Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells
Background: Coagulation factor 7 (FVII) is a vitamin-k-dependent serine protease with an essential role in initiation of extrinsic pathway of coagulation cascade. Extrinsic pathway is the major mechanism of clot formation in physiologic conditions. Congenital deficiency of FVII is a recessively inhe...
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Isfahan University of Medical Sciences
2017-11-01
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| Series: | مجله دانشکده پزشکی اصفهان |
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| Online Access: | http://jims.mui.ac.ir/index.php/jims/article/view/7914 |
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| author | Amir Mashayekhi Shirin Shahbazi Mirdavood Omrani |
| author_facet | Amir Mashayekhi Shirin Shahbazi Mirdavood Omrani |
| author_sort | Amir Mashayekhi |
| collection | DOAJ |
| description | Background: Coagulation factor 7 (FVII) is a vitamin-k-dependent serine protease with an essential role in initiation of extrinsic pathway of coagulation cascade. Extrinsic pathway is the major mechanism of clot formation in physiologic conditions. Congenital deficiency of FVII is a recessively inherited bleeding disorder caused by F7 gene mutations. The use of recombinant FVII is a therapeutic intervention for treatment of FVII deficiency and hemophilia diseases. The first step of production of recombinant FVII is cloning of F7 gene in an appropriate expression vector and transfection of the vector into cells capable of efficient processing of the expressed protein.
Methods: Complete human F7 cDNA was isolated from HepG2 cell using real-time polymerase chain reaction (RT-PCR) method. The cDNA was inserted into pcDNA3.1/neo expression vector and CHO-K1 cells were transiently transfected with the resulting construct. After transfection, in order to study the expression of recombinant FVII, conditioned medium of the cells was collected and coagulant activity and antigen level of FVII was assessed using one-stage PT-based and the enzyme-linked immunosorbent assay (ELISA) methods, respectively.
Findings: The recombinant FVII was expressed in CHO-K1 cells successfully and had appropriate coagulant activity.
Conclusion: In this study, we produced functional recombinant coagulation factor 7 with coagulant activity. This suggests that the pcDNA3.1/neo is a suitable vector for expression of FVII protein in mammalian cells and CHO-K1 cells exert correct and efficient post-translational processes on FVII protein and can be used to produce recombinant FVII protein. |
| first_indexed | 2024-03-12T08:34:01Z |
| format | Article |
| id | doaj.art-9432672456d945bf98e7641095883c26 |
| institution | Directory Open Access Journal |
| issn | 1027-7595 1735-854X |
| language | fas |
| last_indexed | 2024-03-12T08:34:01Z |
| publishDate | 2017-11-01 |
| publisher | Isfahan University of Medical Sciences |
| record_format | Article |
| series | مجله دانشکده پزشکی اصفهان |
| spelling | doaj.art-9432672456d945bf98e7641095883c262023-09-02T17:23:10ZfasIsfahan University of Medical Sciencesمجله دانشکده پزشکی اصفهان1027-75951735-854X2017-11-0111114511502630Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian CellsAmir Mashayekhi0Shirin Shahbazi1Mirdavood Omrani2PhD Student, Department of Medical Genetics, School of Medical Sciences, Tarbiat Modares University, Tehran, IranAssociate Professor, Department of Medical Genetics, School of Medical Sciences, Tarbiat Modares University, Tehran, IranProfessor, Department of Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IranBackground: Coagulation factor 7 (FVII) is a vitamin-k-dependent serine protease with an essential role in initiation of extrinsic pathway of coagulation cascade. Extrinsic pathway is the major mechanism of clot formation in physiologic conditions. Congenital deficiency of FVII is a recessively inherited bleeding disorder caused by F7 gene mutations. The use of recombinant FVII is a therapeutic intervention for treatment of FVII deficiency and hemophilia diseases. The first step of production of recombinant FVII is cloning of F7 gene in an appropriate expression vector and transfection of the vector into cells capable of efficient processing of the expressed protein. Methods: Complete human F7 cDNA was isolated from HepG2 cell using real-time polymerase chain reaction (RT-PCR) method. The cDNA was inserted into pcDNA3.1/neo expression vector and CHO-K1 cells were transiently transfected with the resulting construct. After transfection, in order to study the expression of recombinant FVII, conditioned medium of the cells was collected and coagulant activity and antigen level of FVII was assessed using one-stage PT-based and the enzyme-linked immunosorbent assay (ELISA) methods, respectively. Findings: The recombinant FVII was expressed in CHO-K1 cells successfully and had appropriate coagulant activity. Conclusion: In this study, we produced functional recombinant coagulation factor 7 with coagulant activity. This suggests that the pcDNA3.1/neo is a suitable vector for expression of FVII protein in mammalian cells and CHO-K1 cells exert correct and efficient post-translational processes on FVII protein and can be used to produce recombinant FVII protein.http://jims.mui.ac.ir/index.php/jims/article/view/7914Coagulation factor VIIRecombinant proteinsHepG2 cellsCloning |
| spellingShingle | Amir Mashayekhi Shirin Shahbazi Mirdavood Omrani Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells مجله دانشکده پزشکی اصفهان Coagulation factor VII Recombinant proteins HepG2 cells Cloning |
| title | Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells |
| title_full | Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells |
| title_fullStr | Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells |
| title_full_unstemmed | Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells |
| title_short | Expression of Functional Recombinant Human Coagulation Factor VII Protein in Mammalian Cells |
| title_sort | expression of functional recombinant human coagulation factor vii protein in mammalian cells |
| topic | Coagulation factor VII Recombinant proteins HepG2 cells Cloning |
| url | http://jims.mui.ac.ir/index.php/jims/article/view/7914 |
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