Construction And Preliminary Use Of Expression System In Lactic Acid Bacteria

Lactic acid bacteria (LAB) is widely used in food fermentation, biological medicine industry and generally regarded as safe. In recent years, experiments focus on cloning and expression of valuable genes and the construction of food-grade selection markers in molecular biology of LAB. In this report, a constitutive expression vector for LAB using nisI as the selection marker and green fluorescent protein (GFP) as the reporter protein was constructed. This would help us better understand a series of problems including the relationship between LAB and intestinal epithelial cells and how to activate the immune response. The method established here undoubtedly provides a simple and fast way to detect the colonization ability of LAB.In this study, the nisI fragment was amplified by polymerase chain reaetion with pLEB590as template. The nisI fragment was subcloned into pMG36e, resulting in the expression vector pMG36e-nisI Then pMG36e-nisI was electroporation into Lactococcus lactis WH-C1, and the recombined had the resistance of Nisin. In order to apply pMG36e-nisI, gfp gene was subcloned into pMG36e-nisI, resulting in the plasmid pMG36e-nisI-gfp. It was electroporated into Lactococcus lactis WH-C1, Lactobacillus plantarum MA2, Lactobacillus plantarum KF5and Streptococcus thermophilus34, and then the plasmid stability was evaluated. Results showed that the plasmid pMG36e-nisI-gfp had good stability and can express GFP in LABs.In the animal experiment, the mice were inoculated with10CFU of L. lactis WH-C1/pMG36e-nisI-gfp. After6h, recombined L.lactis WH-C1presented in all samples of the bowel contents and the maximum concentration was reached after42h. Three days later, the amount of bacteria remained at around104CFU/g which indicated L.lactis WH-C1could exist in the gastrointestinal tract for a long time.