[Home ] [Archive]   [ فارسی ]  
:: Main :: About :: Current Issue :: Archive :: Search :: Submit :: Contact ::
Main Menu
Home::
Journal Information::
About the Journal
Editorial Board
Aims& Scopes
Journal News
Articles archive::
All Issues
Current Issue
For Authors::
Call for Papers
Submission Instruction
For Reviewers::
Reviewers Section
Subscription::
Registration Information
Subscription Form
Contact us::
Contact Information
Contact us
Site Facilities::
Site map
Search contents
FAQ
Top 10 contents
Inform to friends
Webmail::
::
Search in website

Advanced Search
..
Receive site information
Enter your Email in the following box to receive the site news and information.
..
Journal Citation Index

 

Citation Indices from GS

AllSince 2019
Citations91005498
h-index3825
i10-index238139

 
..
Central Library of Kurdistan University of Medical Sciences
AWT IMAGE
..
Vice-Chancellery for Research and Technology
AWT IMAGE
..
SCImago Journal & Country Rank
:: Volume 28, Issue 2 (Scientific Journal of Kurdistan University of Medical Sciences 2023) ::
SJKU 2023, 28(2): 12-27 Back to browse issues page
Bicistronic and Stable Expression of Human Coagulation Factor IX and Enhanced Green Fluorescent Protein in Suspension-Adapted Chinese Hamster Ovary Cells
Fahimeh Ghasemi1 , Fatemeh Nikumanesh2 , Alireza Zomorodipour 3
1- Assistant professor, Cell and molecular research center, Birjand university of medical sciences, Birjand, Iran
2- Assistant professor,Infectious Diseases Research Center, Birjand University of Medical Sciences, Birjand, Iran
3- Professor, Department of Molecular Medicine, Institute of Medical Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB),Tehran, Iran. , zomorodi@nigeb.ac.ir
Abstract:   (828 Views)
Background and Aim: The current treatment for hemophilia B is replacement therapy, which involves the intravenous infusion of human coagulation factor IX (hFIX) purified from plasma or a recombinant form produced in mammalian cells. In this study, using a bicistronic expression system, the stable expression of the hFIX in a serum-free and suspension-adapted Chinese hamster ovary cell line (CHO-s) was investigated.
Materials and Methods: A DNA fragment consisting of hFIX, Internal Ribosome Entry Site (IRES) and Enhanced Green Fluorescent Protein (EGFP) nucleotide sequences was cloned into pcDNA3.1 expression plasmid under the control of Cytomegalovirus (CMV) promoter. The bicistronic plasmid was then linearized using BglII restriction enzyme and transfected into CHO-s cells. The transfected cells were treated with geneticin for 14 days. The culture medium of the stable cells was then collected and the expression level of the hFIX were examined using western blotting and ELISA. The coagulation activity was also evaluated by the chromogenic method.
Results: The recombinant CHO-s cells resistant to geneticin were observed under a fluorescence microscope in green color, which indicated the expression and accumulation of the EGFP in the cytoplasm of the cells. The results of Western blotting confirmed the expression and secretion of the hFIX into the culture medium. The amount of the secreted hFIX was 150 ng/mL/106cells with a coagulation activity of 5.6 ±0.2 mU/mL.
Conclusion: Our findings demonstrated that this bicistronic expression system could simultaneously produce EGFP and hFIX in CHO-s cells. This expression system facilitates selection and isolation of hFIX-expressing cells.
Keywords: Human coagulation factor IX, Chinese hamster ovary cell, Enhanced green fluorescent protein, Bicistronic expression.
Full-Text [PDF 886 kb]   (215 Downloads)    
Type of Study: Original Research | Subject: Biotechnology
Received: 2020/10/1 | Accepted: 2021/12/28 | Published: 2023/05/28
References
1. 1.Giangrande P. Haemophilia B: christmas disease.Expert Opin Pharmacother. 2005;6(9):1517-24. [DOI:10.1517/14656566.6.9.1517] [PMID]
2. Camerino G, Grzeschik KH, Jaye M, De La Salle H, Tolstoshev P, Lecocq JP,et al. Regional localization on the human X chromosome and polymorphism of the coagulation factor IX gene (Hemophilia B locus). Proc Natl Acad Sci USA. 1984;81(2):498-502. [DOI:10.1073/pnas.81.2.498] [PMID] []
3. Ghasemi F, Zomorodipour A, Karkhane AA, Khorramizadeh MR. In silico designing of hyper-glycosylated analogs for the human coagulation factor IX. J Mol Graph Model.2016;68:39-47. [DOI:10.1016/j.jmgm.2016.05.011] [PMID]
4. Chia J, Louber J, Glauser I, Taylor S, Bass GT, Dower SK, et al.Half-life-extended recombinant coagulation factor IX-albumin fusion protein is recycled via the FcRn-mediated pathway. J Biol Chem. 2018;293(17):6363-73. [DOI:10.1074/jbc.M117.817064] [PMID] []
5. Franchini M, Frattini F, Crestani S, Sissa C, Bonfanti C.. Treatment of hemophilia B: focus on recombinant factor IX. Biologics. 2013;7:33-38. [DOI:10.2147/BTT.S31582] [PMID] []
