3/2017

Author

page: 3-12 pp
DOI: 10.11134/btp.3.2017.1
Imanbekova M.K. 1, Stybayeva G.S. 1,2, Revzin A. 2, Zholdybaeva E.V. 1
1National Center for Biotechnology,
13/5, Korgalzhyn road, Astana,010000, Kazakhstan
2Department of Physiology and Biomedical Engineering, Mayo Clinic,
200 1stStreet SW, St-11-14, Rochester, MN, 55905

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Abstract

Aptamers are synthetic nucleic acids developed to bind specific targets, such as amino acids, drugs, proteins, and other molecules. In nature, aptamers exist as riboswitches, which are genetic regulatory elements. Aptamers can be generated using a method called SELEX, an iterative process of adsorption, separation, and amplification, followed by sequencing of a specific oligonucleotide. The entire SELEX process takes between a week to a month to complete and consists of 8–20 rounds. Aptamers are an excellent alternative to monoclonal antibodies, which are expensive and immunogenic. Advantages that aptamers offer over antibodies include stability, low cost, lack of toxicity and immunogenicity, minimized batch-to-batch variation, and the ability to develop them against a variety of targets. The ease in which aptamers can be synthesised and modified allow for the creation and application of aptamers for use in various fields of biology and medicine, including diagnostics, targeted drug delivery, discovery of new biomarkers, biovisualization, and molecular therapy.

Keywords: aptamer, SELEX, aptasensors, diagnostics, targeted delivery, bioimaging.

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Author

page: 13-20 pp
DOI: 10.11134/btp.3.2017.2
Iskakova A.N.1, Aitkulova A.M.1, Sikhayeva N.S.1,2, Romanova A.A.1, Maratkyzy L.3, Akanov Zh. A.3, Zholdybayeva E.V.1
1National Center for Biotechnology,
13/5,Korgalzhyn road, Astana, 010000, Kazakhstan
2L.N. Gumilyov Eurasian National University,
5,Munaitpasov str., Astana, 010000, Kazakhstan
Asfendiyarov Kazakh Natoinal Medical University, Department Diabetes Center,
94,Tole bi str., 050000, Kazakhstan

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Abstract

Type 2 diabetes is a common disease worldwide. Control of blood glucose level is important for effective therapy in patients with type 2 diabetes. Inhibitors of dipeptidyl peptidase-4(DPP-4) regulate blood glucose levels by increasing incretin hormone activity by blocking the enzyme DPP-4. Many drugs have negative side effects resulting inadverse drug reactions, which can ultimately lead to ineffective therapy. Drugs do not always work the same way for everyone, yet many currently available drugs are designed as “one size fits all” solution. Predicting who will benefit from a medication and who will not respond at all, or who will experience negative side effects is difficult. Pharmacogenomics is the study of how a person’s genes affect their response to drugs. Here, we identify and examine molecular genetic markers affecting the efficiency of DPP-4 inhibitor therapy. We identified CYP1A2 rs762551 and UGT2B7 rs7668258 to be associated with type 2 diabetes risk. Furthermore, we showed that CYP1A2 rs762551 and UGT2B7 rs7668258 can be usedas prognostic markers for complications, and as sensitivity markers for DPP-4 inhibitor therapy. Moreover,  ABCB1 rs1128503 and NAT2 rs1041983 were identified as molecular markers of DPP-4 treatment effectiveness and NAT2rs1041983 is responsible for the cardioprotective effects of DPP-4 inhibitors, irrespective of therapy effectiveness.

Keywords: DPP4 inhibitors, SNP, diabetes mellitus, incretins.

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Author

page: 21-24 pp
DOI: 10.11134/btp.3.2017.3
Zhumabek A.T., Zhylkibayev А.А., Kairzhanova А.D., Eskendirova S.Z., Manabayeva Sh.А., Shevtsov А.B.
National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

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Abstract

Chlamydia is a significant disease in animals and birds. Chlamydia results in important economic damage by causing the death of animals, pathology of reproductive organs, spontaneous abortion, and the bearing of dead offspring. The prevalence of chlamydia pathogens in nature among domestic and wild animals, including birds, presents a constant threat for people professionally engaged in agriculture. Great attention is paid to diagnosis when performing anti-epizootic activities associated with the prevention and elimination of chlamydia. According to the instructions of the World Organization for Animal Health (OIE), cell culture is the main diagnostic method for the identification of chlamydia causative agents. However, the duration and laboriousness of cell culture methods, along with the risk of staff infection, make laboratory diagnostics very difficult. In guidance to OIE diagnostic tests and vaccines prescribed the use of more than five modifications of PCR test systems for the diagnosis of chlamydia. A large proportion of animals with pathological Chlamydia have Chlamydia abortus. Here, we describe how we designed and synthesized a plasmid containing the conserved C. abortus outer membrane protein A (ompA) gene. After two-stage PCR synthesis, we obtained a 357bp long amplicon consisting of the C. abortus ompA gene flanked by BamHI and SacI restriction sites. The ompA gene was then cloned into thepUC57 plasmid, which was subsequently used to transform competent cells. Sequencing of transformant clones resulted in the identification and selection of a clone containing the ompA gene. The plasmid vector described here, containing the C. abortus ompA nucleotide sequence, can be used as a positive control in the development of PCR test systems for the diagnosis of animal chlamydiosis and for testing the sensitivity of PCR protocols.

