Based Of Lactobacillus Brevis SlpA Promoter Constitutive Expression Vector And Its Activity Analysis

Lactic acid bacteria (lactic acid bacteria, LAB) are a group of Gram-positive, can ferment carbohydrates, themajor metabolite is lactic types of bacteria collectively, with a variety of human and animal health, it is widely usedin food fermentation industries. A variety of lactic acid bacteria prebiotic characteristics also make it an idealcandidate to host the exogenous gene expression and oral vaccine developed.Lactic acid bacteria expression system compared with other prokaryotic or eukaryotic expression system isrelatively immature, mainly exogenous protein expression lower lactobacillus certain genetic background is notclear enough, the operation is relatively cumbersome expression condition is not stable enough. The lactobacillusExpression system exogenous protein in the amount of lactic acid bacteria expression vector promoter activity areclosely related. Related studies show lactobacillus host to the expression vector promoter requires more stringentthan the Escherichia coli (E.coli), highly selective exogenous promoter, therefore, rarely used in the lactobacillusexpression system. Currently lactobacillus expression vector can be used to construct a relatively small number ofpromoter, wherein the promoter activity of the most lactobacillus expression vector is not high, and requiresinduction, which not only reduces the efficiency of expression of the exogenous protein, so that the operationbecomes complicated, and moreis important to limit the practical application of the lactobacillus. Induced by aconstitutive promoter does not require special conditions to bacterial survival continuous expression of foreigngene, and therefore suitable for application in the actual production. Lactobacillus low expression and induciblepromoter limitation in the practical application, this study constructed a lactobacillus constitutive promoter probevectors, and the separation and determination of the Lactobacillus brevis is widely used in the food andpharmaceutical Slayer protein promoter promoter activity, the expression vector, expanding the range ofapplications of the lactobacillus expression vector laid the foundation for further construction of a constitutivepromoter of Lactobacillus brevis.This study in order to study the promoter activity of the S-layer of Lactobacillus brevis, Lactobacillus breviswhole genomic DNA as a template, the size of the amplified357bp fragment of the promoter fragment of the Slayer, pPG612as an expression vector, the TGEV N gene and SLSF of gene fragment constructed recombinantlactic acid bacteria expressing plasmid pPG612-SlpA-N/pPG612-SlpA-N was transformed to the four types oflactic acid bacteria-Lactobacillus casei L. casei393, Lactobacillus plantarum L.plantarum, Lactobacillus paracasei L.paracasei, the Lactococcus lactis L.lactis in,SDS-PAGE results showed that three recombinant strain ofLactobacillus casei, Lactobacillus plantarum and Lactobacillus paracasei were about the size of54ku and58ku theprotein, the size of the expressed proteinconsistent with the theoretical value; Lactococcus lactis is not theexpression of the target protein.Western blot analysis results show that the three kinds of recombinant lactobacilliexpressed proteins were TGEV monoclonal antibody supernatant, and LTB monoclonal antibody supernatantidentified, confirmed that the two target protein expression did get three Lactobacillus. Proved that the S-layerhaving a promoter in the three Lactobacillus activity, whereas no activity in Lactococcus lactis.To investigate the the S layers promoter activity differences in different host bacteria, the experiments in astatic culture model, respectively, sampled at different times (8h,12h,16h,20h,24h), western blot identification,indicating that the promoter canactivated protein expression, and the best activity at16h, the expression amount.The same time, in the time of the optimal expression of three different host bacteria were sampled, westernblot identification, the results show that the S-layer promoter activity is best in Lactobacillus plantarum,Lactobacillus casei in activity somewhat less in paracaseiLactobacillus the weakest activity; promoter of theS-layer does not show activity in Lactococcus lactis. The above results indicate that the promoter of the S-layer ofthe Lactobacillus brevis has strict host selectivity, affinity closer Lactobacillus relatively good activity.As in the stationary culture model will produce lactic acid, lactic acid bacteria during the growth, is a declinein the pH within the medium, the peracid environment leading to the promoter activity of the S layer is suppressed,so in the experiment we have taken the fermenter culture method is cultured, the pH was adjusted to a fixed valueby western blot identification, the results showed that the pH5.5when the the S layers promoter activity bestover-acid and alkaline environment it is possible to inhibit the activity of the promoter, and alkalinethe mediumenvironment more than the peracid environment can be suppressed the activity of the promoter.This study on the basis of previous studies, we source other than the functional protein as a reporter gene,constitutive promoter was constructed recombinant Lactobacillus expression system, good activity and thepromoter, the promoter for further research in different hostbacteria in the regulation of expression of exogenousprotein function, and to lay the foundation for identification and screening constitutive promoter.