6_2020(en)

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 6, 2020
Р. 58-63, Bibliography 17, English
Universal Decimal Classification: 619:616.98-078:578.842.2:577.2.08:636.4
https://doi.org/10.15407/biotech13.06.058

DEVELOPMENT OF RECOMBINANT POSITIVE CONTROL FOR AFRICAN SWINE FEVER VIRUS PCR DETECTION

M. Kit, O. Zlenko, O. Solodiankin, V. Bolotin, A.Gerilovych

National Scientific Center “Institute of Experimental and Clinical Veterinary Medicine” of the National Academy of Agrarian Sciences of Ukraine, Kharkiv

Recombinant plasmids containing target sequences are widely used as positive controls for PCR laboratory diagnostics. The aim of the study was development of recombinant positive control containing a fragment of B646L gene of African swine fever virus. The sequence of interest encodes targets of all the PCR assays for African swine fever laboratory diagnostics recommended by World Organisation for Animal Health. A plasmid containing 1763 bp insertion was cloned in E .coli DH5? strain. After purification, the plasmid ten-fold serial dulutions were used as a positive control while PRC testing. A minimal detectable copy number was 20 copies per reaction for both conventional and real-time PCR assays. The developed plasmid could be used as a safe and effective positive control while ASF laboratory diagnostics by PCR.

Key words: African swine fever virus, molecular cloning, PCR, positive recombinant control.

References

1. Galindo I., Alonso C. African Swine Fever Virus: A Review. Viruses. 2017, 9 (5), 103. https://doi.org/10.3390/v9050103

2. Revilla Y., P?rez-N??ez D., Richt J. A. African Swine Fever Virus Biology and Vaccine Approaches. Advances in Virus Res. 2018, V. 100, P. 41–74. https://doi.org/10.1016/bs.aivir.2017.10.002

3. Listed Diseases 2020: OIE ? World Organisation for Animal Health. Available at: https://www.oie.int/animal-health-in-the-world/oie-listed-diseases-2020/

4. Beltran-Alcrudo D., Arias M., Gallardo С., Kramer S., Penrith M. L. African Swine Fever: Detection and Diagnosis – A Manual for Veterinarians. Rome: FAO. 2017, 92 p.

5. OIE World Animal Health Department. Global situation of ASF. Report № 17 (2016?2019). Retrieved October 05, 2020. https://www.oie.int/fileadmin/Home/eng/Animal_Health_in_the_World/docs/pdf/Disease_cards/ASF/Report_17._Global_situation_of_ASF.pdf

6. World Organization for Animal Health. Manual Of Diagnostic Tests And Vaccines For Terrestrial Animals, 8^th edition. Chapter 3.8.1. African Swine Fever (Infection With African Swine Fever Virus). OIE . 2019, P. 1?18.

7. Aguero M., Fernandez J., Romero L., Sanchez Mascaraque C., Arias M., Sanchez-Vizcaino J. M. Highly Sensitive PCR Assay for Routine Diagnosis of African Swine Fever Virus in Clinical Samples. J. Clin. Microbiol. 2003, 41 (9), 4431–4434. https://doi.org/10.1128/jcm.41.9.4431-4434.2003

8. Fern?ndez-Pinero J., Gallardo C., Elizalde M., Robles A., G?mez C., Bishop R., Heath L., Couacy-Hymann E., Fasina F. O., Pelayo V., Soler A., Arias M. 2012. Molecular Diagnosis Of African Swine Fever By A New Real-Time PCR Using Universal Probe Library. Transboundary And Emerging Diseases. 2013, 60 (1), 48?58. https://doi.org/10.1111/j.1865-1682.2012.01317.x

9. King D. P., Reid S. M., Hutchings G. H., Grierson S. S., Wilkinson P. J., Dixon L. K., Bastos A. D. S., Drew T. W. Development of a TaqMan® PCR assay with internal amplification control for the detection of African swine fever virus. J. Virological Methods. 2003, 107 (1), 53–61. https://doi.org/10.1016/S0166-0934(02)00189-1

10. Syzykova T. Ye., Melnikova Ye. V., Manoshkin A. V., Petrov A. A., Melnikov D. G., Pantyukhov V. B., Lebedev V. N., Borisevitch S. V. The application of external and internal control objects in case of using of polymerase chain reaction and reverse transcription of polymerase chain reaction. Mikrobiologiya. 2013, Nо 3, P. 41?44. (In Russian).

