Prepareation Of Protoplast And Construction Of Serpin Mutant Strains Of Bifidobacteria

Serpin (serine protease inhibitor) is the general term of peptide-like serine protease inhibitor, consisting of350-500amino acids. In2006the Swiss Nestle Research Center found that Serpin from B. longum protected it against exogenous proteolysis and thus provided pre-conditions for the adhesion in intestinal tract. Recently, the biological effect of serpin on host by Bifidobacteria is not completely clear. In our previous study, B. infantis WBAN07with Serpin-like protein was screened out from11Bifidobacteria strains. The purified Serpin protein of Bifidobacteria was shown to efficiently inhibit eukaryotic a-chymotrypsin and pancreatic elastase, and enhance the adherence of Bifidobacteria to HT-29cells. For further investigation of the function of Serpin, a gene knock-out strain of Bifidobacterium longum was planned to construct by importing a replacement-type targeting vector to realize the homologous recombination. Due to the thick cell wall as well as the difficulty for enzyme digestion of Bifidobacterium, the transformation rate of Bifidobacterium is quite low. Therefore, in this study, we try to prepare high active protoplast of Bifidobacterium, subsequently, acquire serpin mutant strains by transforming the serpin gene knock-out vector into cells of Bifidobacterium by protoplast PEG transformation.In this study, six factors were investigated, which is the critical factor on the preparation and regeneration of protoplast of Bifidobacteria longum, including phase of growing cells, mutanolysin concentration, enzymolysis time, enzymolysis temperature and osmotic stabilizers. The optimum conditions were as follows:middle log phase (OD6oo-2.0), mutanolysin concentration5μg/mL, enzymolysis time30min in SMM buffer. The protoplast formation and regeneration rate reached to81%and48%, respectively.Once the protoplast was prepared, the serpin gene knock-out vector was transformed into cells of Bifidobacterium by protoplast PEG transformation, subsequently, serpin mutant strains were acquired. Further study was performed to ascertain their adhesion function, we found that there was no significant difference on the adhesion ability between the wild strains and the serpin mutant strains.In summary, a serpin gene knock-out system was set up by constructing a protoplast transformation system. This work provided a methodology for studying the surface protein of probiottcs in the future.