous cell cultures that are in the depository of the National Center for Biotechnology. Was carried out PCR with species-specific primers to various types of mammals. The experiments were carried out to optimize the conditions for PCR with the concentration of MgCl2 and primers, annealing temperature. The studies found that the optimization of the conditions to determine the presence in a sample of 10 cells/ml. Was revealed that the species-specific primers used showed full compliance with the original test cell cultures mammalian species.
Keywords: cell lines, species identity of cell lines, PCR, primer.

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Author

page: 36-42pp
DOI: 10.11134/btp.1.2014.5
Zhambakin K.J., Shamekova M.H., Volkov D.V., Zatybekov A.K.
Institute of Plant Biology and Biotechnology, 45, Timirjazeva str., 050040, Almaty, Kazakhstan
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Abstract

The incidence of people with Opisthorchiasis in Kazakhstan is one of the highest among the CIS countries. In the disease control system the main role is given to the early diagnosis. Effectiveness of the existing diagnostic methods for Opisthorchiasis is rather low. Nowadays diagnostic test-systems developed on the basis of ELISA have promising outlook. Most suitable antigen for serological diagnosing Opisthorchiasis is excretory-secretory antigen (ES-AG). Unfortunately, immunoassays based on the use of this antigen are not enough practical because there are great difficulties in obtaining standardized metabolite of parasite. In this regard, the great interest is the using antigen’s "internal image", i.e. anti-idiotypic antibodies (AIAT) against Fab-fragments that are specific to definite epitopes.

The aim of research was to obtain antibodies to idiotypes of monoclonal antibodies (Mab) specific for Opisthorchis felineus ES-AG epitope and to determine the possibility of using anti-idiotypes as an antigen in ELISA for serological diagnosis of Opisthorchiasis.

Mab 4B3D9 against helminth’s antigen have been used as immunogen for obtaining AIAT. Two hybrid strains designated as 3H10A4 and 4H10D8 producing anti-idiotypes with "internal image" of Opisthorhis felineus’ antigen  were created by hybridoma technique. Anti-idiotypes produced by hybridomas belonged to IgG1 and IgM, characterized by sufficient affinity with respect to the used immunogen. As expected, in sandwich immunoassay AIAT of both hybridomas, mimicking ES-AG epitope, were recognized by antibodies of dog, infested with parasite. The results of research suggest the possibility of using anti-idiotypes of hybridomas as specific antigen in the development of immunoassays for the diagnosis of Opisthorchiasis.

Keywords: opisthorchiasis, diagnostics, excretory-secretory antigen, ELISA, hybridoma, monoclonal antibodies, anti-idiotypic antibodies.

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Author

page: 43-48pp
DOI: 10.11134/btp.1.2014.6
K.O. Karamendin, A.I. Kydyrmanov, S.E. Asanova, A. Seidalina, E.Ja. Khan, K.D. Daulbaeva, E.T. Kasymbekov, S.A. Suleimenova, K.H. Zhumatov, M.H. Sayatov
Institute of Microbiology and Virology, 103 Bogenbaybatyr str., 050010, Almaty, Kazakhstan
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Abstract

In 2013in a poultry farm in South-Eastern Kazakhstan, an outbreak of acute infectious disease has occurred among 30-days-old domestic chickens. Positive results were obtained at screening of the collected materials in PCR and of specific bands of 132 bp were detected, indicating the presence of Newcastle disease virus RNA in samples.

After isolation of the virus in embryonatedchicken embryos, its subsequent purification, concentration and RNA extraction, sequencing of the gene encoding the fusion protein (F- protein) was performed.. In the result of sequencing, the nucleotide sequence of 999 bp was decoded, which includes the cleavage site of the fusion protein that is important to identify its pathogenicity.

The results of phylogenetic analysis of Newcastle disease virus strain isolated from sick chickens are shown on the basis of sequencing of the F- gene. It is shown that on the fusion protein gene, all the studied viruses classified in two phylogenetically distinct classes - 1 and 2. Kazakhstan APMV-1/chicken/Almaty/41/2013 strain was in a group of viruses belonging to genotype VII in Class 2. This group is formed by the APMV -1 viruses, containing polybasic amino acids KRQKR in the sequence of the fusion protein, that indicates their high pathogenicity to birds. Territory of circulation of this highly pathogenic genotype virus covers vast territory on the Asian subcontinent spaces, affecting a significant number of avian fauna. Supposition of an exogenous origin of the virus was proposed, as poultry in this farm wasimmunized with a vaccine containing lentogenic (avirulent) Newcastle disease virus strain La Sota.

It is known that the use of a vaccine containing any genotype of Newcastle disease virus protects poultry against mortality and clinical signs of disease, but the virus release into environmentwas not stopped, that may contribute to the continuous spread of infection especially in cases where the introduced virus has divergence in the genetic structurewith vaccine strain.

Keywords: paramyxovirus, Newcastle disease, PCR, primer, phylogeny, genotype, cluster.

