Cloning, Expression of Hirudin Gene from Leech (Hirudo orientalis) in BL21(DE3) Strain

Authors

  • Adnan Al-Badran University of Basrah, College of Science, Biology department, Basra, Iraq
  • Sabaa Al-Fadal University of Basrah, College of Pharmacy, Pharmacognosay department, Basra, Iraq

Keywords:

Hirudin, Hirudo orientalis, Real-Time PCR, DEAE Sepharose, SP Sepharose.

Abstract

The use of leeches in bloodletting therapy is one of the most important techniques in ancient medicine from Greek time up to now. Leeches saliva contain anticoagulant hirudin which facilitate blood flow, this is one reason for using leeches to save thousands of severed fingers, noses and ears in recent years. In this study, hirudin gene successfully cloning and expressed in BL21(DE3) strain. Leech type was detected as H. orientalis by using two types of genes 18SrDNA and CO-I genes, Hirudin gene was amplified by specific primers from H. orientalis cDNA. Furthermore, Hirudin gene was expressed in BL21(DE3) strain under the control of T7 promoter in pET-16b vector, constructed vector pET-16-HR vector was extracted and used to amplify hirudin gene by specific primers, then hirudin gene band was appeared after digestion of extracted plasmid with Hind III and NdeI restriction enzyme. Hirudin expression was established by Real-time PCR. Production of hirudin established in LB medium and purified by IMAC column, DEAE Sepharose and SP Sepharose. Concentration of produced hirudin within its solution was measured by ELISA kit which reached to 1.35ng, thrombin titration method was used to determine hirudin activity which showed Hirudin protein required 360µl from thrombin for clot formation.

