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 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 [30g/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<0.001]
Conclusion: Findings of the analysis of the recombinant protein expression by Baculovirus expression system indicated its production in a larger scale than similar eukaryote and prokaryote expression systems
ABSTRACT
Rh [Rhesus] is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blood, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study. Total RNAs were extracted from cord blood [O[+]]. The quality of RNA was determined by electrophoresis. In order to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The isolated RhD gene was cloned to pUCIS vector and transformed to DH5alpha. The confirmed construct was sub cloned into expression vector, pBADgIII/A, and expressed in Top 10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available human anti-RhD polyclonal antibody with peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane. The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applicable for production anti- D antibody in an animal model
Subject(s)
Fetal Blood , Gene Expression Profiling , Genetic Vectors , Isoantibodies , Models, Animal , RNAABSTRACT
Rh [Rhesus] is a highly complex blood group system in man which plays an important role in transfusion medicine. The aim of this study was the isolation of RhD protein from the membrane of RBCs. In this experimental study immunoprecipitation method with human anti-RhD polyclonal antibody was utilized for the isolation of RhD antigen from Rh[+] human blood samples Proteins of RBCs were characterized by SDS-PAGE and Western blot analysis. Antigenicity of the RhD protein was assessed by ELISA using commercially available human anti-RhD polyclonal antibody with peroxidase conjugated goat anti-human as a secondary antibody. The results show that RhD protein has successfully been isolated by immunoprecipitation method. The expected size of RhD protein was confirmed by Western blot analysis. RhD antibody reacted with RhD antigen prepared from ghost with polyclonal antibody in ELISA, but no reaction was observed in Western blot analysis with monoclonal antibody: It is necessary to mention that this is the primary report of relative purification of RhD and further studies are recommended. The RhD may be helpful to further investigate the molecular basis of RhD protein and could be applicable for production of anti-D antibody in an animal model