Detection A Strong Constitutive Promoter Based On Secreted Protein Of Lactic Acid Bacteria And Analysis Its Functions

Lactic acid bacterium is a Gram-positive lactic fermentation of sugars main product.the genericname of Bacillus bacteria. Currently Lactic acid bacterium can be divided into at least18genera, atotal of200species.With few exceptions, most of which is essential for the body and has importantphysiological functions flora.widely present in the intestine, play an important role in enhancingintestinal function, improve the intestinal environment, lactic acid bacteria is a kind of probiotics. afood safety grade microorganism, it is because of the role of lactic acid bacteira such characteristicsand physiological function, in the early food additive process and the recent biological engineeringand pharmaceutical fields, lactic acid bacteria also become antibacteiral，expression of the exogenousprotein and oral immunization worth exploring emerging host genus.Efficient promoter sequence can ipmrove the level of expression of exogenous protein in Lacticacid bacterium，strong promoter sub-normal circumstances a large number of mRNA transcribedAfter the translation, produce large amounts of target protein. Based on this, of the experiment is to beadopted for the detection and analysis of protein secreted by the lactic acid bacteirum itself, filter outthe high expression amount needed induced constitutive secretory expression of the promotersequence of the protein, expression vectors for lactic acid bacteria, the exogenous protein in a lacticacid bacterium highly eiffcientexpression.Lactobacillus casei supernatant total protein secretion two-dimensional electrophoresis analysis,mass spectrometry sequencing to find cheese lactic acid bacteria in the natural secretion of themaximum amount of protein secretion supernatant peptidoglycan hydrolase (PPH). Further inLactobacillus casei in the coding sequence and upstream regulatory sequences from the database ofthe U.S. National Center for Biotechnology (NCBI), Suitable for prokaryotic promoter predictionsoftware BDGP Neural Network Promoter Prediction peptidoglycan hydrolase gene upstream ofapproximately2000bp this sequence analysis and forecasting, ultimately determine the peptidoglycanhydrolase gene upstream522bp of its promoter region, the peptidoglycan hydrolase amino acidsequence analysis after with SignalP4.0software to find a signal peptide sequence for the first31amino acids. Then through the design of specific primers with specific restriction sites fromLactobacillus casei genome amplified the peptidoglycan hydrolase promoter and signal peptidenucleic acid sequence, in order to veriyf both the promoter found in the experiment whether as a strong promoter, a known strong promoter was cloned from the genome of Lactobacillus casei also b\their specific primers were designed and lactate dehydrogenase (Lactose dehydrogenase) promoterconstruct plasm id LDH-pPG612transformed into Lactobacillus casei, the same method of detectionof reporter gene expression.The recombinant Lactobacillus casei PPH-pPG612/L.casei and LDH-pPG612/L.caseirespectively expand cultured for48hours, and centrifuged to obtain the supernatant of the culture,carried a TCA50times concentrated SDS-PAGE and Western blot analysis, observation recombinantLactobacillus casei PPH-pPG612specific object at the80kD band Description the predictedpeptidoglycan hydrolase522bp fragment having a constitutive promoter active in Lactobacillus caseiAddition to the qualitative detection gusA the expression in this expeirment, in order to comparepeptidoglycan hydrolase promoter, lactate dehydrogenase start start of the strength of the promoterand the vector inducible xylose, culturing the host bacteria with three different promoter plasmid, thesonicated broth, go DNA, total RNA was extracted after, then the inversion rate of the obtained cDNAas a template designed for use in fluorescent quantitative PCR primers specific.Chloramphenicolresistance gene of the plasmid itself as the reference gene CT value calculated according to theefficiency of the three promoter is the peptidoglycan promoter:Lactate dehydrogenase promoter:inducible xylose promoter is (K55r0J22:1.Thus determined by the method used in the presentexperiment we cloned a constitutive strong promoter, and at the same time having a boot exogenousprotein secretion signal peptide sequence.