Mahshid Mohammadipoor; Kazem Parivar; MohammadAli Shokrgozar; Naser Masrori; Ahmad Reza Kamyab; raheleh Halabian; Mehryar Habibi Roudkenar
Volume 15, Issue 3 , September and October 2008, , Pages 152-157
Abstract
Background and purpose: Factor VII is one of the important coagulation factors in extrinsic blood coagulation pathway which can resolve the use of FVIII and FIX for hemophilia patients by activating FX. Recombinant expression of this factor can eliminate the potential problems in preparing those factors ...
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Background and purpose: Factor VII is one of the important coagulation factors in extrinsic blood coagulation pathway which can resolve the use of FVIII and FIX for hemophilia patients by activating FX. Recombinant expression of this factor can eliminate the potential problems in preparing those factors from plasma and the risk of transferring hematological diseases. Therefore the present study intended to investigate the expression of recombinant FVII at a higher level using Gateway technology and TOPO cloning. Methods and Materials: In this experimental study Factor VII cDNA was isolated from HepG2 cell line by PCR and cloned to prokaryote TOPO vector by TOPO cloning reaction. The recombinant vector was extracted for bacterial colonies after screening and was used in Gateway adapted Baculovirus DNA by LR recombination reaction. The recombinant virus was transfected onto insect cell line and the expression of the protein was analyzed after necessary screening. Findings of the protein expression via ELISA were presented in triadic (Mean ± SD); the differences across the three groups were investigated using Student t-test. Results: Cloning and recombination reaction analysis by PCR determined cloning of rFVII in high accuracy (≥90%) in the vectors. High level expression of recombinant FVII was confirmed by SDS-PAGE ELISA and Western blot analysis (30μg/ml). The highest expression level was produced on the 7th day after transfection (1.960±0.076). Determined by ELISA this result was negatively significant in the transfected sample (P