4/2016

Author

page: 3-11 pp
DOI: 10.11134/btp.4.2016.1 
Khapilina O.N., Kupeshev Z.S., Danilova A.N., Kalendar R.N.
National Center for Biotechnology
13/5, Korgaldjin st., Astana, 010000, Kazakhstan
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Abstract

We present the results of our study on in vitro culture of the valuable medicinal plant Rhodiola rosea (Rhodiola rosea L.). We collected sample plant materials from the Kazakh Altai territory. The essential stages of in vitro cultivation techniques for R. rosea were optimized. We determined the most effective sterilization protocol for different types of explants of Rhodiola and optimized stages of organogenesis and formation of adventitious shoots. We found that aseptic cultures of Rhodiola necessitate the use of multi-stage sterilization protocol using different types of antiseptics. Apexes of shoots and rhizome buds were the most effective explants. High concentration of exogenous cytokinin in the culture medium induced adventitious organogenesis in R. rosea. Maximum adventitious shoots (up to 34 explants) were observed usingMSZ1 medium supplemented with zeatin, indole-3-acetic acid, and gibberellic acidat concentrations of 2, 0.1, and 0.5 mg/L, respectively. The formation of the root system wasdependent on the agar concentration in the medium. Morphometric characteristics were higher in regenerated Rhodiolagrown on a medium containing 0.6% agar.

Keywords:golden root, Rhodiola rosea L., in vitro culture, explants, adventitious organogenesis, micropropagation.

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Author

page: 12-20 pp
DOI: 10.11134/btp.4.2016.2
Kairzhanova A.D.1, Kamalova D.K.1, Abisheva G.D.1, Amirgazin A.O.1, Shvedyuk V.B.1, Popova O.A.2, Kim G.M.2, Shevtsov A.B.1
National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2PLL «Astana ECOLIFE»
22, Alash road, Астана, 010000, Казахстан

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Abstract

Male infertility plays a significant role in reproduction. Although medical therapy can successfully treat ejaculation disorder and impotence,a majority of genetic disorders remain therapeutically intractable. Y-chromosome deletions are the most common causes of male infertility. The distal end of the Y-chromosome long arm bears the locus of azoospermia factor (AZF), which carriesgenes essential for spermatogenesis. Microdeletions inthe Y-chromosome long arm are associated with spermatogenesis disorder and are the most common genetic cause of oligospermia(5-10%) and azoospermia(10-15%). In our study, we analysed microdeletions of the AZF locus, using sixSTS markers (recommended by the European Association of Andrology),two gene controls for reaction inhibition, and a Y-chromosome control.

We tested DNA samples of 138 male subjects with the developed testsystem. The distribution results of microdeletions of the Y-chromosome AZF locus were obtained for samples from the Kazakh population. Deletion of the AZF locus was reported in11% of males with oligospermia and 16% males (7 of 45 subjects)with azoospermia.

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

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Author

page: 21-27 pp
DOI: 10.11134/btp.4.2016.3
Tarlykov P.V.1, ShevtsovA.B.1, Ogryzko V.V.2, Ramanculov E.M.1
1National center of biotechnology
13/5, Korgaldjin st., Astana, 010000, Kazakhstan
2Institut Gustave Roussy
CNRS UMR 8126, 114 Rue Edouard Vaillant, 94805, Villejuif, France

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Abstract

One of the most promising areas of biology is the systematic approach to the study of chromatin, using new-generation sequencing and mass spectrometry. Our study proposes the use ofa previously developed technique, based on biotinylation of proteins that are inclose spatial proximitywith each other, for the study of DNA fragments co-precipitated during the pull-down of biotinylated proteins. We performed co-expression of the nuclear membrane protein emerin fused tobiotin ligase and hybrid histone mH2A fused to biotin acceptor (peptide specifically biotinylated by biotin ligase in the vicinity of the protein of interest). Using new-generation sequencing, we studied the chromatin in the vicinity of the nuclear envelope protein.This method has several advantages such as theability to utilize histone variants associated with specific functional states (e.g. active or repressed chromatin) and the possibility to perform pulse-chase experiments to monitor DNA sequences identified in proximity with lamin‑associated domains. Our work may throw light on the cellular mechanisms of chromatin remodelling and has important relevance for the understanding of different nuclear domains.

Keywords: emerin, chromatin, biotin-ligase, biotinylation, western-blot, sequencing, lamin‑associated domain.

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Author

page: 28-35 pp
DOI: 10.11134/btp.4.2016.4
Azhіmahan M.A.1, Varisev. U.A.2, Drenova N.V.3, Khasanov V.T.1, Dzhaymurzina A.A.4, Tuleeva A.K.1, Umiralieva Zh.Z.4
1JSC «S.Seifullin Kazakh Agro Technical University»
Pobedy, 62, Astana, 010000, Kazakhstan
2SSI All-Russian research institute of potato farming by A.G.Lorh
villageKorenevo, str. Lorch, 23, Moscow Region, 140052, Russian Federation
3SSI «All-Russian Plant Quarantine Centre»
Ramenskoye district, villageBykovo,str. Border, 32, Moscow Region, 140150, Russian Federation
4LLP «Kazakh Research Institute of plant protection and quarantine»
Karasai district, village Rakhat, str. Kazybekbi, 1, Almatyregion, 040924, Kazakhstan
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Abstract

We designed a test system for the immunofermental analysis of Erwinia amylovora, the causative agent of fire blight in fruit crops. We isolated bacteria from fruit samples cultivated in the Uigur district of Almaty, Kazakhstan. For the selection of causative agent to the studied bacterium on different nourishing environments, the best a Levanovaya environment appeared of environments further is an environment of КingaB In and potato agar. We isolated bacteria and identified their species, using real-time polymerase chain reaction (RT-PCR). Immunization of laboratory animals’ suspension of bacterial cages was carried out (anti-gene). For the receipt of specific antiserums used the strain of СFBP 1430 (Bielefeld, France, isolated в1972, author SamsonR.) from collection of E. amylovora FGBU ‘VNIIKR’.

