Screening Of Bifidobacteria Strains Yielding Serpin And Construction Of Serpin Mutant Stratins

Bifidobacteria are the predominant species in human intestine, which play an important role in maintaining microecology balance, anti-inflammatory and promoting a healthy gastrointestinal tract by adhering and colonizing intestinal epithelial cells and thus forming biological barriers together with production organic acids and lower redox potential to prevent invasion of pathogens.Adherence and immunity were the major basis for the physiological function of Bifidobacterium sp. However, the mechanisms of Bifidobacterium adhering to the intestinal epithelial and its relevant substances involving into host immunostimulation and immunomodulation were not totally clear. Currently studies on the adhesion and immunity of probiotics were mainly focused on out-membrane proteins of Bifidobacteria. In 2006 the Swiss Nestle Research Center found that Serpin (serine protease inhibitor, about 350-500 amino acids in size) from B. longum was shown to efficiently inhibit eukaryotic elastase-like proteases, which protected Bifidobacteria against exogenous proteolysis. In recent four years, serpins from procayotes attracted a number of researchers; no much progress was carried out in Bifidobacteria.Two strategies were used in this study of screening Serpin, namely,1. There is a serine proteases domain in plasminogen (Plg) which could bind with Serpin. Therefore, we conjugatedμPlg with magnetic beads, and therewith a 60 kD protein in the cell surface/extracellular was captured when incubated with Bifidobacterium; 2. Due to the weak conserved serpin gene in different Bifidobacteria strains, it is hard to detect serpin through PCR by primers of the conserved sequences. Therefore, anti-Serpin polyclonal antibodies were prepared. The Serpin-like proteins were screened out from Bifidobacteria strains by colony immunoblotting. The results indicated that Serpin-like protein had been found in B. infantis WBAN07 from 11 Bifidobacteria strains.In addition, insertional inactivation vector was constructed with the aim to damage the function of targeted serpin of B. longum WBL001 by their own homologous recombination. Further study will be performed to ascertain the functions of serpin mutant strains, and thus lay the foundation for studying the biological effect of serpin.In conclusion, the whole work provided a methodology for screening out functional proteins from proboitics and might open our vision for understanding the adhesion mechanism of probiotics e.g. Bifidobacterium...