11. Chan M., Jiang B., Tan T.-Y. Using Pooled Recombinant Plasmids As Control Materials For Diagnostic Real-Time PCR. Clinical Laboratory. 2016, 62 (10), 1?11. https://doi.org/10.7754/clin.lab.2016.160114

12. World Organization for Animal Health. Manual Of Diagnostic Tests And Vaccines For Terrestrial Animals, 8^th edition. Chapter 2.2.3 Development and optimisation of nucleic acid detection assays. OIE. 2018, P. 195?205.

13. Qiagen. Critical factors for successful real-time PCR. Real-Time PCR Brochure. 2010, Nо 07, P. 1–63. https://www.gene-quantification.de/qiagen-qpcr-sample-assay-tech-guide-2010.pdf

14. James H. E., Ebert K., Mcgonigle R., Reid S. M., Boonham N., Tomlinson J. A., Hutchings G. H., Denyer M., Oura Ch. A. L., Dukes J. P., King D. P. Detection of African swine fever virus by loop-mediated isothermal amplification. J. Virological Methods. 2010, 164 (1?2), 68–74. https://doi.org/10.1016/j.jviromet.2009.11.034

15. Stegniy B. T., Gerilovych A. P., Goraychuk I. V., Solodyankin O. S., Bolotin V. I., Vovk S. I. Development of regulations for production of positive DNA control for the PCR diagnosis of ASF. Veterynarna medytsyna. 2012, Nо 96, P. 60?62. (In Ukrainian).

16. Wo?niakowski G., Fr?czyk M., Kowalczyk A., Pomorska-M?l M., Niemczuk K., Pejsak Z. Polymerase cross-linking spiral reaction (PCLSR) for detection of African swine fever virus (ASFV) in pigs and wild boars. Scientific Reports. 2017, V. 7, P. 42903. https://doi.org/10.1038/srep42903

17. Wang A., Jia R., Liu Y., Zhou J., Qi Y., Chen Y., Liu D., Zhao J., Shi H., Zhang J., Zhang G. Development of a novel quantitative real-time PCR assay with lyophilized powder reagent to detect African swine fever virus in blood samples of domestic pigs in China. Transbound Emerg. Dis. 2020, 67 (1), 284?297. https://doi.org/10.1111/tbed.13350

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ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 6, 2020
Р. 50-57, Bibliography 23, English
Universal Decimal Classification: 577.112:57.083.3
https://doi.org/10.15407/biotech13.06.050

POLYCLONAL ANTIBODIES AGAINST HUMAN PLASMINOGEN: PURIFICATION, CHARACTERIZATION AND APPLICATION

T. A. Yatsenko, S. M. Kharchenko

Palladin Institute of Biochemistry of NAS of Ukraine

The plasminogen/plasmin system plays a crucial role in fibrinolysis and regulation of cell functions in a wide range of normal and pathological processes. Investigation of plasminogen/plasmin functions requires the availability of well-characterized and effective molecular tools, such as antibodies. In the present work, the isolation and characterization of rabbit polyclonal antibodies against human plasminogen are described and approaches for the identification of plasminogen and its fragments using the purified antibodies are demonstrated. For the antibodies isolation, standard animal immunization and blood collection procedures, serum isolation, protein salting out and affinity chromatography were performed. For the antibodies characterization and application, the following methods were used: enzyme linked immunoassay (ELISA), Western blotting, FITC-protein conjugation, flow cytometry and spectrofluorometry. The obtained polyclonal rabbit anti-human plasminogen antibodies interacted with human Glu- and Lys-plasminogen, kringles 1-3 and 1-4 of plasminogen, mini-plasminogen, the heavy and light chain of plasmin. We propose the application of anti-plasminogen antibodies for the direct ELISA, Western blot analysis, and for flow cytometry and spectrofluorometric analysis of plasminogen binding with cells. The obtained anti-plasminogen antibodies are promising tools for the investigation of plasminogen/plasmin system functions, either fibrinolytic or signaling.

Key words:. plasminogen, rabbit polyclonal antibodies, affinity chromatography, ELISA, Western blotting, FITC-coupling, flow cytometry, spectrofluorometry.