References

[1] Apakupakul, K., Siddall, M. E. and Burreson, E. M. Higher Level Relationships of Leeches (Annelida: Clitellata:Euhirudinea) Based on Morphology and Gene Sequences. Molecular Phylogenetics and Evolution , 1999, 12(3):1–10. ]
[2] Petrauskiene, L., Utevska, O. and Utevsky, S. Can different species of medicinal leeches (Hirudo spp.). interbreed?. Invertebrate biology, 2009,128(4):324-331
[3] Abdullah, S., Dar, L.M., Rashid, A. and Tewari, A. Hirudotherapy/Leech therapy: Application and Indications in Surgery Archives of Clinical Experimental Surgery, 2012,1(3):172-180
[4] Patel, R. K . Hirudin-Thrombin complex : An Approach To Prepare A blood Compatible Surface. MSc. thesis. Department of Pharmaceutics and Pharmaceutical Chemistry. The University of Utah.1995.
[5] Greinacher, A. and Warkentin, T. E. The direct thrombin inhibitor hirudin. Thrombosis and Haemostasis, 2008, 99(5):819–829.
[6] Nisio, M. D., Middeldorp, S. and Buller, H. R. Direct Thrombin Inhibitors. The New England Journal of Medicine , 2005, 353:1028-40
[7] Mille, B., Condamines, O., Herbert, J.M., Maftouh, M., Picard, C., Dussossy, D. and Pau, B. Two-Site Immunoassay of Recombinant Hirudin Based on Two Monoclonal Antibodies.Clinical Chemistry,1994, 40(5):734-739
[8] Elliott, J. and Kutschera, U. Medicinal leeches: historical use, ecology, genetics and conservation. Freshwater Reviews,2011, 4(1):21–41.
[9] Novagen. Competent cells user manual. Novagen Catalog. 2011
[10] Sambrook, J., Frtish, E. and Maniatis, T. Molecular cloning, a Laboratory Mannual. 2nd ed. Gold Spring Harbor Laboratory Press.USA, 1989
[11] Santos, C.A., Beloti, L.L., Toledo, M.A., Crucello, A., Favaro, M.T., Mendes, J.S., Santiago, A.S., Azoni, A.R. and Souza, A.P. A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coli. Protein Expression and Purification, 2012, 82: 284-289
[12] Markwardt, F. Hirudin as an Inhibitor of Thrombin. Method Enzymolog , 1970, 69:924-932
[13] Trontelj, P. and Utevsky, S.Y. Celebrity with a neglected taxonomy: Molecular systematics of the medicinal leech (genus Hirudo). Molecular Phylogenetics and Evolution, 2005,34:616–624.
[14] Meyer, A., Todt, C., Mikkelsen, N. T. and Lieb, B. Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity. Biomed central evolutionary biology, 2010, 10:70
[15] Wu, S., Xiong, J. and Yu, Y. Y. Taxonomic resolutions based on 18S rRNA Genes: A case study of subclass Copepoda. PLoS ONE Public library of science, 2015,10(6):1–19
[16] Hebert, P.D.N., Ratnasingham, S. and Waard, J. Barcoding animal life?: cytochrome c oxidase subunit 1 divergences among closely related species Barcoding animal life?: cytochrome c oxidase subunit 1 divergences among closely related species. Proceeding of Royal Society of London, 2003, 270: S96–S99
[17] Laufer, A. S., Siddall, M. E. and Graf, J. Characterization of the digestive-tract microbiota of Hirudo orientalis, a European medicinal leech. Applied and Environmental Microbiology, 2008, 74(19): 6151–6154.
[18] Qiagen. RNeasy® Plus Mini Handbook. 2014
[19] Padmanabhan, S., Banerjee, S. and Mandi, N. Screening of Bacterial recombinants: strategies and preventing false positive. Molecular Cloning – Selected Applications in Medicine and Biology,2011,pp1-19. Avaliable at http://www.intechopen.com/books/molecular-cloning-selected-applications-in-medicine-and-biology/screening-of-bacterial-recombinants-strategies-and-preventing-false-positives
[20] Joseph,B.C., Pichiamuthu, S., Srimeenakshi, S., Murthy, M., Selvakumar, K., Ganesan, M. and Manjunath, S. R. An overview of the parameters for recombinant protein expression in Escherichia coli. Cell Science and Therapy, 2015, 6(5): pp.1–7
[21] Promega. Subcloning Notebook. Promega Corporation. 2004
[22] Ginzinger, D. G. Gene quantification using real-time quantitative PCR?: An emerging technology hits the mainstream.Experimental Hematology, 2002, 30:503–512.
[23] Fraga, D., Meulia, T. and Fenster, S. Real-Time PCR. Current Protocols Essential Laboratory Techniques, 2014,64(2):10.3.1-10.3.40.
[24] Life Technologies. Life TechnologiesReal-time PCR handbook. Available at: http://find.lifetechnologies.com/ Global /FileLib/qPCR/ Real. 2012
[25] Bornhorst, J. A. and Falke, J. J. Purification of proteins using polyhistidine affinity tags. Methods in enzymology, 2002, (326): 245–254.
[26] Promega. HisLink™ Protein Purification Resin. Promeg Catalog. Promega corporatio. 2013
[27] Terpe, K. Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology Biotechnology,2003, 60:523–533
[28] Young, C. L., Britton, Z. T. and Robinson, A. S. Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnology Journal,2012,7: 620–634.
[29] Acikara, Ö. B. Ion exchange chromatography and its applications. Column chromatography, 2013,p 31–58.
[30] Jun, W. L., Jun, L., Bin, W., Feng, L. X., Ning, Y. S., Liang, Z. and Tong, W. W. Optimization for purification and charicterization of recombinant Hirudin III from E. coli. Chinese pharmaceutical sciences, 2005, 14(2):79-85.
[31] GEHealthcare. Ion exchange columns and media. selection guide. 2008.
[32] Chen, H. Y., Zhang, J. H., LU, J., Xu, Q. J., Qi, X. H. and Zhu, D. C. Simple and efficient methods for isolation and activity measurement of the recombinant hirudin variant 3 from Bacillus subtilis. African Journal of Biotechnology, 2011,10(44): 8889–8894.

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Published

2017-01-02

How to Cite

Al-Badran, A., & Al-Fadal, S. (2017). Cloning, Expression of Hirudin Gene from Leech (Hirudo orientalis) in BL21(DE3) Strain. American Scientific Research Journal for Engineering, Technology, and Sciences, 27(1), 25–42. Retrieved from https://www.asrjetsjournal.org/index.php/American_Scientific_Journal/article/view/2511

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