[Cloning and expression of n-teminal domain of pseudomonas aeruginosa flagellin and evaluation of antibodies raised against it on motility inhibition of pseudomonas aeruginosa]

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe and lethal infections in immunocompromised individuals. This bacterium possesses a single polar flagellum. Flagellum and its subunit Flagellin play important roles in the pathogenesis of P. aeruginosa. Flagellin induces immune responses by interaction of its N-terminal domain with TLR-5. Our main aims of this study were cloning and expression of N-terminal domains of flagellin and evaluation of antibodies raised against it on motility inhibition of P. aeruginosa. The DNA sequence coding for the first 161 amino acids of flagellin was PCR amplified and cloned into a pET-28a expression vector. Recombinant protein was over expressed in BL-21[DE3], and purified by Ni-NTA resin. The immune reactivity of recombinant truncated flagellin was evaluated by Western blotting. The recombinant protein was injected into a rabbit and antibodies raised against it were evaluated for the cell motility inhibition of P. aeruginosa 8821M. The N-terminal domain of Flagellin was successfully overexpressed in Escherichia coli BL-21[DE3] host strain. Anti-native and anti-N-terminal flagellin antibodies reacted with the recombinant protein. Motility inhibition assay demonstrated that polyclonal antiserum against N-teminal flagellin is able to inhibit the motility of P. aeruginosa 8821M. The N-terminal domain of flagellin may be used for development of a new recombinant vaccine against P. aeruginosa infections

Infection of MSCs by adenovirus expression system expressing NRF2 as a cytoprotective factor

Poor viability of Mesenchymal Stem Cells [MSCs] following transplantation is one of the major challenges in their therapeutic application. Manipulation of MSCs by the genetic engineering method is one of the strategies used to protect the cells against cytotoxic microenvironment. However, maintaining multi differentiation capacity of MSCs following manipulation is important. We investigated if the manipulation of MSCs with NRF2 affects the multi differentiation capacity. MSCs were isolated from bone marrow. NRF2 was isolated and TOPO cloned into the pENTR vector. The recombinant vector was transferred into pAD/CMV/V5-DEST vector by gateway technology. Recombinant adenovirus was produced in AD293 cells, followed by being infected into MSCs. Expression of NRF2 was verified by RT-PCR. The NRF2 engineered MSCs were exposed to stress conditions followed by the evaluation of the cells viability and apoptosis. Finally, NRF2 expressing MSCs differentiation into osteoblast and adipocyte lineages was studied. NRF2 was successfully expressed in MSCs. NRF2- MSCs differentiation into osteoblast and adipocyte lineages indicating overexpression of NRF2 does not affect the differentiation property of MSCs. Expression of NRF2, a well known cytoprotective factor, by using adenovirus expression system does not intervene in the differentiation capacity of MSCs. NRF2-MSCs might be applicable for stem cell-based cell therapy in future

Effects of polygonum aviculare herbal extract on proliferation and apoptotic gene expression of MCF-7

One of the most common malignancies in women is breast cancer. Although several treatments for breast cancer are available, application of herbal medicine as a supplementary treatment is a new option to help curing the disease. In this study anticancer effects of Polygonum avicular herbal extract was investigated. Polygonum avicular extract was obtained by methanol. MCF-7 cell line was treated with different concentrations of Polygonum avicular [50, 100, 150, 200, 250, 300,350 400 ng/ micro l] for different time lengths [6, 12, 24, and 48 hrs]. MTT assay and Flow Cytometry were used to evaluate cell proliferation and apoptosis, respectively. RT-PCR was also carried out to evaluate the expression of apoptotic genes. Results showed that Polygonum avicular induced cytotoxicity in MCF- 7 cell line at concentrations higher than 300 ng/ micro l and this was confirmed by the highest rate of cell death as measured by Trypan Blue and MTT assays. RT-PCR results showed up-regulation of P53 and down-regulation of Bcl-2 proteins which implied the ability of Polygonum avicular to induce apoptosis in MCF-7 cells and confirmed its anticancer property. Further studies are required to evaluate effects of the extract on other apoptotic genes

[Application of expired platelets in the preparation of platelet gel and study of proliferative effects of expired platelet derived growth factor on variety of cell lines]

There is growing evidence indicating that growth factors derived from platelets can be used in wound healing. This study aimed to investigate whether old platelets can be used as the main material for preparation of platelet gel and as substitute for FBS and FCS in cell culture medium. In this exprimental study, platelets were prepared from voluntary blood donors by centrifugation. To prove the hypothesis that the platelet gel and the growth factor derived from expired platelets are able to propagate different cells, platelet derived factors were prepared from both new and expired platelet-rich plasma. The concentration of platelet-derived growth factors was measured by ELISA and cell proliferation was measured by MTT assay. The results showed the high quality of platelet gel obtained from old platelets. Our results also revealed that old platelets released growth factors similar to those released by new platelets. The growth factors derived from old and new platelets had the same proliferation effects on MSC, CHO, and Fibroblast cell lines Old platelets released the same growth factors that new platelets did; this showed that old platelets as valuable constituents of blood are cost effective to be used