Keywords: vector, ompA gene synthesis, animal chlamydia, Chlamydia abortus, PCR test system.

 

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Author

page: 25-32 pp
DOI: 10.11134/btp.3.2017.4
Kairzhanova A.D. 1, Karibaev T.B. 2, Shvedyuk V.B. 1, Tyulegenov S.B. 2, Zharova M.K. 2, Shevtsova E.S. 1, Kuibagarov M.A. 1, Shevtsov А.Б. 1
1National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2National Reference Center for Veterinary Medicine
22/3, 150 let Abaya str., Astana, 010000, Kazakhstan

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Abstract

Determining the genetic stability of the causative agent of anthrax requires analysis of highly variable regions of DNA, including tandem repeats. Analysis of 25 loci with tandem repeats (MLVA-25) is a highly discriminating method used to genotype field strains of Bacillus anthracis, and allows the geographical distribution of genotypes to be tracked. This method can be easily applied to control the genetic stability of vaccine strains and strain identification. However, to date, there is no data about the genotypes of MLVA-25 vaccine strains used for vaccination in countries of the Commonwealth of Independent States. Here, using MLVA-25, we genotyped vaccine strains of B. anthracis STI-1 and B. anthracis 55-VNIIVViM that were deposited by three individuals from Kazakhstan. MLVA profiles of the individual vaccine strains obtained were identical, irrespective of the source. B.anthracis STI-1 and B. anthracis 55-VNIIVViM differed only in a single tandem repeat at the pXO1aat locus. Comparison of MLVA profiles obtained in this study with those obtained by in silico analysis of whole genome data revealed a discrepancy at the Bam22 and Bam23 loci in both B. anthracis STI-1 and B. anthracis 55-VNIIVViM, and at the Bam5 and Bam24 loci of B. anthracis STI-1.This study shows that MLVA analysis is highly discriminatory and can be used for quality control analysis of vaccine preparations. Moreover, standardization of the procedure for commercial application would require the analysis of typical crops.

Keywords: Bacillus anthracis, MLVA typing, VNTR alleles, vaccine strains, anthrax.

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Author

page: 33-43 pp
DOI: 10.11134/btp.3.2017.5
Amirgazin A.O.1, Shvedyuk V.B.1, Kuibagarov M.A. 1, Karibaev T.B.2, Shevtsov A.B.1
1National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
2National Reference Center for Veterinary Medicine
22/3, 150 let Abaya str., Astana, 010000, Kazakhstan

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Abstract

Polymerase chain reaction (PCR) is a multifunctional tool used extensively in molecular biology. PCR is modified in numerous ways for specific purposes, all of which rely on proper efficiency, specificity and sensitivity. Despite the general acceptance of the "classical" principle, there is no unified optimization scheme that takes into account the diversity of PCR applications. Such an optimization scheme would guide researchers toward the best optimization approaches for their specific PCR application. Currently, researchers are guided only by data from previously published authors who submitted their own PCR optimization schemes. Here, we describe a PCR protocol optimization algorithm for the detection of microorganisms using Pasteurella multocida as an example. P. multocida, which we have use dextensively in the past, allows us to achieve the necessary PCR specificity and sensitivity to demonstrate the application of our algorithm. Our scheme differs from others as it uses an inductive method to learn the specificity of the protocol being developed. Our approach uses real-time PCR, with SYBR Green I, to monitor amplification during the optimization process. Furthermore, the simplicity of this approach means that it can be used to develop and optimize diagnostic PCR for a wide range of researchers and applications. As a proof of concept, PCR conditions were optimized for two pairs of primers which were tested on a collection of samples comprising DNA from 92 species of bacteria, three eukaryotic species, and with the sensitivity of at least five P. multocida genomic copy equivalents.

Keywords: PCR, optimization, Pasteurella multocida.

