3/2014

Author

page: 4-11pp
DOI: 10.11134/btp.3.2014.1
N.S. Sikhayeva1, 2,  А.А. Iskakova1,  А.М. Aitkulova1,  Е.V. Zholdybayeva1, K.T. Momynaliev1,  Е.М. Ramanculov1
1National Centre for Biotechnology, 13/1, Valikhanov str., Astana, 000001, Kazakhstan
2L.N. Gumilev Eurasian National University, 5, Munaitpasov str, Astana, 000001, Kazakhstan
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Abstract

For understanding genetic predisposition to sports activities it is necessary to pay  attention to some genes, such as ACTN3, PPARD, LPL, EDN1, MMP3, INSIG2, PPARGC1A, APOE, SHBG, COL5A1, LEPR, LOXL1, PTPN22, TRAF1. Polymorphisms of these genes are associated with muscular strength, endurance, muscle fiber size and composition. Genetic differences in any of these genes can influence athletic performance. The allele frequency of these genes have been well studied in the European population, but the population of Central Asia has not been extensively studied. It should be noted that the allele frequency may depend on race and ethnicity. The frequency of alleles and genotypes of 19 SNPs were obtained in the Kazakh population (n = 365). The allele frequency (MAF – Minor Allele Frequency) of investigated genes for the Kazakh population were as follows: rs1815739 (С>T) 0,47; rs2016520 (T>C) 0,24; rs328 (C>G) 0,07; rs5370 (G>T) 0,29; rs679620 (С>T) 0,47; rs7566605 (C>G) 0,38; rs8192678 (С>T) 0,52; rs429358 (T>C) 0,14; rs7412 (C>T) 0,05; rs10033464 (G>T) 0,30; rs6258 (C>T) 0,18; rs12722 (C>T) 0,41; rs2025804 (A>G) 0,50; rs2165241 (C>T) 0,38; rs2200733 (C>T) 0,31; rs2476601 (G>A) 0,05; rs3761847 (A>G) 0,48; rs5934505 (T>C) 0,34; rs6457617 (T>C) 0,48.

Keywords: single nucleotide polymorphism, allele frequency, sports genetic, genotype distribution.

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Author

page: 12-21pp
DOI: 10.11134/btp.3.2014.2
A.T. Kulyyassov1, G.S. Zhubanova1, E.M. Ramanculov1, V.V. Ogryzko2
1Republican State Enterprise “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
2Institut Gustave Roussy, CNRS UMR8126, 94805, Villejuif, France, 39 Rue Camilles Desmoulin
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Abstract

Protein-protein interactions (PPI) play a key role in many processes within the cell, and their affinity and specificity are fine-tuned with respect to the functions they perform. Disturbances in the signal transduction pathways associated with PPI lead to the development and progression of cancer. Study of protein-protein interactions not only improve our understanding the mechanism of the processes inside the cell, but also help in the search of small molecule modulators of PPI for the treatment of various types of tumors.

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 its 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 HP1a and transcription factor KAP1.

In experiments on protein expression of BAP and BirA fusions of HP1a and KAP1 in HEK293T cells we found elevated levels of biotinylation due to in vivo interaction of proteins BAP-HP1a and BirA-wtKAP1. The ratio of biotinylation between BAP-HP1a in samples with BirA-mutKap1 and BirA-wtKap1 was 0,43±0,086.

In reciprocal experiments, the level of biotinylation was higher in case of interaction between proteins BAP-wtKap1 and BirA-HP1a. The ratio of biotinylation of BAP-mutKap1 and BAP-wtKap1 in the presence of BirA-HP1 was 0,2±0,04.

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

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Author

page: 22-27pp
DOI: 10.11134/btp.3.2014.3
S.A. Garbar, M.V. Fomenko, O.A. Ten, D.S. Balpanov
The Branch of «National Center for Biotechnology», the MES in Stepnogorsk, microrayon 7, building 21, p/b 114, Stepnogorsk, Akmolinskaya oblast, 021500, Kazakhstan
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Abstract

The functional food product «Biolakt», consisting of a probiotic consortium of fermented milk cultures Bifidobacterium bifidum, Lactobacillus bulgaricus and Lactobacillus acidophilus was selected as an object for research. We have conducted the studies to determine the number of viable cells, and their antagonist activity and moisture content of the product at different stages of storage.

Antagonistic activity of probiotic consortium was studied by the method of agar blocks by submerged way. The culture Staphylococcus aureus was used as the test culture.

The amount of viable cells during storage of the probiotic consortium was determined by limiting dilution on a nutrient medium Blaurock for the growth of probiotic Bifidobacterium bifidum culture and semisolid nutrient medium №1 for the growth of lactic acid cultures Lactobacillus bulgaricus and Lactobacillus acidophilus.