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Author

page: 49-56pp
DOI: 10.11134/btp.1.2014.7
Mukantayev K.N., Shustov A.B., Sydyknabi I., Bigalyeva A., Raiymbek G., Mukanov K.K.
National Center for Biotechnology, Sh.Valikhanov str., 13/1, Astana, 010000, Kazakhstan
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Abstract

Glycoprotein gp51 and the main structural protein p24 leucosis virus of livestock animals are the main diagnostic antigens. In this case, the most significant part of the eresearch is devoted to obtaining recombinant antigens. As a source of gp51 and p24 antigen is a virus which the cell lines derived from fetal lamb kidney (FLK) that, chronically infected with the virus. The current method is inefficient and time-consuming, also, very low yield of the final product. In the last decades, many studies have been done on the expression of Env glycoprotein gene in heterologous systems such as Escherichia coli, Saccharomyces cerevisiae, as well as in the system of the recombinant vaccinia virus and more recently, in the baculovirus system.

In previously published work we had described recombinant gp51 antigen based on the expression vector pET32 that gene-carrying thioredoxin. However, the use of the resulting antigen gp51 + thioredoxin in diagnostic test systems proved undesirable due to significant cross-reactions to thioredoxin, which is unacceptable in the development of high-performance test systems, such as immunochromatographic or enzyme immunoassays.

In this case, the purpose of the work was to produce recombinant gp51 protein of livestock leucosis in E. coli without thioredoxin expression.

In the process used immunological, biochemical, serological and biotechnology research.

As a result of this study, the gene of hexahistidine tags was added on the 3 'end of the viral protein gp51 gene of livestock leucosis, after then, the gene was cloned into an expression vector E3.pET22. The transformation of the vector which carrying the gen of leucosis virus gp51. Identified the parameters of the gp51 protein expression strains that transformed by microorganisms and proved various analytical and immunochemical methods for expression of the desired protein.

Keywords: virus, leukemia, gp51, recombinant antigen ELISA, the reaction diffusion precipitation.

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Author

page: 57-61pp
DOI: 10.11134/btp.1.2014.8
G.D. Abisheva1, A.D. Kairzhanova1, E.S. Shevtsova1, T.B. Karibaev2, A.S. Dzhailbekova2, I.I. Sytnik2, S.B. Tjulegenov2, A.B. Shevtsov2, K.K. Mukanov1

1National Center for Biotechnology, c. Astana, 13/1 Valikhanov str., 010000, Kazakhstan
2
National Reference Center on Veterinary, 223, 150 let Abaya str., (Koktal), 010000, Kazakhstan
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Abstract

Despite significant progress in monitoring and control of infectious diseases, brucellosis remains one of the most important zoonotic infections with a vast distribution area. Efficiency of anti-brucellosis campaign depends on the effectiveness of diagnostic procedures that must be focused on early detection of infection. In Kazakhstan, serological methods are used for mass confirmatory analysis of brucellosis. These methods include complement fixation (CF) and enzyme-linked immunosorbent assay (ELISA). High infection rate among domestic animals gives rise to widespread distribution of the disease among people. The aim is to develop a PCR protocol for application in veterinary laboratories. As a result of this study, the new protocol for PCR detection of Brucella is presented in this article. Sensitivity of the PCR protocol in detection of Brucella DNA was 21 genome equivalents. Determination of the specificity of PCR by analyzing a DNA sample 78 showed its specificity level of about 100%.

Keywords: Brucellosis, PCR, diagnosis, protocol, strain, specificity.

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Author

page: 62-70pp
DOI: 10.11134/btp.1.2014.9
Y.B. Yevtykhova, D.V. Silaev, A.J. Baltabekova, A.V. Shustov
National Center for Biotechnology, 13/1 Valikhanov str., Astana, 010000, Kazakhstan
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Abstract

One of the main challenges for the Kazakhstan's livestock and fodder industry is improvement of food supply and increase in feed efficiency. A cost effective way to improve the quality of animal feed is to mobilize bound phosphate from indigestible plant components. Particular feed additives, namely phytases are able to solve this problem. Phytases are enzymes that break down the phytic acid, which is main storage form of phosphorus in plants. This paper describes a method of producing a recombinant thermostable phytase.

Phytase Nov9x is a derivative of E.coli phytase AppA with improved thermostability which is an important industrial quality. Compared to the parental phytase AppA, the Nov9x sequence has eight mutations.

Synthetic gene for recombinant thermostable phytase Nov9x was designed in silico and synthesized de novo from an array of long oligonucleotides. The assembled gene was sequence-verified. Expression constructs were obtained based on the Novagen vectors for bacterial expression, pET22 and pET32. An E.coli strain BL21(DE3) transformed with the pET22/Nov9x plasmid did not produced significant amounts of the recombinant protein. Efficient expression of the recombinant protein was observed for same strain transformed with the pET32/Nov9x plasmid. In the latter case the expression product is a fusion protein bearing a sequence of E.coli thioredoxin on the N-terminus and a sequence of phytase (Nov9x) at the C-terminus. Recombinant phytase was purified by metal chelate chromatography. The yield of purified recombinant phytase was 2,1 milligram from 200 ml of induced culture. Specific enzymatic activity of the purified phytase was 3344 FTU per 1 mg of protein.

