Molecular cloning, expression and In-silico characterization, immunogenicity, Salmonella typhimurium, polymerase chain reaction, isolates, immunity
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Date
2012-01
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
The present investigation was undertaken to isolate and characterize the immunogenic Omp28 protein from S. Typhimurium. The Genus-specific PCR assay was standardized with aview to amplify specific region of 16S rDNA gene and subsequently subjected for molecular characterization for 28 different field isolates of Salmonella from different sources. Theisolates were S. Abortuseqaui, S. Anatum, S. Agona, S. Bareilly, S. Bonariensis, S. Bredeney,S. Berta, S. Bergen, S. Choleraesuis, S. Derby, S. Enteritidis, S. Gallinarum, S. Greenside, S. Heidelberg, S. Illinois, S. Infantis, S. Java, S. Kentucky, S. Newport, S. Paratyphi-B, S. Pollurum, S. Rubislaw, S. Saintpaul, S. Stanley, S. Seftenberg, S. Typhi, S. Typhimurium and S. Weltevreden along with standard Salmonella Typhimurium culture (MTCC-3214). All the samples revealed a specific banding pattern of amplified PCR products with a presence of specific 574bp band, for genus Salmonella. In order to determine Salmonella genus specific Omp28 gene, PCR condition was standardized, gene was amplified and sequenced. All these serovars were also subjected to PCR for determination of presence of immunogenic Omp28 gene in the form of specific amplified products size specifically of 341bp. The outer membrane protein was analyzed using SDS-PAGE which revealed the presence of low molecular weight of 12.32kDa main protein as outer membrane protein of Salmonella Typhimurium. The Immunogenic Omp28 gene of S. Typhimurium was successfully cloned in cloning Vector(pUC19) and confirmed by nucleotide sequencing. The construct pUC19-Omp28 was further Sub-cloned in expression vector pET32a(+). The expression construct pET32a(+)-Omp28 was then used for expression of protein through induction with IPTG and expressed fusion recombinant protein was further characterized through SDS-PAGE analysis, which shows about 29.59 kDa fusion expressed recombinant proteins, consisting of about 17.27 kDa fusion tag of pET32a(+) expression vector and about 12.32 kDa of expressed protein of Omp28 gene of S. Typhimurium which was inserted in expression vector. The sequenced Omp28 gene of S. Typhimurium, S. Enteritidis and S. Paratyphi-B was subjected to in-silico characterization and structure determination (primary, secondary and 3D structure of amino acid sequence derived from sequenced gene). Further phylogenetic tree analysis, B-cell epitope, MHC I, II binding prediction with Allele HLA (Human) and homology modeling was also carried out with predicted amino acid of proteins. Analysis of Pfam showed that the protein belong to Pfam A family, hdeB super family [PRK11566]. The immunogenicity of protein was computed through antigenic plot and found antigenicity index of Omp28 protein were about 2.2 (0.1-2.2) for S. Typhimurium and S. Enteritidis and for S. Paratyphi-B about 2.1(0.1-2.1). More than 1.2 antigencity index suggests that all isolates possessing Omp28 gene protein can trigger the immune system and produce antibody in vivo. i.e. this expressed protein from Omp28 gene could be immunogenic in nature.
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Thesis-PhD
Keywords
molecular markers, cloning, gene effects, in-silico, characterization, immunogenicity, Salmonella typhimurium, polymerase chain reaction, isolates, immunity