The study of the microbiological purity of the drug at different storage periods was determined by microscopic control and seeding on a number of culture media (MPA, MPB).

Moisture content was determined by the method of drying up to constant mass at a temperature of (100-105)°C in a laboratory baker. 

The data on the study of conservation of specific activity and stability of characteristics during the storage were submitted. .

The practical significance of the work is in the introduction of the technology of producing functional food product, preserving the biological activity within 3-6 months into production.

Keywords: lactic acid bacteria, probiotic activity, antagonistic activity, microbiological purity, probiotic consortium. 

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Author

page: 28-36pp
DOI: 10.11134/btp.3.2014.4
S.S. Bekkuzhina, S.N.Borovikov, I. Rahimbaev
1S. Seifullin Kazah Agro Technical University,62Victory Square, Astana, 010011, Kazakhstan
2Institute of Plant Biology and Biotechnology, 42 Timiryazevst., Аlmaty, 050040, Kazakhstan
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Abstract

The Article presents the world publications review on theoretical and applied aspects of haploid technologies, in particular the ways to increase pollen embryogenesis efficiency. The analytical review discusses in details the issues of donor plants hormonal pretreatment role in induction of microspore sporophytic development pattern.

Phytohormone balance is one of the key mechanisms of microspores shifting from gametophytic to sporophytic development pattern. In addition, the Article specifies the impact of various stressors on microspore reprogramming from gametophytic to sporophytic development pattern. In discussing this issue all plant pretreatments being in use are distributed into new and old pretreatment methods before in vitro introduction.The result of our experimental research is to create an effective biological system , that allows switching development of microspores from the gametophytic type to the sporophytic development type based on pretreatment of cut plants hormone of auxin cut type as - IAA, and 2,4-D with contents of endogenous IAA in plants. The developed method improves the efficiency of pollen embryogenesis to 8%. Conventional methods of pretreatment are that hormonal induction factor is added to the environment, and plants cut were subjected while pre-treatment in our experiments.

Кeywords: embryogenesis, androgynies, haploid,dihaploids pollen, gormon regulation, stress, microspores, sporophyt, gametophyte, selection.

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Author

page: 37-43 pp.
DOI: 10.11134/btp.3.2014.5
A.S. Nizkorodova, B.K. Iskakov
Institute of Molecular Biology and Biochemistry named after Aytchozhin M.A., Science Committee of Ministry of Education and Science, 86 Dosmuchamedova str., Almaty, 050012, Kazakhstan
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Abstract

One of the most promising directions in creation of transgenic cold resistant plants is constitutive expression of transgenic RNA-chaperons. RNA-chaperons are proteins that bind nucleic acids (single stranded RNA and DNA) and destabilize their secondary structures which are formed by the decrease of temperature. Cold-shock proteins (CSP) are RNA-chaperons which present in all living organisms. The main cold-shock protein of E. coli CSPA is consist of 70 amino acids and binds single stranded RNAs and DNAs nonspecifically. We have obtained several lines of transgenic tobacco Nicotiana tabacum var. Samsun constitutively expressing protein EcCSPA in cytoplasm. There were nine transgenic tobacco lines carrying transgen EcCspA (marked as “A” with number) and three transgenic lines carrying transgen EcCspA which had 5’-untranslated region (UTR) of potato virus Y upstream (marked as “YA” with number). 5’-UTR of potato virus Y is well known translational enhancer; we used it for expression enhancement of goal protein in transgenic plants. We explored tolerance of transgenic plants to chilling and freezing. Three transgenic lines demonstrated 100% survival under the conditions of cold shock (lines A-20, YA-17 and YA-20). Two of transgenic lines namely YA-20 and A-27 showed statistically valid increase in raw weight of plants green part after the cold shock. The raw weight gain was 30% (YA-20) and 57% (A-27) in compare with control plants. Thus, we constant economical expediency of creation of cold-resistant agricultural plants transgenic by EcCspA.

Keywords: cold shock proteins (CSP), EcCSPA, transgenic plants, protein expression, cold resistance.

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Author

page: 44-48 pp
DOI: 10.11134/btp.3.2014.6
I.V. Charykova, N.I. Nekrasova, D.S. Balpanov, O.A. Ten
Scientific and Analytical Center "Biomedpreparat", building 3, microdistrict 9, p/b 94, Stepnogorsk, Akmola region, 021500, Kazakhstan
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Abstract

In various natural and economic zones of Kazakhstan there live about 270 species of locusts. About 15-20 species are of great danger to agricultural lands. 

The search for natural entomopathogenic hyphomycetes active against pest species of locusts, possessing increased productivity of viable conidia and promising for the development of a biological insecticide preparation has been performed.

