High folate production by naturally occurring Lactobacillus sp. with probiotics potential isolated from dairy products in Ilam and Lorestan provinces of Iran

Rapidly proliferating cells require large amounts of folate to support efficient DNA replication, repair and methylation indicating the importance of folate in human metabolism. Milk products are good sources of such vitamins which are produced by probiotics. In order to find suitable strains capable of high folate production, isolation and identification of Lactobacilli in traditional fermented milk from two different provinces located in the west of Iran were carried out. Lactobacillus  bacteria were isolated according to the ISO 7889 standard procedure. The isolated bacteria were characterized phenotypically and were screened for their ability to produce folate during fermentation of skim milk. Folate production by the selected strains was between 2.8 to 66.6 μg/l. Two strains with the highest folate production were then selected. The 16SrRNA genes from these two strains were amplified and sequenced and a phylogenetic tree constructed. The sequencing results in combination with phenotypic and biochemical properties showed that both strains were similar to Lactobacillus crustorum. Therefore, two new strains with an ability of high folate production were isolated and identified. These could be used as probiotics in the dairy industry. Hence, exploiting natural food-grade microorganisms for the production of nutritive dairy products is possible.Keywords: Folate, Lactobacillus, probiotic, traditional dairy products, IranAfrican Journal of Biotechnology Vol. 9(33), pp. 5383-5391, 16 August, 201

Development of quantitative competitive PCR for determination of copy number and expression level of the synthetic glyphosate oxidoreductase gene in transgenic canola plants

Background: For successful in vitro plant regeneration, plant cell lines with multiple transgene integration and low transgene expression levels need to be ruled out. Although real-time polymerase chain reaction (real-time PCR) is a rapid way to accomplish this, it is also expensive and typically limits the size of the target sequence. Quantitative competitive PCR (QC-PCR) is proven to be a safe and accurate method for determination of both copy number and quantification of transcript levels of synthetic transgenes in transformed plants.Results: The glyphosate oxidoreductase gene was chemically synthesized and used to transform Brassica napus L. via Agrobactrium -mediated transformation. A construct containing the mutated form of a synthetic glyphosate oxidoreductase (gox) gene (internal standard) was prepared. Gene copy number was estimated in nine independent transgenic lines using QC-PCR as well as the standard method of Southern blot analysis. By quantitative competitive reverse transcriptase PCR (QC-RT-PCR), transcript levels were also determined in these lines. High (> 3), medium to high (2.2-3), medium to low (1-2.2), and low (< 1) levels of transcript were detected. Conclusions: No direct relationship was found between copy number and transgene expression levels. QC-PCR method could be implemented to screen putative transgenic plants and quickly select single T-DNA inserts. QC-PCR methods and the prepared competitor construct may be useful for future quantification of commercial transgenic food and feed

Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

Objective(s): Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli) system and then biological activity of this protein was evaluated in animal wound healing. Materials and Methods: E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Conclusion: Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability

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Improvement of Large-scale PRP production by Haemophilus influenzae typeb, using modified CY medium

Background and Objective: Haemophilus influenzae type b (Hib) is a gram negative bacterium and one of the most common causative agents of acute meningitis in infants and less than 5 years old children worldwide. The production of Hib capsular polysaccharide; polyribosyl ribitolphosphate (PRP) is important for the production of conjugate vaccines against Hib infections. The aim of this study is the improvement of Large-scale PRP production by Hib. Materials and Methods: Haemophilus influenzae type b standard strain ATCC10211 was cultivated in 2L fermentors contain 1.5L CY (casaminoacid yeast extract) medium with normal or modified concentrations of glucose, yeast extract, hemin and NAD (nicotinamide adenine dinucleotide). Seed culture of two fermentors was inoculated to 50 L fermentor, separately and range of PRP production and Dry cell weight (DCW) were studied. Results: Cultivation of Hib in 50L fermentor contained modified CY medium with 6gl-1 Glucose, 2.5 gl-1 Yeast extract, 0.03 gl-1 Hemin and 0.015 gl-1 NAD , with controlled pH at 7.3 and 30% Dissolved oxygen tension (DOT) resulted to about 5.1 gl-1 DCW and 1.16 gl-1 PRP , that was significantly higher than normal CY medium. Conclusion: In conclusion, by modification in some medium components of CY medium, control of Dissolved oxygen tension and pH, the Large-scale production of PRP is improved. Improvement of PRP production leads to reduce the final cost of Hib conjugate vaccines

