[Induction of heme oxygenase -1 by lipocalin 2 mediated by NF-kB transcription factor]

Effect of lipocalin 2 on the expression of heme oxygenase I, II and NF-kB transcription factor was the purpose of this survey. Lcn2 was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing lipocalin 2. The presence of lipocalin 2 gene in these cells was confirmed by using through RT-PCR and western blot analysis. Expression of Lcn2 was also down-regulated by siRNA in A549 cell line. Expression of heme oxygenase I, II and NF-kB transcription factor were determined in both ectopic expression Lcn2 cells and Lcn2 down regulated cells by using of RT-PCR and western blot analysis. The results showed that the expression of heme oxygenase I and those of NF-kB were higher in cells expressing recombinant lipocalin2 compared with the control cells. On the other hand, expression of heme oxygenase I and NF-kB in siRNA transfected cells was down-regulated. These findings indicate that lipocalin2 induces the expression of HO-1 and suggest Lcn2 through NF-kB induces HO-1 expression

Isolation, cloning and expression of recombinant human FVII in baculovirus expression system

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

[Isolation and detection of RhD antigen from red blood cell [RBC] membrane by immune- precipitation method]

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