15A-2 |
Application of thin aggregative fimbriae of Salmonella enteritidis |
K. B. SONG1, S. Kim, and S. Lee. (1) Dept. of Food Science & Technology, Chungnam National Univ., College of Agriculture, 220 Gungdong, Yusungku, Taejon, 305764, South Korea Salmonella enteritidis is a serious food-borne enteric pathogen in human and poultry. It expresses three morphologically distinct fimbriae. The Salmonella enteritidis fimbriae (SEF) 17 is tightly coiled and comprised primarily of AgfA. Its excellent antigenic nature implies a potential for the development of vaccine. Diagnostic tests using fimbriae or specific antibodies against them have been applied to the rapid identification of Salmonella infections. Our objective was to study the characteristics of recombinant thin aggregative fimbriae of Salmonella and to overproduce the agfA subunit for a vaccine development. DNA fragment of AgfA was cloned into an E. coli expression vector, pMalE-CR1 encoding IPTG-inducible maltose binding protein (MBP). The 393 base pair of DNA was amplified by PCR using synthetic primers and chromosomal DNA as a template. The production of MBP-AgfA fusion protein was induced in the presence of 1 mM IPTG for 5 h. MBP-AgfA was purified using amylose cross-linked to agarose resin. The purity of MBP-AgfA was detected by SDS-PAGE. The fusion protein was used for the preparation of antibody. The immunogenicity was examined using the Western blot. The secondary structure of AgfA was elucidated from difference CD spectra. MBP-AgfA fusion protein was overproduced in E. coli and purified using amylose resin. The purified MBP-agfA subunit fusion protein had a molecular mass of 60 kDa with more than 95% purity on SDS-PAGE. The immunogenicity was examined using the Western blot. The estimation of secondary structure of agfA by difference CD spectra shows that the protein mainly consists of ет-sheet structure. This result shows that MBP-AgfA fusion protein was overproduced in E. coli as a soluble form in large amounts and it suggests the possible development of a vaccine for salmonella infections.
Session 15A, Biotechnology
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