<em>In silico</em> analysis of chimeric subunit vaccine containing HER-2-MUC1 against breast cancer

224-233Breast cancer is a leading cause of cancer-related deaths in women worldwide. Although tumorectomy, radiotherapy, chemotherapy and hormone replacement therapy have been used for the treatment of breast cancer, there is no effective therapy for patients with invasive and metastatic breast cancer. Targeting tumors using cancer vaccine therapeutics has several advantages including the induction of long-term immunity, prime boost strategies for additional treatments and reduced side effects compared to conventional chemotherapeutics. However, one problem in targeting tumor antigens directly is that it can lead to antigen loss or immune editing. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu- (480–620) and seven tandem repeats of MUC1 (VNTR) that represents a three-dimensional epitope. The construct was analyzed by bioinformatics softwares. Linear and discontinuous B-cell epitopes, MHC class I and II binding peptides of chimeric protein were predicted. Results suggest that the construct can be an appropriate vaccine candidate against breast cancer

Simultaneous expression of 5-enol pyruvyl shikimate 3-phosphate synthase (epsps) and glyphosate oxidoreductase (gox) in transgenic canola plants towards enhancing resistance to glyphosate herbicide

Canola (Brassica napus L.) is known as one of the most important oil-producing plants worldwide that has a high food value. Today, expansion of planting area of this plant has been highly considered. The presence of weeds in canola fields causes a significant loss in crop yield and quality. So far, the most widely herbicide used to manage weeds is the broad spectrum glyphosate that targets 5 enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme.  In this study, with the aim of identification of new strategies to develop herbicides-resistant plants, Glyphosate Oxidoreductase (gox) and epsps genes under the control of CaMV 35S promoter were transferred to canola seedlings with pBI121 expression vector, to develop new plants with higher herbicide resistance level. Acquired seedlings were screened and then subjected to herbicide resistance bioassay. Molecular analysis of transgenic lines through PCR and RT-PCR showed successful integration and expression of the transgene, respectively. Result showed the higher relative resistance of the transgenic lines expressing two gene cassettes compared to single gene cassette lines. This study suggests that simultaneous application of two different strategies can lead to more glyphosate-resistance to develop new genetically modified crops specifically in oilseed plants such as canola

Comparison of immunization potential from recombinant proteins EIT and Stx2B nanoparticulated based on chitosan in animal model, as nanovaccin candidate against E. coli O157:H7

Introdution: E. coli O157:H7 with its ability of quick implantation inside gastrointestinal tract that causes hemolytic uremic and bloody diarrhea in humans is an important subject to investigate. One of the effective ways to prevent these infections is using nanovaccines prepared from immunogenic recombinant protein from bacterial virulence factors. In this research, the chitosan encapsulated by recombinant protein of EIT with and without Stx2B were introduced to BALB/c mice and immunogenicity was evaluated and compared. Methods: In this experimental study the recombinant protein of EIT and Stx2B were nanoparticulated with chitosan through ionic gelation method after expression and purification with Ni-NTA column and approval by Western blotting. After Vaccinations antibodies against rStx2B and rEIT in the mice’s serum and feces were detected by ELISA. Challenge test for neutralization and protection against Stx2 toxin was applied with immunized serum by Fisher's exact test. Results: Evaluation of IgA and IgG antibody levels in the mice’s serum and also the IgA level in their feces showed more appropriate immunity oral-injection groups with the both nanoparticulated antigens. The protection challenge study show that, the mice group which received rEIT+ rStx2B could neutralize the toxin activity significant difference (P<0.05) between test and control groups of mice was recorded. Conclusion: The data indicate these multiple vaccine candidates with both nanoparticulated recombinant proteins EIT and Stx2B with chitosan, provide more effective protection against of E. coli O157:H