References

1. Lijnen H. R. Elements of the fibrinolytic system. Ann N Y Acad Sci. 2001, V. 936, P. 226?236. https://doi.org/10.1111/j.1749-6632.2001.tb03511.x

2. Deryugina E., Quigley J. P. Cell surface remodeling by plasmin: a new function for an old enzyme. J. Biomed. Biotechn. 2012, V. 2012, Art IDn564259, 21 p. https://doi.org/10.1155/2012/564259

3. Miles L. A., Castellino F. J., Gong Y. Critical role for conversion of glu-plasminogen to Lys-plasminogen for optimal stimulation of plasminogen activation on cell surfaces. Trends Cardiovasc. Med. 2003, 13 (1), 21?30. https://doi.org/10.1016/s1050-1738(02)00190-1

4. Tykhomyrov A. A., Shram S. I., Grinenko T. V. Role of angiostatins in diabetic complications. Biomed. Khim. 2015, 61 (1), 41?56. (In Russian). https://doi.org/10.18097/pbmc20156101041

5. Miles L. A., Parmer R. J. Plasminogen Receptors: The First Quarter Century. Semin. Thromb. Hemost. 2013, 39 (4), 329–337. https://doi.org/10.1055/s-0033-1334483

6. Suelves M., L?pez-Alemany R., Llu?s F., Aniorte G., Serrano E., Parra M., Carmeliet P., Mun?oz-Ca?noves P. Plasmin activity is required for myogenesis in vitro and skeletal muscle regeneration in vivo. Blood. 2002, 99 (8), 2835?2844. https://doi.org/10.1182/blood.v99.8.2835

7. Palumbo J. S., Talmage K. E., Liu H., La Jeunesse C. M., Witte D. P., Degen J. L. Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency. Blood. 2003, 102 (8), 2819?2827. https://doi.org/10.1182/blood-2003-03-0881

8. Guti?rrez-Fern?ndez A., Gingles N. A., Bai H., Castellino F. J., Parmer R. J., Miles L. A. Plasminogen enhances neuritogenesis on laminin-1. J. Neurosci. 2009, 29 (40), 12393?12400. https://doi.org/10.1523/JNEUROSCI.3553-09.2009

9. Heissig B., Salama Y., Takahashi S., Osada T., Hattori K. The multifaceted role of plasminogen in inflammation. Cell Signal. 2020, V. 75, P. 109761. https://doi.org/10.1016/j.cellsig.2020.109761

10. Petrenko O. M., Tykhomyrov A. A. Levels of angiogenic regulators and MMP-2, -9 activities in Martorell ulcer: a case report. Ukr. Biochem. J. 2019, 91(1), 100-107. https://doi.org/10.15407/ubj91.01.100

11. Deutsch D. G., Mertz E. T. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970, 170 (3962), 1095?1096. https://doi.org/10.1126/science.170.3962.1095

12. Norrman B., Wall?n P., R?nby M. Fibrinolysis mediated by tissue plasminogen activator. Disclosure of a kinetic transition. Eur. J. Biochem. 1985, 149 (1), 193?200. https://doi.org/10.1111/j.1432-1033.1985.tb08911.x

13. Tykhomyrov A. A., Yusova E. I., Diordieva S. I., Corsa V. V., Grinenko T. V. Production and characteristics of antibodies against k1-3 fragment of human plasminogen. Biotechnol. acta. 2013, 6 (1), 86?96. (In Ukrainian). https://doi.org/10.15407/biotech6.01.086

14. Guidelines for the Production of Polyclonal and Monoclonal Antibodies in Rodents and Rabbits. Research Animal Resource Center ? Memorial Sloan-Kettering Cancer Center ? Weill Cornell Medical College. 2015, 13 p.

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18. Roka-Moya Y. M., Zhernossekov D. D., Grinenko T. V. Plasminogen/plasmin influence on platelet aggregation. Biopolym. Cell. 2012, 28 (5), 352?356. https://doi.org/10.7124/bc.000077

19. Abad M. C., Arni R. K., Grella D. K., Castellino F. J., Tulinsky A., Geiger J. H. The X-ray crystallographic structure of the angiogenesis inhibitor angiostatin. J. Mol. Biol. 2002, 318 (4), 1009?1017. https://doi.org/10.1016/S0022-2836(02)00211-5

20. Tykhomyrov A. A., Zhernosekov D. D., Grinenko T. V. Plasminogen modulates formation and release of platelet angiogenic regulators. Ukr. Biochem. J. 2020, 92(1), 31-40. https://doi.org/10.15407/ubj92.01.031

21. Radziwon-Balicka A., Moncada de la Rosa C., Zielnik B., Doroszko A., Jurasz P. Temporal and pharmacological characterization of angiostatin release and generation by human platelets: implications for endothelial cell migration. PLoS One. 2013, 8 (3), e59281. https://doi.org/10.1371/journal.pone.0059281

