Antigenic characterization of major cytotoxic proteins of Salmonella Weltevreden
| dc.contributor.advisor | Ambwani, Tanuj | |
| dc.contributor.author | Sonwane, Arvind Asaram | |
| dc.date.accessioned | 2019-06-24T11:09:58Z | |
| dc.date.available | 2019-06-24T11:09:58Z | |
| dc.date.issued | 2005-08 | |
| dc.description.abstract | In spite of years of research, Salmonella has remained to be one of the most important pathogens of humans as well as animals. Salmonellosis is the major foodborne illness world over. The menace of Salmonella can only be contained by ensuring its in-time detection in suspected food/feed. For this purpose, a rapid, sensitive and specific test based upon genus specific bacterial toxin-protein antigen may be of great value. Prior to exploring the use of these toxins as diagnostic antigens they must be studied in some detail for their antigenic characters. Meager information is available on such aspect. Considering this, the present investigation was undertaken to study the toxic/cytotoxic preparations obtained from cefotaxime extract (CE) of Salmonella Weltevreden with the help of SDS-PAGE, western blotting and circular dichroism (CD) spectroscopy. From CE, the toxic fraction viz. - 40-70% DCP (desalted and concentrated protein) was obtained by differential salt fractionation with the help of ammonium sulfate precipitation and then eventually desalting and concentrating with spin columns. Also, the sub-fractions of 40-70% DCP viz. - 40-50% DCP, 50-60% DCP and 60-70% DCP were prepared. The cytotoxic fraction from among the 40-70% DCP was separated as 2nd major peak (cytotoxic peak/CP) after elution on Sephadex G-100 column. Studies for cytotoxicity of these proteins were carried out on chicken embryo fibroblast (CEF) cell culture. The cytotoxicity shown by 2nd peak contents (CP) was characteristic. The SDS-PAGE analyses of 40-70% DCP; 40-50%, 50-60% & 60-70% DCPs and CP revealed 25, 9, 15, 22 and 7 polypeptides respectively of different molecular weights ranging from 106.3 kDa to 16.7 kDa. The toxic/cytotoxic preparations used in this experiment were highly immunogenic. On western blot analysis, 40-70% DCP revealed 14 immunogenic polypeptides in the range of 106.3 kDa to 35.1 kDa while, CP revealed 6 immunogenic bands in the range of 98.8 kDa to 52.0 kDa. This indicates that not all the proteins from these immunogenic toxic/cytotoxic preparations were immunogenic. Also, their individual immunogenicities were variable. The polypeptide, identified as SP-2 (Salmonella Polypeptide-2) was the most immunogenic one. The polypeptides in the descending order of their individual immunogenicities were SP-2, SP-9, SP-4, SP-3, SP-15, SP-1, SP-5, SP-6, SP-8, SP-16, SP-17, SP-18, SP-19 and SP-20. Secondary structure studies of the major immunogenic proteins those were observed with help of western blotting of CP were tried using CD spectroscopy. No conclusive results were obtained, which may be attributed to the probable partial denaturation of proteins during sample processing and/or the use of a CD spectra analysis database that is incompatible for the proteins of the present study. On the basis of above study it is concluded that, spin column method is superior over conventional dialysis tubing method; the CEF cell culture can be used for the cyotoxicity studies of Salmonella; the cytopathic effects induced by 2nd peak contents may be considered as „characteristic‟ for such studies in CEF cell culture system; there is a need to study the major immunogenic polypeptides obtained in this study for their immunological and molecular characters by purifying them to their homogeneity and with a more rational approach, Viz.- adopting enhanced protein purification processes and/or using proper CD data analysis database, CD spectroscopic studies should be performed for these proteins to analyze their secondary structures. | en_US |
| dc.identifier.uri | http://krishikosh.egranth.ac.in/handle/1/5810109793 | |
| dc.keywords | antigens, characterization, cytotoxicity, proteins, Salmonella | en_US |
| dc.language.iso | en | en_US |
| dc.pages | 125 | en_US |
| dc.publisher | G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand) | en_US |
| dc.research.problem | Salmonella | en_US |
| dc.sub | Animal Biotechnology | en_US |
| dc.subject | null | en_US |
| dc.theme | Antigens | en_US |
| dc.these.type | M.V.Sc. | en_US |
| dc.title | Antigenic characterization of major cytotoxic proteins of Salmonella Weltevreden | en_US |
| dc.type | Thesis | en_US |