Isolation of species specific proteins of Salmonella and its characterization
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Date
2006-08
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Present study was conducted to find genus specific proteins of Salmonella and to study cytotoxic effects of cytotoxic preparations of Salmonella on VERO, MDBK and BHK 21 cells. It was also aimed to standardize polyclonal ELISA based test for detection of Salmonella in foods of animal origin. Cell lysates of Salmonella serovars (12), E.coli (1), Shigella flexneri and Klebsiella spp.(1), were prepared by ultrasonication. The protein concentration of cell lysate obtained from Salmonella serovars were found to range between 1.4 to 4.6 mg/ml. On the basis of consistent cytotoxic activity indicated by Salmonella Typhimurium cell lysates, PDP and CFCS of S.Typhimurium were prepared for comparative study. Cell lysates of different Salmonella serovars were tested for cytotoxicity on MDBK, BHK 21 and VERO cells. Partial purification of Salmonella protein preparations was achieved using Sephacryl S-200 HR and Sephadex G-100, which revealed four and two distinct peaks, respectively. On gel filtration through Sephacryl S-200 HR, the cell lysate of Klebsiella spp, E.coli and Shigella flexneri yielded 2,3 and 4 distinct peaks respectively. PDP and PDCL revealed two separate peaks. Pooled and concentrated peak contents were tested for cytotoxic activity on BHK 21, VERO and MDBK cells. The first and second peak content showed mild cytotoxicity, where as fourth peak content showed moderate cytotoxicity. On SDS-PAGE Cell lysate revealed 31 polypeptides. Number of polypeptides kept on decreasing as the purification steps advanced. PDCL and PDP revealed 24 and 10 polypeptide, respectively with different mol wt ranging from 126.9 to 11.2 kDa. The first, second and fourth peak contents obtained after eluting the cell lysate and Second peak of PDP of S. Typhimurium through Sephacryl S200 HR showed 14, 7, 3 and 2 bands, respectively. Immunological characterization of cytotoxic preparations was done by performing ELISA using antiserum raised against cell lysate of S. Typhimurium. On the basis of the results obtained in the present investigation it seems possible to isolate species specific cytotoxic protein which can later be used as important diagnostic tool for detection of salmonella in foods of animal origin. The difference in the chromatographic profile of Salmonella, E. coli and Klebsiella spp clearly indicate that cytotoxic fourth peak content may be specific to salmonellae except of Shigella flexneri. The cytotoxic protein of these two organism need to be studied immunologically. Further it appears from the present study that in-vitro assay model like BHK 21 and MDBK cells are quite promising and it may be used for future studies.
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