Fieldwork to find entomopathogenic fungi was carried out in the steppe areas of Akmola region, around Stepnogorsk city and in Kostanai region in 2012: in the foci of mass reproduction, as well as when examining the steppe zones, populated by single individuals of nongregarious locusts. The main place where dead insects were gathered was the soil in the place of concentration of nongregarious locusts.

As a result of the directional variation of productivity of the most active culture isolated from natural sources, the strain of the fungus Beauveria bassiana (Va1s.) Vuі11 B-3 which has a high potential: high activity of enzymes and virulence against larvae of the Asian migratory locust (Locusta migratoria L.), good sporulation, viability and productivity has been obtained.

Keywords: entomopathogenic fungi, screening, Beauveria bassiana, biological activity.

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Author

page: 49-53 pp
DOI: 10.11134/btp.3.2014.7
I.V. Charykova, N.I. Nekrasova, D.S. Balpanov, O.A. Ten
Scientific and Analytical Center "Biomedpreparat", Stepnogorsk, p/b 94, microdistrict 9, building 3, Akmola region, 021500, Kazakhstan
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Abstract

Fungal diseases play an important role in the spread of insects, sometimes significantly reducing their number in the wild. Biopreparations based on entomopathogenic fungi Beauveria bassiana are considered to be the most promising ones, because at optimal conditions the application of the pathogen on any site, affected by the pests, results in massive propagation and dissemination of B. bassiana, the result of which is the epizootic. In addition, a single introduction of the pathogen ensures the preservation and maintenance of the amount at the site for one season and even for several years. A wide range of entomopathogenic activity of B. bassiana and safety in relation to vertebrate animals and plants allows extensive and effective use of biopreparations based on B. bassiana in agriculture. Taking into consideration all facts, mentioned above, the study of issues related to the cultivation of entomopathogenic fungi is of particular relevance.

A modified Czapek medium has been obtained, which helps to increase the virulence of conidia of entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin strain B-3 due to the introduction of nutrient yeast broth into the medium. As a result of further modification with the introduction of salts of rare metals: potassium iodide and zinc sulfate, the virulence of conidia has significantly increased, resulting in a shorter time of the death of the test - insects.

A simplified nutrient media has been developed for practical application, consisting of sucrose, fodder yeast, sodium nitrate and salts of rare metals that enhances greater virulence of conidia of entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin strain B-3.

Keywords: entomopathogenic fungi, Beauveria bassiana, cultivation, virulence, conidia.

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Author

page: 59-65pp
DOI: 10.11134/btp.2.2014.8
A.B. Shevtsov1, A.D. Kairzhanova1, G.D. Abisheva1, E.S. Shevtsova1, D.K. Kamalova1, A.S. Dzhailbekova 2, T.B. Karibaev2, I.I. Sytnik2, A.E. Ahmetova2, K.K. Mukanov1
1National Center for Biotechnology, 13/1, Valikhanov str., (Koktal) Astana, 010000, Kazakhstan
2National Reference Center on Veterinary, 223, 150 let Abaya str., (Koktal) Astana, 010000, Kazakhstan

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Abstract

At this time, the laboratories responsible for diagnostics of campylobacteriosis and evaluation of the animal products contamination by campylobacteria use “golden standard” – extraction and identification of clean cultures. However, campylobacteria microaerophiles grow on complex growth medium and require high qualified staff. The diagnostics is also complicated due to diversity of clinical campylobacteriosis. As a result there is an underestimation of the true campylobacteriosis situation in the structure of the human and animal infectious diseases. Introduction of the PCR tests into diagnostic laboratories may simplify and increase the level of camplylobacteriosis infections diagnostics. The aim of the work was development of high PCR test for identification of species differentiation of C. coli, C. jejuni and C. fetus in clinical material and animal products. In the research the primers selected to the unique DNA markers Cc01460c for C. coli and Cj0339 for C. jejuni were used; for species differentiation of C. fetus primers for rpoB gene were selected. The conditions were optimized by real-time PCR using intercalating dye SybrGreen, which allowed evaluating the maximum efficiency on basis of minimum value of threshold cycle (Ct). As a result the protocol with optimized composition of reaction mixture and PCR amplification program was developed. Sensitivity evaluation on two-fold diluted DNA samples has allowed identifying analytical sensitivity threshold in 108 genomic equivalents for C. coli and C. fetus and 13.7 genomic equivalent for C. jejuni. The sensitivity evaluation of the PCR test on DNA of 10 camplylobacteria types, DNA of genetically related H. pylori, as well as DNA of the bacteria that cause human and animal infectious diseases similar to the clinical picture, has proved high test specificity. High analytical sensitivity and specificity allow to use the developed test in express diagnostics of campylobacteriosis as well as to analyze contamination of animal products by C. coli, C. fetus and C. jejuni.