22. Miles L. A., Ginsberg M. H., White J. G., Plow E. F. Plasminogen interacts with human platelets through two distinct mechanisms. J. Clin. Invest. 1986, 77 (6), 2001?2009. https://doi.org/10.1172/JCI112529

23. Montague S. J., Andrews R. K., Gardiner E. E. Mechanisms of receptor shedding in platelets. Blood. 2018, 132 (24), 2535?2545. https://doi.org/10.1182/blood-2018-03-742668

24. Tangen O., Berman H. J. Gel Filtration of Blood Platelets: A Methodological Report. In: Platelet Function and Thrombosis. Advances in Experimental Medicine and Biology, vol 34. Mannucci P. M., Gorini S. (eds). Springer, New York, NY. 2012, 1 p. https://doi.org/ 10.1007/978-1-4684-3231-2_13

25. Walkowiak B., Kralisz U., Michalec L., Majewska E., Koziolkiewicz W., Ligocka A., Cierniewski C. S. Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods. Thromb. Res. 2000, 99 (5), 495?502. https://doi.org/10.1016/s0049-3848(00)00282-6

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 6, 2020
Р. 41-49, Bibliography 34, English
Universal Decimal Classification: 616.11
https://doi.org/10.15407/biotech13.06.041

PROSPECTS FOR APPLICATION OF BOVINE PERICARDIAL SCAFFOLD FOR CARDIAL SURGERY

A. A. Sokol, D. A. Grekov, G. I. Yemets, N. V. Shchotkina, A. A. Dovghaliuk, N. M. Rudenko, I. M. Yemets

State Institution "Scientific - Practical Medical Center of Pediatric Cardiology and Cardiac Surgery of the Ministry of Health of Ukraine", Kyiv

The aim of the study was to estimate the properties of the scaffold obtained by decellularization of bovine pericardium with a 0.1% solution of sodium dodecyl sulfate. The experiment included standard histological, microscopic, molecular genetic, and biomechanical methods. Scaffold was tested in vitro for cytotoxicity and in vivo for biocompatibility. A high degree of removal of cells and their components from bovine pericardium-derived matrix was shown. Biomechanical characteristics of artificial scaffold were the same as those of the native pericardium. With prolonged contact, no cytotoxic effect on human cells was observed. The biointegration of the scaffold in laboratory animals tissues was noted, which confirms the potential possibility of the implant applicationin cardiac surgery.

Key words:. scaffold, decellularization, cardioimplant, tissue engineering.

References

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6. Xiu-Fang Xu, Hai-Ping Guo, Xue-Jun Ren, Da Gong, Jin-Hui Ma, Ai-Ping Wang, Hai-Feng Shi, Yi Xin, Ying Wu, Wen-Bin Li. Effect of carbodiimide cross-linking of decellularized porcine pulmonary artery valvular leaflets. Int. J. Clin. Exp. Med. 2014, 7 (3), 649–656.

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10. Naso F., Gandaglia A. Different approaches to heart valve decellularization: A comprehensive overview of the past 30 years. Xenotransplantation. 2017, 25 (1), 1?10. https://doi.org/10.1111/xen.12354

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12. Ott H. C., Matthiesen T. S., Goh S. K., Black L. D., Kren S. M., Netoff T. I., Taylor D. A. Perfusiondecellularized matrix: using nature’s platform to engineer a bioartificial heart. Nat. Med. 2008, V. 14, P. 213–221. https://doi.org/10.1038/nm1684

13. Sarig U., Au-Yeung G. C. T., Wang Y., Bronshtein T., Dahan N., Boey F. Y. C., Venkatraman S. S., Machluf M. Thick acellular heart extracellular matrix with inherent vasculature: a potential platform for myocardial tissue regeneration. Tissue Eng. Part A. 2012, V. 18, 2125–2137. https://doi.org/10.1089/ten.tea.2011.0586

14. Zhou J., Fritze O., Schleicher M., Wendel H.-P., Schenke-Layland K., Harasztosi C., Hu S., Stock U. A. Impact of heart valve decellularization on 3-D ultrastructure, immunogenicity and thrombogenicity. Biomaterials. 2010, 31 (9), 2549–2554. https://doi.org/10.1016/j.biomaterials.2009.11.088

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ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 6, 2020
Р. 30–40, Bibliography 20, English
Universal Decimal Classification: 577.21
https://doi.org/10.15407/biotech13.06.030

