Screening, Cloning And Expression Of Adhesion Proteins From Bifidobacterium Spp And Preliminary Study Of Their Functions

Bifidobacteria play an important role in balance of intestinal microecosystem, inhibition of pathogen invasion, anti-tumor, anti-mutant and anti-aging. Adhesion and colonization to intestinal epithelium is critical for Bifidobacteria to function probiotic effects, and also the most important characteristics for microecological modulator evaluation.In this study, an adhesion protein named BIF (binding inhibition factor) was cloned and expressed from B. longum, and BIF-like proteins were screened from four strains of Bifidobacteria, adhesion proteins from B. longum were isolated by using magnetic nanobeads as the carrier. Biological effects of adhesion proteins on intestinal epithelium were studied preliminarily.The thesis contains the following parts:Adhesion is an important step for probiotics and pathogens to function in human bodies, mechanisms and biological effects of bacteria adhesion to intestinal epithelium were discussed. Remarkably, competitive adhesion of probiotics and pathogens to epithelium was reviewed, and the application and fundamental reaseach tendency were prospected.The distributions of BIF in Bifidobacteria were rarely reported, therefore, we carried out detailed research on BIF. A pBIF-GEX expression plasmid was successfully constructed and transformed to Eschericha coli DH5a, the expressed BIF was purified with GST affinity column. Adhesion of BIF protein to HT-29 epithelium was observed by labeling BIF with quantum dots, then BIF was found to present remarkable concentration-dependent inhibition effect on adhesion of Listeria monocytogenisis and E. coli O 157:H7. BIF with a concentration of 50μg/mL could inhibit adhesion of Listeria monocytogenesis from 41.6% to 8.6% and E. coli O 157:H7 from 44.3% to 10.4%., respectively. Moreover, BIF had no influence on the adhesion of B. longum and Lactobacillus planturam. Results showed that BIF had concentration-dependent effects on IL-4 and IL-8 producing, but no effect on TNF-a.Anti-BIF antiserum was prepared and purified to screen BIF-like proteins from Bifidobacteria by dot blot. Bifidobacteria producing BIF-like proteins were confirmed by PCR using primers bif-F1and bif-R1. Bif-like genes from B. infantis WBIN03, B. infantis WBAN07, B. adolescentis WBAD08 and B. lactis WLABO9 had homology rates of 89.71%,82.53%,90.75% and 82.91%, respectively, comparing with bif from B. longum. Similarly, four of the above proteins were cloned and expressed, and three out of four purified BIF-like proteins were found to adhere to HT-29 epithelium with quantum dots labeling.It is a challenging work to search for adhesion relevent proteins from Bifidobacteria. In the present study, epithelium debris coupled with magnetic beads were used to isolate the adhesion-relevant components from extracted surface layer proteins. Four adhesion relevant proteins were identified by mass spectrography, namely PP1, PP2, PP3 and PP4. Plasmids were constructed for above protein expression, purified proteins were labeled with QDs and three out of four of the screened proteins were observed to adhere to HT-29 epithelium cell line. Meanwhile, the four proteins were found to have concentration-dependent effects on the producing of IL-4, IL-8 and TNF-a.In conclusion, searching for adhesion proteins and coding genes from probiotics, studying their probiotic effects on adhesion inhibition and cytokines producing in epithelium cell level, are promotive in the area of probiotics and microccology. The method of screening and functional analysis of adhesion relevant proteins by using magnetic beads and QDs was creative in functional probiotics study, providing new strategies in the future reseach on probiotics.