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Author

page: 43-48 pp
DOI: 10.11134/btp.3.2017.6
Korotetskiy I.S.1, Shilov S.V.1, Shvidko S.V.1, Jumagaziyeva A.B.1, Suldina N.A.1, Korotetskaya N.V.1, Ilin A.I.1, Reva O.N.2
1 Scientific Center for Anti-infectious Drugs,
75V al-Farabi Ave., Almaty, 050060, Kazakhstan
2Department of Biochemistry, Centre for Bioinformatics and Computational Biology, University of Pretoria,
Pretoria, South Africa

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Abstract

The aim of this study was to evaluate transcriptional regulation in a model multidrug resistant Staphylococcus aureus (MRSA) culture, S. aureus ATCC® BAA-39™, after exposure to a new antibacterial drug, FS-1. Growth media were supplemented with FS-1 at the end of the lag phase and in the middle of the exponential growth phase. To investigate the immediate effects of the iodine-containing antibacterial complex FS-1, 24 RNA samples were obtained 5 min after exposure to the drug.RNA sequencing revealed substantial changes in gene expression in the lag phase and, to a lesser degree, in the exponential growth phase. Analysis of the regulated genes suggested that FS-1 primarily targeted microbial cell wall proteins. The effect of FS-1 on S. aureus gene expression was similar, but not identical, to differential gene regulation profiles caused by several cell wall-disrupting antibiotics including bacitracin, D-cycloserine, and oxacillin. However, the gene regulation profiles were not completely congruent, suggesting that FS-1 displays a significant level of antimicrobial activity specificity. Therefore, it is possible that combining FS-1 with other anti-staphylococcal antibiotics will result in a cumulative synergetic effect.

Keywords: S. aureus, antibiotic resistance, FS-1, RNA, gene expression

 

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Author

page: 49-55 pp
DOI: 10.11134/btp.3.2017.7
Amenov A.A., Kalendar R.N., Abeldenov S.K., Musakhmetov A.S., Li P.K., Kiribayeva A.K., Khassenov B.B.
National Center for Biotechnology
13/5, Korgalzhyn road, Astana, 010000, Kazakhstan

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Abstract

Infection of cattle with bovine leukaemia virus (BLV) and Brucella spp. result in economic losses due to reduced productivity, reduced milk production, and early culling. Furthermore, BLV and Brucella spp. can potentially infect humans. This study was conducted to develop Xtreme chain reaction (XCR) and loop-mediated isothermal amplification (LAMP) assays for the rapid and efficient detection of leukaemia and Brucella spp. pathogen infections in cattle. Brucella spp. XCR and LAMP assays targeted the host specific antigen gene and IS711 repeats from the transposase gene. To detect BLV proviral DNA, the BLV long terminal repeat region and the Env gene were used to develop XCR and LAMP assays. The results of LAMP assays applied to field samples were compared with those of XCR/LAMP and serological tests for BLV and Brucella spp. The results of the XCR and LAMP assays showed a high level of agreement with those of serological methods, and accurately detected the target sequences with no cross-reactions observed. Therefore, the XCR and LAMP assays described here are highly sensitive and specific tests to detect and differentiate between BLV and Brucella spp. and could help with the detection of infection in the early stages.

Keywords: leukaemia, brucellosis, polymerase, LAMP, XCR, genome, diagnosis.

 

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Author

page: 56-60 pp
DOI: 10.11134/btp.3.2017.8
Nurmagambetova A.B.1, Akhmetova D.G.2, Khassenov B.B.1, Baltin K.K.1
1National Center for Biotechnology
13/5 Korgalzhyn road, Astana, Z05K8D5, Kazakhstan
2 Republican Diagnostic Center
HighVill B-1, 3 Akhmet Baitursynov, Astana, 020000, Kazakhstan

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Abstract

Recombinant protein A is widely used in biotechnology for the purification of immunoglobulin G antibodies. In this study, truncated protein A (protein A (5), encoding only five IgG binding domains (E, D, A, B, and C) and lacking signal sequence S and cell-wall anchoring region X M, was cloned into pMBP his parallel 2 and expressed in BL21 (DE3) cells. The gene encoding protein A was inserted downstream of the maltose binding protein (MBP) encoding malE gene. MBP is often fused with other proteins to improve their solubility, enhance stability, and increase the final yield. Proteins expressed using this system can be found in the soluble fraction, which is likely due to the solubilizing properties of MBP. A strain producing recombinant protein A (5) fused with MBP was obtained. The recombinant protein was purified and concentrated by gradient affinity chromatography. The weight of the protein fused with MBP was approximately 77.3 kDa, and without MBP was approximately 37 kDa. The IgG binding specificity of the recombinant protein was assessed using the agar gel immunodiffusion test with the target protein, bull, mouse, rat, and rabbit IgGs. The antigen antibody interaction showed a pattern not character to that described previously.

Keywords: protein A, cloning, expression, purification, spa, SpA.

 

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