1/2016

Author

page: - pp
DOI: 10.11134/btp.1.2016.1
Dаnlybayeva G.A., Akhmadeyeva Z.T.
National Center for Biotechnology
13/5, Korgalzhyn Road, Astana, 010000, Kazakhstan
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Abstract

Cell culture is a major tool in biomedical research that has been developing since the middle of the last century.

Healthy cells isolated from body tissues have specific requirements for the composition of artificial culture media. There are a number of synthetic growth media (for example, 199, 703 and Eagle), which contain amino acids, vitamins, and mineral salts, among other additives. Serum is added to all synthetic media, as the main source of growth factors, minerals and proteins, for the cultivation of high-grade cell cultures. In addition to standard synthetic media, there are a various specialised media for efficient propagation of cells with low proliferative potential; however, the high cost of these specialised media prohibits their use in mass-production applications.

To obtain the required amount of material for experimental or commercial purposes, cell proliferation is usually necessary. A major factor in ensuring sufficient cell proliferation is the choice of optimal culture media, which may involve the use of cell-conditioned media, among various other components. Therefore, the addition of growth factors, antioxidants or vitamins to standard media (at non-physiological concentrations) can be very attractive.

This article presents details of the effects of four antioxidants and vitamins on the viability, proliferation activity and expression of proteins in diploid cell culture. Our results reveal that addition of minimal non-toxic concentrations of vitamins and antioxidants to cell culture media has differing effects on the proliferation of continuous cell cultures, ranging from stimulation to suppression of cell proliferation.

Keywords: vitamins, antioxidants, vitamin C, retinoic acid, coenzyme Q10, Dihydroquercetin, diploid human fibroblasts, continuous cell culture MCF-7 and HeLa.

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Author

page: - pp
DOI: 10.11134/btp.1.2016.2
Zhigailov A.V., Kislitsin V.Y., Iskakov B.K.
M.Aitkhozhin Institute of Molecular Biology and Biochemistry
86, Dosmukhamedov str., Almaty, 050012, Kazakhstan
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Abstract

In eukaryotic cells, phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α), including by protein kinase R (PKR), is a basic mechanisms of protein synthesis inhibition under various stress conditions. Such phosphorylation inhibits the conversion of GDP to GTP on peIF2α, which is catalysed by eIF2B, causing rapid suppression of mRNA translation initiation. In plants, there is no biochemical or genetic equivalent of eIF2B and the mechanism of mRNA translation regulation via phosphorylation of eIF2α in plant molecular biology remains unclear.

Since there is no PKR equivalent in plants, we evaluated the use of heterologous mammalian mPKR, which is activated by double-stranded (ds)RNA, as a tool for controlled phosphorylation of eIF2α in plant systems in vitro and in vivo.
In this study, a cDNA encoding human dsRNA-activated PKR (HsPKR) was cloned into the pET23c vector. The gene was expressed in Escherichia coli cells, and recombinant HsPKR protein isolated by immobilised metal ion affinity chromatography (IMAC). The activity of HsPKR in an in vitro plant system was confirmed by evaluation of its ability to phosphorylate wheat TaeIF2α in the presence of [γ-33P]ATP, as well as by immunoblotting using antibodies against phosphorylated eIF2α.

Active HsPKR may be useful for in vitro experiments to study molecular mechanisms of mRNA translation regulation in plants through peIF2α phosphorylation, while, HsPKR cDNA has potential for use in production of transgenic plants resistant to viruses.

Keywords: eIF2 factor, phosphorylation, PKR, cloning, recombinant protein.

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Author

page: - pp
DOI: 10.11134/btp.1.2016.4
Karalnik B.V.1, Zhunussova G.B.1, Denisova T.G.1, Ponomareva T.S.2, Deryabin P.N.2, Atshabar B.B.2, Turuzhanova A.A.1, Tugambayev T.I.2, Zakaryan S.B.2
1H. Zhumatov Scientific Hygiene and Epidemiology Center
34,Makataev str., Almaty, 050002, Kazakhstan
2M. Aykimbayev Kazakh Scientific Center of Quarantine and Zoonotic Diseases
14, Kapalskaya str., Almaty, 050054, Kazakhstan
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Abstract

The aim of this study was to analyse the specificity of an immunological reagent developed to identify lymphocytes with receptors for the dominant antigen (F1) of plague bacteria (lymphocytes with F1 receptors; LfR). Rabbits were used to test immunogens, consisting of live and inactivated plague vaccine, and inhibitors of LfR. Interactions between immunological reagents, consisting of suspensions of three inactivated Yersinia species (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis); antigens (F1 and lipopolysaccharide of Y. pestis); and the immunomodulators, betaleukin (recombinant interleukin 1β) and polyoxidonium were also investigated. Intercell adhesion methods were used to detect LfR and determine their inhibition. The specificity of the reagent developed was experimentally determined using homologic and heterologic LfR antigens. The clonal nature of LfR and the development of weakly expressed, non-specific factors during the early phase of the adaptive immune response were confirmed after immunisation of the plague vaccination model, by inhibition of LfR identification using various antigens. The expression of a weak, non-specific response did not affect the specific detection of LfR, indicating the specificity of the developed immunological reagent as a clone of LfR.

Keywords: plague, early phase of adaptive response, immunological reagent, specificity.