Keywords: campylobacter, PCR diagnostics, specificity, sensitivity, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus.

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Author

page: 61-65pp
DOI: 10.11134/btp.3.2014.9
S. Abeldenov, S. Kirillov, A. Nurmagambetova, A. Kiribayeva, D. Silayev, B. Khassenov
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
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Abstract

Development of fodder and increase of feed efficiency is an urgent task for livestock and poultry in Kazakhstan. An important characteristic of feed is the content of bioavailable phosphorus. Traditional source of phosphorus in feed is the mineral phosphate. New and cost-effective way to improve the quality of feed is to mobilize bound phosphate from indigestible plant components.

Development of pig and poultry production has increased the cost of traditional sources of organic phosphorus feed (fish and meat and bone meal). At the same time, the limitations of the world's reserves of phosphorus-containing minerals will lead to the progressive increase of the price of animal feed with mineral additives.

Relatively new and cost-effective way to address the shortage of bioavailable phosphorus for livestock, poultry and fish farming is the use of feed additives - phytase. Phytase - a group of enzymes phosphohydrolases cleaving the ester bond in the molecule of phytic acid and releasing one or more residues of phosphoric acid.

In this work gene appA of Escherichia coli BL21 (DE3) was cloned into plasmid vector under T7 promoter control and was expressed resulting in high purity recombinant phytase AppA. Optimal cultivation temperature and induction conditions, cell lysis conditions, purification conditions of recombinant enzyme AppA were determined. Phytase activity assay was performed for recombinant enzyme.

Work has absolute novelty for the biotechnology industry in Kazakhstan.

Keywords: phytase, polymerase chain reaction, Escherichia coli, recombinant protein.

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Author

page: 66-71pp
DOI: 10.11134/btp.3.2014.10

A. Turgimbayeva, M. Baltabekova, A. Yelyubay, A. Ibrayeva, A. Mussakhmetov, S. Abeldenov, B. Khassenov
National Center for Biotechnology, Valikhanov str. 13/1, Astana, 010000, Kazakhstan
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Abstract

The aim of this study is to get polyclonal antibodies against GFP and mCherry recombinant proteins. Bacterial strain producers of fluorescent proteins were obtained due to transformation of Escherichia coli with the prepared expression vectors: pET-28c/gfp and pET-28c/mcherry. After 16 hours of induction GFP and mCherry were extracted by method of metal-chelate affinity chromatography from bacterial cultures. The extracted proteins possessed 98% of electrophoretic degree of purity, as well as the high concentration of the proteins in fractions provides intense glow of the proteins under ultraviolet light. Rabbits were immunized with the recombinant proteins GFP and mCherry, from serum of which corresponding immunoglobulins were purified by 35% of ammonium sulfate precipitation. The concentration of antibodies after purification was 4.4 µg/µl. The optimal dilution and the specificity of anti-GFP and anti-mCherry antibodies were confirmed by total proteins extracts of bacterial, yeast-derived and HEK293 cells.

Keywords: GFP, mCherry, recombinant proteins, polyclonal antibodies, Western-blotting.

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Author

page: 72-77pp
DOI: 10.11134/btp.3.2014.11
A.E. Esenbaeva, O.A. Ten, D.S. Balpanov
The Branch of “National Center for Biotechnology”, the MES in Stepnogorsk, microdistrict 7, building 21, p/b 114, Akmola region, 021500, Kazakhstan
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Abstract

Organic fertilizers (manure, litter and non-litter bird droppings, sapropel, etc.) help to increase the humus content in the soil and are an essential tool to restore and to improve the soil fertility. Litter bird droppings is a valuable high-performance, full fertilizer. Considering the impact on the crop yields, the substances that make up the bird droppings, can be compared with analogous substances of mineral fertilizers. Microorganisms introduced into the soil in the composition of complex fertilizers and fertilizers based on monocultures, enhance biological activity of the soil; improve the soil from plant pathogens; promote activation of plant-microbe interactions, help to obtain high yields of organic farming; enhance the recovery of microbiocenosis of the soils, upset due to the human impact. As a result of the work performed, the optimal conditions of fermentation of litter bird droppings, the effectiveness of the introduction of micro-organisms at different stages of fermentation of litter bird droppings was assessed. It is shown that the biologically active microorganisms are retained when added to mature compost. The experimental batch based on highly active strains of fungicide microorganisms Bacillus subtilis S-2 and the phosphate-mobilizing bacteria Bacillus megaterium F-1 immobilized on the fermented bird droppings is promising to be used as a mineral complex organic bio-fertilizer.

Keywords: Antifungal activity, phosphate-mobilizing activity, aerobic fermentation of the bird droppings, anaerobic fermentation of the bird droppings, immobilization, introduction, elimination.

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