Agricultural Research Management Information System

Home
Research Summary
Search
About Us
- ARMIS
- Brochure
Contact Us
Report
User Request
Data Input
Help
- Operation Manual
  - PDF
  - Video
- Program Area & Commodity
We have reached 37600 number of research entries at this moment.

- Logout

Research Detail

Home
Research
Detail

Comparison of Protein Patterns Among Some Salmonella Serovars and E.coli by Two-dimensional Polyacrylamide Gel Electrophoresis

F. Begum
Animal Health Laboratory, School of Agriculture, Ibaraki University, Chuou, Ami,Ibaraki 300-0393, Japan

Y. Adachi
Animal Health Laboratory, School of Agriculture, Ibaraki University, Chuou, Ami,Ibaraki 300-0393, Japan

M. S. R. Khan
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University,Mymensingh

Abstract/ Executive Summary:

The study was conducted to compare the protein patterns among some Salmonella serovars and E. coli using Two Dimensional Polyacrylamide Gel Electophoresis. The Two Dimensional Polyacrylamide Gel Electophoresis showed a 37.81 kDa well separated protein spots with all Salmonella serovars at the same time with E. coli a 36.5 kDa protein. However, these protein spots of Two Dimensional Polyacrylamide Gel Electophoresis were further tested with Immunoblotting analysis with specific antiserum against Salmonella typhimurium infected chicks. All selected Salmonella serovars successfully identified a common 37.81 kDa protein whereas E. coli spots identified as 36.5 kDa protein instead of 37.81 kDa. As a further monitoring of these proteins as to check the homogeneity and heterogeneity for N-terminal amino acid sequencing, the specific protein bands from all Salmonella serovars and E. coli were excised, purified and subjected to sequence analysis. The amino acid sequence alignment showed the 37.81 kDa proteins of some Salmonella serovars were identical or homologous among the Salmonella serovars. The N-terminal amino acid alignments of the 37.81 kDa proteins were determined as alanineglutamine- valine-isoleucine-asparagine-threonine-asparagine. On the other hand, the N-terminal amino acid alignment of the 36.5 kDa protein of E. cloi ACLD2201 was found to be heterologous as alanine-proline-lysine-aspartic acid-aspararginethreonine- tryptophan. The findings of this study can be concluded that the 37.81 kDa protein of some Salmonella serovars and 36.5 kDa protein of E. coli were completely different though there is some identity of these organisms due to the presence of Enterobacterial common antigen.

Keywords: Salmonella, 2D-PAGE, Amino acid sequence

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Cattle

Objectives:

To get more precise information about a specific protein by 2D-PAGE and its IB

Methodology:

Salmonella serovars S. typhimurium L1338, S. cerro A12, S. johannesburg A28, S. virchow A53, S. liverpool A32, S. meleagridis A36, S. newport A39, S. worthington A54 and E. coli ACLD2201 were used for 2D-PAGE. These organisms were cultured according to the procedure described by Begum (2005). The work was performed during the period of July 2003 to June 2004 in Animal Health Laboratory, School of Agriculture, Ibaraki University, Chuou, Ami, Ibaraki 300-0393, Japan. Out of eight Salmonella serovars, four Salmonella serovars namely S. typhimurium L1338 , S. cerro A12, S. johannesburg A28, and S. virchow A53 were further tested for 37.81 kDa protein bands after excising from the SDS-PAGE gel for further purification and characterization according to the procedure described by Miyazaki et al., (1994) with slight modification. The target proteins of 4 serovars of Salmonella were separated by SDSPAGE with a 10% (w/v) gel. The gel containing the 37.81 kDa band was excised and extracted from gel fragments into a running buffer (0.1% SDS, 10mM Tris-HCL) electrically. For protein precipitation, double volumes of cold (-20ºC) acetone were added to the running buffer in which the 37.81 kDa protein was dissolved. The mixture was placed overnight at -20ºC and then centrifuged at 15,000 rpm for 15 min at 4ºC. The supernatant was removed. After air-drying the precipitated protein pellet, the precipitated protein was dissolved in the dye buffer and subjected to SDS-PAGE and IB for the confirmation of the target protein band. The target protein precipitated and confirmed and was used for 2D-PAGE. The 2-D PAGE gel was also subjected to IB. 2D-PAGE was carried out as described previously (Begum, 2005, Jiang et al., 2004, Toda, 1997, and O, Farrell, 1995). IB was carried out as described previously (Towbin et al., 1979).

Source of Information: Bangl. J. Vet. Med. (2008). 6 (2): 127–138

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Therefore, only the whole cell lysate was used for 2D-PAGE followed by IB. The results of IB and the amino acid sequencing in each step were found to be identical (Table 1). A further experiment was performed to analyze a difference between the 36.5 kDa protein of some Enterobacteria and the 37.81 kDa protein of Salmonella serovars. The sera of chicks experimentally infected with S. typhimurium specifically detected the 37.81 kDa spot in Salmonella serovars and the 36.5 kDa spot in Enterobacteria with IB after 2DPAGE. However, the N-terminal amino acid sequence aligment of the 36.5 kDa protein of E. coli ACLD2201 was different from those of the 37.81 kDa proteins of Salmonella serovars. The protein spot patterns of S. typhimurium L1338, S. cerro A12, S. johannesburg A28 , S. virchow A53, S. liverpool A32, S. meleagridis A36, S. newport A39, S. worthington A54, and E. coli ACLD2201 were investigated by 2D- PAGE and / IB. The results showed that there was the distinction between Salmonella serovars and E. coli and that the N-terminal amino acid sequence of the 37.81 kDa protein of some Salmonella serovars was clearly distinct from that of the 36.5 kDa protein of E. coli ACLD2201.

Knowledge Management: Journal