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Author

page: - pp
DOI: 10.11134/btp.1.2016.5
Tabynov K. K, Assanzhanova N.N., Ryskeldinova Sh.Zh., Kozhamkulov Y.M., Inkarbekov D.A., Kydyrbayev Zh.
The Research Institute for Biological Safety Problems
Gvardeiskiy, Kordaiskiy rayon, Zhambulskaya oblast, 080409, Kazakhstan
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Abstract

We developed a novel live modified viral vaccine for specific prophylaxis of equine influenza virus subtype H3N8, based on the reassortant cold-adapted strain, A/HK/Otar/6:2/2010. Previously we performed all stages of vaccine development, from a battery of laboratory studies, to isolating the vaccine strain, up to evaluation of vaccine safety and effectiveness in horses. The next important step is to summarise the laboratory studies and to confirm compliance of the properties of the developed vaccine with the requirements (specification) of the normative and technical documentation (NTD) draft for vaccine preparation. The results of this study will determine the potential for use of the vaccine in compliance with the NTD for approval by the authorized state body in the field of veterinary medicine.

The process of manufacturing and quality control of the finished vaccine was carried out in accordance with an approved program, which was based on the draft NTD for the vaccine. The studies demonstrated that a laboratory series of vaccine (13,400 vials or 134,000 doses), prepared according to the developed methodology, met the requirements of the draft NTD completely with regards to quality parameters including appearance, the presence of impurities, vacuum, solubility, pH, mass fraction of humidity, sterility, infectious activity, safety and immunogenicity. The vaccine, therefore, withstood the commission trials. The results of these commission trials of a novel live modified cold-adapted viral vaccine against equine influenza will subsequently be presented for agreement and approval, according to the draft NTD.Keywords: equine influenza, vaccine, cold-adapted strain, normative and technical documentation, physical properties, safety, immunogenicity, commission trials.

Keywords: equine influenza, vaccine, cold-adapted strain, normative and technical documentation, physical properties, safety, immunogenicity, commission trials.

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Author

page: - pp
DOI: 10.11134/btp.1.2016.6
Yessenbayeva A.E., Ten O.A., Balpanov D.S.
Branch of  The National Center for Biotechnology"
21, microdistrict 7, p/o 114, Stepnogorsk, Akmolinskaya oblast, 021500, Kazakhstan
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Abstract

In the agricultural sector the VAM is a natural alternative to application of a great amount of fertilizers, primarily the phosphorous ones and can be used for the remediation of the disturbed natural ecosystems. According to the literature data the arbuscular mycorrhizae have a stimulating effect which results in a significant increase of the yield of agricultural crops. The VAM has a therapeutic effect by protecting the plants against the root pathogens. The VAM decreases a disease rate by means of the action of the following 2 mechanisms: 1) elimination of a pathogen by means of synthesis of the antibiotics or under a substrate competition; 2) induction of the immune reactions of a host plant. The VAM-fungi have a high adaptability to various environmental conditions. They develop in a wide range of the soil acidity, temperature, humidity and aeration.

The article describes the method of isolation of the vesicular-arbuscular mycorrhizal fungi from the soil samples of the northern Kazakhstan. We obtained the enrichment cultures of the VAM fungi associated with the roots of sorghum. A VAM fungi-based fertilizer was developed on the basis of the obtained cultures and tested on one of the cereal grain cultures – barley. The efficiency of the VAM is determined by a plant response to a mycorhization expressed in a yield increase.

This article describes the investigation and isolation of the VAM fungi from the soil samples of Kazakhstan.

Keywords: vesicular-arbuscular mycorrhizal fungi, VAM fungi, arbuscules, vesicles, hyphae, enrichment cultures, centrifugation, Glomus, inoculation material.

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Author

page: - pp
DOI: 10.11134/btp.1.2016.3
BulashevA.K., SuranshievZh.A.,ZhumalinA.Kh., TursunovK.А.
S. Seifullin Kazakh Agro-Technical University
2 Altynsarin Str., Astana, 010011, Kazakhstan

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Abstract

The effectiveness of brucellosis eradication measures depends on the timely diagnosis and isolation of infected animals. Commercial ELISA-kits for diagnosis of brucellosis use lipopolysaccharides (LPS) as target antigens. It is well known that use of these cell wall components can result in false-positive results due to cross-reaction of antibodies against Brucella spp. with other gram-negative bacteria. Outer membrane proteins (OMP) are of great interest, as they are specific, not only to the Brucella genus, but also for particular species. OMP extracted from Brucella abortus 19 and Brucella melitensis Rev-1 were more antigenic than LPS by agglutination and complement fixation tests using serum samples from cows positive for brucellosis. B. abortus OMP also had significantly greater diagnostic value than an antigenic recombinant protein with a molecular weight of 26 kDa (rBP26). A noticeable advantage of OMP over rBP26 was also established by serological investigation of cows vaccinated with the rough strain, B. abortus RB-51. The results suggest that in the development of ELISA-kits for diagnosis of bovine brucellosis not a single protein, but several recombinant proteins, with distinct antigenic properties should be used. Among OMP protein fractions from both Brucella species, those with the greatest potential as diagnostic antigens had molecular weights of 19 and 15 kDa, in addition to a B. melitensis protein, with a molecular weight of 12 kDa. These proteins demonstrated antigenicity in western blot analysis using serum antibodies from cows positive for brucellosis by serological tests and PCR.

Keywords: brucellosis, Brucella abortus, Brucella melitensis, outer membrane proteins, antigenicity, ELISA.

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