Biochemical investigations with the recombinant phytase included determination of the pH- and temperature-dependence of activity and description of thermostability.

Obtained phytase demonstrated highest activity in a pH range of 4,5-6,0. Particularly, the recombinant phytase retains at least 20% from maximum activity at pH 1,5 corresponding to most acidic parts of the stomach. The recombinant phytase has high thermal stability: it retains 75% of activity after incubation at 60°C for 2 hours, or 29% of activity after incubation at 70°C for 30 min. Addition of protectant to the probes further improves the thermostability of phytase: in presence of 1 mg/ml gelatine the recombinant phytase retains 50% of activity after incubation at 70°C for 2 hours.

The described gene of thermostable phytase, bacterial strain transformed with expression plasmid and the recombinant enzyme, all have prospects of utilization in production of additives for the livestock and poultry fodder.

Keywords: phytase, Nov9x, enzyme, feed additive, plasmid, gene design, strain E.coli, gene synthesis.

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Author

page: 71-78pp
DOI: 10.11134/btp.1.2014.10
Kokhmetova A.M., Sapakhova Z.B., Madenova A.K., Yessenbekova G.T.
Institute of Plant Biology and Biotechnology, 45 Timiryazev str., Almaty, 050040, Kazakhstan
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Abstract

Wheat rust diseases are a major cause of yield losses of this crop. Yellow (Puccinia striiformis f. sp. tritici) and leaf (Puccinia recondita Rob.et Desm f. tritici Eriks) rusts are of the most widespread and dangerous diseases of wheat and are the major factor that adversely affects wheat yield and quality and causes considerable economic damage. Use of genetic host resistance is the most effective, economical and environmentally safe method of controlling stripe rust that allow to eliminate the use of fungicides and minimize crop losses from this disease. Due to the threat of development epiphytoties rust disease it is necessary to identify new donors of resistance to yellow and leaf rust and creation on their basis of wheat breeding material. As a result phytopathological assesses susceptibility to rust on infectious background we have selected a number of samples resistant to Puccinia striiformis and Puccinia recondita f. sp. tritici. In the present study, attention was drawn to the part of the effective resistance genes to yellow and leaf rust – Yr5, Yr10 Yr15, as well as gene complex Lr26/Sr31/Yr9/Pm8 and Yr18/Lr34, which were identified in the process of molecular screening of wheat germplasm. Using molecular markers gene Yr5 identified in 1 sample, Yr10 – in 5, Yr15 – in 2, complex of resistance genes Lr26/Sr31/Yr9/Pm8 – in 3 and gene complex Yr18/Lr34 – in 6 samples. The most valuable donors of rust resistance are cultivars Mereke 70 and Akdala with 2 identified rust resistance genes. In cv. Mereke 70 highly efficient genes Yr10 and Yr18/Lr34 and in cv. Dastan Yr10 and Yr15 were identified. The results are used in Kazakhstan to create yellow and leaf rust resistant wheat varieties using MAS breeding. Our results provide an opportunity to move the breeding process in Kazakhstan to a new scientific level through the use of technology Marker Assisted Selection.

Keywords: wheat, resistance genes, yellow rust, leaf rust, molecular markers.

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Author

page: 79-80pp
DOI: 10.11134/btp.1.2014.11
Balpanov D.S., Ten О.А., Essepbay G.E., Barbasova S.K.
Scientific and Analytical Center "Biomedpreparat”, 3, microdistrict 9, building 3, p/b 94, Stepnogorsk, Akmolinskaya oblast, 021500, Kazakhstan
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Abstract

In modern economic environment practical production of fodder in our country doesn’t have sufficient range of affordable, cheap and efficient domestic preserving additives and mixtures to improve the conditions of ensiling green mass of plants, as well as the composition and quality of the prepared silage.

The reason of the study is to obtain high-quality silage from the straw, the basis of the research are the strain of lactic acid culture Lactobacillus plantarum F-1 and the strain of propionic culture Propionibacterium acidopropioni F-5, because silage prepared together with lactic acid and propionic acid bacteria is of high quality. To achieve the goal of the research, the following work has been carried out:

The modes of cultivation of lactic culture Lactobacillus plantarum F-1 and propionic culture Propionibacterium acidopropioni F-5 have been selected.

Optimal mode of concentration has been selected.

The optimal mode of drying of biomass of lactic acid culture Lactobacillus plantarum F-1 and propionic culture Propionibacterium acidopropioni F-5 in semiindustrial conditions has been selected.

The optimal ratio of dry biomass of bacteria in the silage ferment has been selected.

Keywords: coarse stem forage, silage, strains, Lactobacillus plantarum F-1, Propionibacterium acidopropioni F-5.

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