[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

[Effect of growth factors of platelet gel on proliferation and differentiation of mesenchymal stem cells]

Mesenchymal stem cells [MSCs] are bone marrow populating cells, which posses an extensive proliferation potential. In isolation and expansion protocols for clinical scale production of MSCs, fetal bovine serum [FBS] is used as a supplement with potential risk for infections as well as immunological reactions. Autologous platelet gel is made from a natural component of the patient's own blood. Activated platelets release growth factors are mitogenic for MSCs. In vitro studies have indicated that concentration of growth factor varies according to platelet concentration, methods of preparation and mechanism of platelet growth factors release. The aim of our study was to investigate the effect of platelet growth factors on the proliferation and differentiation of human mesenchymal stem cells. Mononuclear cells of bone marrow were collected in 10% FBS growth medium. The expanded cells were characterized by flow cytometric analysis of specific surface antigens. Analysed markers included CD45, CD34, CD166, CD105, CD90, and CD44. The gel is formed by adding calcium and thrombin to platelet rich plasma [PRP]. Treated PRP was incubated for 30 min, 6, 24, 48 and 72 hours in incubator. Growth factors concentrations in supernatants were determined by ELISA. Human mesenchymal stem cells were cultured in the complete medium that supplemented with 10% FBS or Platelet growth factors for 8 days. The rate of proliferation was evaluated by MTT assay. Expanded cells were seeded on calcium phosphate scaffold. Cells growth and morphology on scaffold were analyzed by SEM. Isolation and expansion of MSCs in the complete medium supplemented with platelet growth factors were successful and morphology of cells was compatible with that of FBS. Cells were highly positive for CD90, CD166, CD44 and CD105 and negative for CD34, CD45. There was no significant difference between expression of markers on cells expanded with platelet growth factors and FBS. We demonstrated that platelet growth factors provide a significantly higher proliferative effect on MSCs than those of FBS. MSCs cultured in the presence of growth factors maintain their osteogenic differentiation properties. Osteogenic differentiation was indicated by deposition of mineralized matrix stained with Alizarin red and increased expression alkaline phosphates. Platelet growth factors can be used in place of FBS to provide a safer and more effective culture condition to expand MSC for clinical purposes. MSCs cultured in the presence of platelet growth factors maintain their osteogenic properties

Expression of green fluorescent protein [GFP] using in vitro translation cell free system

One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. pIVEX2.3-GFP plasmid was cloned to E. coli and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification

Isolation, cloning, expression and purification of recombinant RhD antigen from cord blood

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

Presence of antioxidant in vitro maturation medium and its effects on glutathione level, spindle area and rate of in vitro fertilization

Effect of different doses of cysteamine on rate of in vitro maturation [IVM], in vitro fertilization [IVF] and glutathione [GSH] level was studied. Metaphase II [MII] spindle area was analyzed for quantification of shape and size of oocytes. Female mice were primed with 5 IU of pregnant mare's stimulating gonadotrophin. Germinal vesicle [GV] oocytes were retrieved 48 hrs later. IVM medium was supplemented with 0, 50, 100, 200 and 500 mM of cysteamine. For IVM and IVF assessment in each group, 150 GV oocytes were used. Experiments also included a group of ovulated oocytes [matured in vivo] after priming with pregnant mare's stimulating gonadotrophin and human chorionic gonadotropin. GSH level was measured by 5, 5-Dithio-bis [2nitrobenzoic acid] DTNB-GR recycling protocol in GV and MII oocytes. For IVF, MII oocytes were inseminated with mature mouse sperm and rate of two-cell embryo was measured. For immunocytochemistry of microtubule and chromosomes, MII oocytes were fixed by methanol and immunostained with alpha-and beta-microtubule antibody and Hoechst. The spindle area was then analyzed. A dose-dependent improvement was observed in IVM and IVF rate. MII development and two-cell embryo formation were increased significantly in group which received 200 micro m cysteamine compare to the control group. GSH level was increased in presence of cysteamine in group which received 200 micro m cysteamine. Spindle area was increased in all groups in vitro except for the group which received 500 micro m cysteamine. The difference between spindle area in 200 micro m cysteamine and in vivo group was not significant [P>0.05]. Administration of cysteamine improves IVM and IVF rate in a dose-dependant manner. Also cysteamine induces glutathione synthesis in MII oocyte and improves microtubule when administered at a dose of 200 micro m. Therefore, addition of cysteamine as an antioxidant can improve IVM and IVF rate by increasing of oocytes quality

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