دوره 6، شماره 2 - ( 2-1397 )
جلد 6 شماره 2 صفحات 35-29 |
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Vatandoost J, Azimifar M A. Expression of Recombinant Factor IX Using the Transient Gene Expression Technique. Res Mol Med (RMM) 2018; 6 (2) :29-35
URL: http://rmm.mazums.ac.ir/article-1-270-fa.html
URL: http://rmm.mazums.ac.ir/article-1-270-fa.html
وطن دوست جعفر، عظیم فر محمدامین. Expression of Recombinant Factor IX Using the Transient Gene Expression Technique. Research in Molecular Medicine. 1397; 6 (2) :29-35
1- دانشگاه حکیم سبزواری ، j.vatan@hsu.ac.ir
2- دانشگاه آزاد سبزوار
2- دانشگاه آزاد سبزوار
چکیده: (2978 مشاهده)
Background: Pilot and large-scale production of recombinant proteins require the presence of stable clones, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes.
Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique.
Materials and methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEK-hFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test.
Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX.
Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and low-yield methods.
Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique.
Materials and methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEK-hFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test.
Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX.
Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and low-yield methods.
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