Antiserum with a specific caption 1: 107 in ELISA was received. Erwinia amylovora specific for immunoglobulin from which they were emitted and conjugates of antibodies with high cleaning peroxidase of horseradish were prepared. The sandwich ELISA exhibited a sensitivity of 8 x 104 cells/mL, which exceeds the sensitivity of the commercial analog. Test systems were compared based on their sensitivity and specificity to various strains of E. amylovora, isolated from the Russian Federation, Kyrgyzstan, Kazakhstan, Moldova, Poland, and France, as well as other species of the genus Enterobacteriaceae from FGBU ‘VNIIKR’.The strain CFBP 1430 was used as a control.

Keywords: burn fruit crops, Erwinia amylovora, antigen, antibody, immunisation, ELISA, testsystem.

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Author

page: 36-42 pp
DOI: 10.11134/btp.4.2016.5
Kirillov S., Silayev D., Abeldenov S., KhassenovB.
National center of biotechnology
13/5, Korgaldjin st., Astana, 010000, Kazakhstan
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Abstract

Phytase, used as an animal feed additive, catalysesphytic acid hydrolysis with sequential release of phosphate and reduces feeding costs. In this work, we amplified the Escherichia coli acid phosphatase gene appA and inserted it into an expression vector pPICZαA. appA was under the control of inducible promoter of alcohol oxidase AOX1 with an α-factor signal peptide, which provides secretory protein expression. The recombinant plasmid was purified and lineariszed with the restriction enzyme PmeI, followed byits transformation into the host strain Pichia pastoris GS115,using electroporation. The transformed P.pastoris GS115 grew well on YPDS containing 100 µg/mL zeocin. PCR results confirmed the integration of appA into the genome of P. pastoris. SDS-PAGE analysis revealed that phytase was over expressed and secreted into the culture supernatant. The maximal extracellular activity and optimum temperature of phytase was reported to be 1510 U/mL and 50°C, respectively. Phytase was active at pH 2.0-7.0, with optimum activity at pH 5.0. The recombinant protein was thermostable and retained 80% of its activity after incubation at 60°C for 10 min. Protein glycosylation was confirmed with endoglycosidase H. The recombinant yeast strain P. pastoris GS115 pPICZαA/AppA exhibits potential industrial applications.

Keywords: phytase, Escherichia coli, Pichia pastoris, heterologous expression.

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Author

page: 43-50 pp
DOI: 10.11134/btp.4.2016.6
Gritsenko D.A.1,2, Kenzhebekova R.T.1, Deryabina N.D.3, Galiakparov N.N.1,2
1Institute of plant biology and biotechnology
Timiryazevstreet, 45, Almaty,050040,Kazakhstan
2Institute of plant protection and quarantine
Kazybekbistreet, 1, Almaty, 050000,Kazakhstan
3Al-Farabi Kazakh National Universit,
Al-Farabi Street, 71,Almaty,050000, Kazakhstan
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Abstract

Thermostable enzymes that lack3′ to 5′ exonuclease activity are able to add deoxyadenosine residues to 3′-overhangs in PCR-amplified products, thereby allowing direct cloning of PCR products into T-vectors. A T-vector for cloning of amplified products was created by inserting two Eam1105I restriction sites in the multiple cloning site of the plasmid pGEM‑3zf(+). The ‘excess’Eam1105I restriction site within theβ-lactamase gene was deleted by point mutation while maintaining the amino acid sequence. Addition of a spacer (282 nucleotides) between the Eam1105I sites increased the cleavage efficiency of the enzyme. The developed T-vector reduces the time and cost for cloning PCR products as compared to other methods involvingT-vector preparation with enzymatic addition of deoxythymidine to 3′-ends, primer design with suitable restriction sites, and restriction enzyme digestion of PCR products. The developed vector was used to clone the open reading frames of different sizes of grapevine virus A.

Keywords: T-vector, PCR products, cloning.

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Author

page: 51-60 pp
DOI: 10.11134/btp.3.2016.7
Manat Е., Sarina N.I., Eskendirova S.Z., Shustov A.V., Mukanov К.К.
National center of biotechnology
13/5, Korgaldjin st., Astana, 010000, Kazakhstan
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Abstract

Despite the significant progress jointly made by scientists and practical veterinary service in the Republic of Kazakhstan, the problem of animal brucellosis has not been completely solved. Consistently high levels of human brucellosis morbidity, which is due to extremely tense epizootic situation and large socio-economic damage, determine the special importance of this infection in the structure of infectious pathologies. One of the potential to increase the sensitivity and specificity of serological diagnostic methods of brucellosis is the use of recombinant analogs of immunodominant proteins of pathogenic Brucella, which diversity has determined and studied at the present time. Periplasmic protein BP26 is a specific antigen which is highly conserved for genus of Brucella. It has high diagnostic value for use in the development of immunoassays and immunochromatographic test kits of brucellosis for the veterinary and medical use.

A highly sensitive and specific immunoassay system for rapid detection of antibrucellar antibodies was developed on the basis of recombinant Brucella antigen BP2. It is intended for serological diagnosis of brucellosis in animals. High diagnostic efficiency of ICA was tested in 962 samples of blood serum of cattle and goats, sheep as well as 16 control reference sera.

Keywords: immunochromatographic assays, recombinant antigen, blood serum, antibody.

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