Aflp Screening Of The Differential Probiotic Genes Of Lactic Acid Bacteria

Lactic Acid Bacteria (LAB) have now attracted more and more attention as an important probiotic. However, long-term administratration of whole bacteria would render the bodies immunotolerant to pathogenic organisms. It is an important issue to decipher and ulitize the probiotics in LAB to build up the immunity of human. The present study, aiming this object, is to screen and clone the differential probiotic genes of LAB, including three kinds of Lactobacilli and one kind of Lactococcus by Amplified Fragment Length Polymorphism (AFLP). The coding protein encoded by one of the differential genes were expressed in E. coli, which would facilitate the furher function establishment.Object:To cast a cluster analysis on the proteomic profile of Lactobacilli by bioinformatic software, and to discern the commonly different proteins of 4 differernt Lactobacilli related to pathogenic bacteria. and to screen genetic differences between probiotic and pathogenic bacteria by the method of AFLP.Methods:1. Protein Cluster Analysis: Gene ontology classes were downloaded by their ID number in the Uniprot database. Four kinds of functioin proteins of lactic acid bacteria were selected, which may be related to immunoloregulation as follows: cell adhesion, establishment of localization, protein secretion, signal transduction. Sequence alignment were conducted by software PROMALS and phylogenic trees were produced by mega.2. AFLP analysis: Genomic DNA were extracted from Lactobacillus plantarum followed by two enzyme-digestion and two-stages PCR. The PCR products were resolved by 6% PAGE. The common polymorphic bands for 4 kinds of probiotic bacteria compared to pathogenic ones were recovered and re-amplified followed by cloning of the products into pGEM-T vector and sequenced.3. Gene cloning: The sequences of the screened fragments were aligned in NCBI website by BLAST program. Retrieved full-length genes were cloned from the corresponding LAB bacteria into pEASY-T3 vector. The coding regions were cloned into expression vector and the positive clones thus produced were applied for protein expression.Results:1. Protein cluster analysis showed that there is a high similarity in the protein sequences selected between the different Lactobacilli.2. Nine commonly different fragments were produced by AFLP screening.3. The coding regions of sugar transport protein and citrate lyase beta chain were cloned and their sequences were confirmed. Sugar transport protein were expressed in prokaryote cells.Conclusion:1. The differential genes possibly act as key factors by which LAB can regulate human immunity.2. AFLP is a reliable approach that can screen differential genes between two populations with conservative genetic background and obvious phynotypic difference.