6. Kundu RK, Sangiorgi F, Wu LY, Kurachi K, Anderson WF, Maxson R, et al. Targeted inactivation of the coagulation factor IX gene causes hemophilia B in mice. Blood. 1998;92(1):168-74. [DOI:10.1182/blood.V92.1.168.413k06_168_174] [PMID]
7. Franchini Mannucci PM. Past, present and future of hemophilia: a narrative review. Orphanet J Rare Dis. 2012;7(1):1-8. [DOI:10.1186/1750-1172-7-24] [PMID] []
8. Santagostino E, Negrier C, Klamroth R, Tiede A, Pabinger-Fasching I, Voigt C,et al.Safety and pharmacokinetics of a novel recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in hemophilia B patients. Blood. 2012;120(12):2405-11. [DOI:10.1182/blood-2012-05-429688] [PMID] []
9. Sankar AD, Weyand AC, Pipe SW. The evolution of recombinant factor replacement for hemophilia. Transfusion and Apheresis Science. Transfus Apher Sci. 2019;58(5):596-600. [DOI:10.1016/j.transci.2019.08.010] [PMID]
10. Jayandharan GR, Srivastava A. Hemophilia: genetics, diagnosis and treatment.J Genet Syndr Gene Ther. 2011;1:1-12. [DOI:10.4172/2157-7412.S1-005]
11. White II GC, Beebe A, Nielsen B. Recombinant factor IX. Thromb Haemost. 1997;78(1):261-5. [DOI:10.1055/s-0038-1657536]
12. Zacchi LF, Roche-Recinos D, Pegg CL, Phung TK, Napoli M, Aitken C,et al.Coagulation factor IX analysis in bioreactor cell culture supernatant predicts quality of the purified product. Communications Biology. 2021;4(1):1-19. [DOI:10.1038/s42003-021-01903-x] [PMID] []
13. Windyga J, Solano Trujillo MH, Hafeman AE. BAX326 (RIXUBIS): a novel recombinant factor IX for the control and prevention of bleeding episodes in adults and children with hemophilia B.Ther Adv Hematol. 2014;5(5):168-80. [DOI:10.1177/2040620714550573] [PMID] []
14. Pegg CL, Zacchi LF, Recinos DR, Howard CB, Schulz BL. Identification of novel glycosylation events on human serum-derived Factor IX. Glycoconjugate Journal. 2020;37(4):471-83. [DOI:10.1007/s10719-020-09922-2] [PMID]
15. Zhong D, Smith KJ, Birktoft JJ, Bajaj SP. First epidermal growth factor-like domain of human blood coagulation factor IX is required for its activation by factor VIIa/tissue factor but not by factor XIa. Proc Natil Acad Sci. 1994;91(9):3574-8. [DOI:10.1073/pnas.91.9.3574] [PMID] []
16. Ghasemi F, Khorramizadeh MR, Karkhane AA, Zomorodipour A. Studying the Expression Efficiencies of Human Clotting Factor IX Analogs, Rationally Designed for Hyper-glycosylation. Iran J Pharm Res. 2021;20(2):523-535.
17. Nielsen FS, Schmidt AS, Kristensen AK, Nielsen AD, Kristensen BK, Palm L. Characterisation of recombinant factor IX before and after GlycoPEGylation. Int J Pharm. 2020;588:119654. [DOI:10.1016/j.ijpharm.2020.119654] [PMID]
18. Samis JA, Ramsey GD, Walker JB, Nesheim ME, Giles AR. Proteolytic processing of human coagulation factor IX by plasmin. Blood. 2000;95(3):943-51. [DOI:10.1182/blood.V95.3.943.003k34_943_951] [PMID]
19. Tuddenham EG, Cooper DN. The molecular genetics of haemostasis and its inherited disorders Oxford University Press, USA; 1994.
20. Kim JY, Kim YG, Lee GM, CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol. 2012; 93(3):917-30. [DOI:10.1007/s00253-011-3758-5] [PMID]
21. Croset A, Delafosse L, Gaudry J-P, Arod C, Glez L, Losberger C,et al.Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells. J Biotechnol. 2012;161(3):336-48. [DOI:10.1016/j.jbiotec.2012.06.038] [PMID]
22. Gurtu V, Yan G, ZhangG. IRES bicistronic expression vectors for efficient creation of stable mammalian cell lines. Biochem Biophys Res Commun. 1996;229(1):295-8. [DOI:10.1006/bbrc.1996.1795] [PMID]
23. Khorshidi S, Zomorodipour A, Behmanesh M, Vatandoost J, Bos MH. Functional expression of the human coagulation factor IX using heterologous signal peptide and propeptide sequences inmammalian cell line. Biotechnol lett. 2012;37(10):1773-1781. [DOI:10.1007/s10529-015-1868-3] [PMID]
Send email to the article author

Add your comments about this article
Your username or Email:

CAPTCHA

XML   Persian Abstract   Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Ghasemi F, Nikumanesh F, Zomorodipour A. Bicistronic and Stable Expression of Human Coagulation Factor IX and Enhanced Green Fluorescent Protein in Suspension-Adapted Chinese Hamster Ovary Cells. SJKU 2023; 28 (2) :12-27
URL: http://sjku.muk.ac.ir/article-1-6344-en.html
Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Volume 28, Issue 2 (Scientific Journal of Kurdistan University of Medical Sciences 2023) Back to browse issues page
مجله علمی دانشگاه علوم پزشکی کردستان Scientific Journal of Kurdistan University of Medical Sciences
مجله علمی دانشگاه علوم پزشکی کردستان Scientific Journal of Kurdistan University of Medical Sciences
Persian site map - English site map - Created in 0.06 seconds with 45 queries by YEKTAWEB 4645