Salmonella entertidis is a foodborne pathogen that causes various diseases in human beings worldwide. The toxin of Salmonella can cause infectious diseases. In this research project, Salmonella was detected through various microbial, biochemical and molecular tests in diverse food samples collected from highly populated, moderately populated and less populated areas of Lahore, Pakistan. Enriched cultures of all food samples such as apples, tomatoes, yogurt and mayonnaise was streaked on violet-red bile glucose agar, Simmon’s citrate agar and eosin-methylene blue agar (EMB). Salmonella isolates were screened for the presence of toxin encoding gene through PCR. 27% apples, 19% tomatoes, 5% mayonnaise and 7% yogurt were found to be positive for INVA genes (invasion protein genes). In medical and pharmaceutical point of views the INVA gene can also help to develop specific medicines against salmonella. The cytotoxin that is protein in nature was confirmed by SDS PAGE in mayonnaise samples. This study illustrates that foods of highly populated areas are reservoir for Salmonella entertidis in Pakistan. There is need to develop specific drugs, precautionary measures to control salmonella and its disease.
Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon + and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~ 25.5% and ~ 6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.
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