EXPRESSION OF NUCLEOCAPSID VIRAL PROTEINS IN THE BACTERIAL SYSTEM OF Escherichia coli: THE INFLUENCE OF THE CODON COMPOSITION AND THE UNIFORMITY OF ITS DISTRIBUTION WITHIN GENE

E. G. Fomina, E. E. Grigorieva, V. V. Zverko, A. S. Vladyko

State Institution "Republican Scientific and Practical Center for Epidemiology and Microbiology", Republic of Belarus, Minsk

A heterologous host has got a unique expression ability of each gene. Differences between the synonymous sequences play an important role in regulation of protein expression in organisms from Escherichia coli to human, and many details of this process remain unclear. The work was aimed to study the composition of codons, its distribution over the sequence and the effect of rare codons on the expression of viral nucleocapsid proteins and their fragments in the heterologous system of E.coli. The plasmid vector pJC 40 and the BL 21 (DE 3) E. coli strain were used for protein expression. The codon composition analysis was performed using the online resource (www.biologicscorp.com). 10 recombinant polypeptides were obtained encoding the complete nucleotide sequence of nucleocapsid proteins (West Nile and hepatitis C viruses) and the fragments including antigenic determinants (Lassa virus, Marburg, Ebola, Crimean-Congo hemorrhagic fever (CCHF), Puumaravala, Hantaan, and lymphocytic choriomeningitis (LHM)). Hybrid plasmid DNAs provide efficient production of these proteins in the prokaryotic system with the recombinant protein yield varying by a factor of 8: from 5 to 40 mg per 1 liter of bacterial culture. No correlation was found between the level of protein expression and the frequency of occurrence of rare codons in the cloned sequence: the maximum frequency of occurrence of rare codons per cloned sequence was observed for the West Nile virus (14.6%), the minimum was for the CCHF virus (6.6%), whereas the expression level for these proteins was 30 and 5 mg/L culture, respectively. The codon adaptation index (CAI) values, calculated on the basis of the codon composition in E. coli, for the cloned viral sequences were in the range from 0.50 to 0.58, which corresponded to the average expressed proteins. The analysis of the distribution profiles of CAI in the cloned sequences indicated the absence of clusters of rare codons that could create difficulties in translation. A statistically significant difference between the frequencies of the distribution of amino acids in the cloned sequences and their content in E. coli was observed for the nucleocapsid proteins of the Marburg, Ebola, West Nile, and hepatitis C viruses.

Key words: recombinant nucleocapsid proteins, expression, rare codons, codon adaptation index.

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Details: Hits: 259

ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)

Biotechnologia Acta V. 13, No 6, 2020
Р. 24-29, Bibliography 46, English
Universal Decimal Classification: 612.17
https://doi.org/10.15407/biotech13.06.024

GASOMEDIATOR H₂S IN THROMBOSIS AND HEMOSTASIS

Druzhyna Nadiya

Department of Pediatrics, University of Texas Medical Branch, Galveston, USA

This review was aimed to briefly summarize current knowledge of the biological roles of gasomediator H₂S in hemostasis and cardiovascular diseases. Since the discovery that mammalian cells are enzymatically producing H₂S, this molecule underwent a dramatic metamorphosis from dangerous pollutant to a biologically relevant mediator. As a gasomediator, hydrogen sulfide plays a role of signaling molecule, which is involved in a number of processes in health and disease, including pathogenesis of cardiovascular abnormalities, mainly through modulating different patterns of vasculature functions and thrombotic events. Recently, several studies have provided unequivocal evidence that H2S reduces blood platelet reactivity by inhibiting different stages of platelet activation (platelet adhesion, secretion and aggregation) and thrombus formation. Moreover, H₂S changes the structure and function of fibrinogen and proteins associated with fibrinolysis. Hydrogen sulfide regulates proliferation and apoptosis of vascular smooth muscle cells, thus modulating angiogenesis and vessel function. Undoubtedly, H₂S is also involved in a multitude of other physiological functions. For example, it exhibits anti-inflammatory effects by inhibiting ROS production and increasing expression of antioxidant enzymes. Some studies have demonstrated the role of hydrogen sulfide as a therapeutic agent in various diseases, including cardiovascular pathologies. Further studies are required to evaluate its importance as a regulator of cell physiology and associated cardiovascular pathological conditions such as myocardial infarction and stroke.

Key words:. hydrogen sulfide, gasomediator, hemostasis, thrombosis, fibrinolysis, platelets, cardiovascular diseases.

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