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:: Volume 6, Issue 1 (Spring 2009) ::
Sci J Iran Blood Transfus Organ 2009, 6(1): 1-11 Back to browse issues page
Isolation, cloning and expression of recombinant
Mehryar Habibi Roudkenar Dr , R Halabian, N Shagerdi Esmaili, A Oodi, N Masroori, N Amirizadeh Dr, K Mousavi Hosseini Dr, A Gharehbaghian Dr, H Rezvan Dr, M.A Jalili Dr
Abstract:   (16949 Views)
Isolation, cloning and expression of recombinant human factor VII in CHO cell line Halabian R.1( MS ), Shagerdi Esmaili N.1( MS ), Oodi A.1( MS ), Masroori N.1(MS), Amirizadeh N.1(PhD), Mousavi Hosseini K.1(PhD), Gharehbaghian A.1(PhD), Rezvan H.1(PhD), Jalili M.A.1(PhD), Habibi Rudkenar M.1(PhD) 1Iranian Blood Transfusion Organization, Research Center, Tehran, Iran Abstract Background and Objectives Factor VII is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. Factor VII plays an important role in the cascade of coagulation. The aim of this study was to clone and express human recombinant factor VII in CHO cell line as an eukaryotic host cell. Materials and Methods In this descriptive study, FVII cDNA was isolated from HepG2 cell line and cloned to pcDNA 30.1(+) vector. The constructs were transfected to CHO cell line. A cell line that permanently expressed recombinant factor VII was established. The expression of recombinant FVII was determined by RT-PCR, ELISA, SDS-PAGE and western blot analysis. Biological activity of recombinant factor VII was determined by prothrombin time assay in factor FVII-depleted plasma. Results The results showed that FVII was successfully cloned and expressed. After 3 weeks stable cell lines were generated in the culture of the CHO cell line in the presence of geneticin. RT-PCR, ELISA, SDS-PAGE and western blot analysis results indicate the expression of FVII in the stable clones. A three- to four-fold decrease of the specific coagulant activity of rFVII was observed that indicates of rFVII being biologically active. Conclusions Four thousands to Six thousands vials of rFVII were imported to our country having high cost for the government. Therefore, production of rFVII through recombinant DNA technology within lab scale is the first step to overcome the problems. Key words: Hemophilia, rFVIIa, CHO, Transfection SJIBTO 2009 6(1): 1-11 Received: 21 Oct 2008 Accepted:21 May 2009 Correspondence: Habibi Roudkenar M., PhD of Biotechnology. Assistant professor of Iranian Blood Transfusion Organization-Research Center. P.O.Box: 14665-1157, Tehran, Iran. Tel: (+9821)88601599 Fax: (+9821)88601599 E-mail: roudkenar@ibto.ir
Keywords: Hemophilia, rFVIIa, CHO, Transfection
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Type of Study: Research | Subject: Hematology
Published: 2014/08/16
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Habibi Roudkenar M, Halabian R, Shagerdi Esmaili N, Oodi A, Masroori N, Amirizadeh N, et al . Isolation, cloning and expression of recombinant . Sci J Iran Blood Transfus Organ. 2009; 6 (1) :1-11
URL: http://bloodjournal.ir/article-1-297-en.html


Volume 6, Issue 1 (Spring 2009) Back to browse issues page
فصلنامه پژوهشی خون Scientific Journal of Iran Blood Transfus Organ
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