Construction And Functional Studies Of Expression Of GFP With Cellulase In Termite Fusion Recombinant Lactobacillus

Cellulose is recognized as the world’s largest number of renewable resources,but due to the special nature of the cellulose structure,it has a lot of limitations for people.Cellulase as a class to cellulose-degrading enzymes which can degradate cellulose into cellobiose and glucose is the most effective way for degradation.With the expanding field of biotechnology development and research,the researchers continued to filter with high efficiency fiber degradation ability of strain from the nature and use of genetic engineering to build a cellulose efficient decomposition of genetically engineered bacteria,there is a great significance for the industrial production of cellulase furthermake full use of cellulose,to solve the current energy crisis□food shortages and environmental pollution problems.Lactic acid bacteria is the essential body flora of human and animal,as a versatile probiotics,there are a number of important prebiotic function in lactic acid,it not only able to maintain the gut microflora balance,but also can mediate mucosal immunity? treatment of diseaseand infection control and other effects.In recent years,there are a lot of emerging reports for lactic acid bacteria as the oral vaccine viable carrier,such vaccines can play the double effect of the expression of exogenous protein and lactic acid bacteria.This experiment is according to the characteristics of Lactococcus lactis expression vector pNZ8149to design specific primers (including restriction sites), cloning of green fluorescent protein gene (GFP) and termites derived cellulase gene (Cell),and using the restriction endonuclease with connections to construct the fusion gene expression vector pNZ8149-GFP-Cell,concentration for5ng/mL Nisin induction,then observed by fluorescence microscopy□SDS-PAGE and Congored staining to detect the activity of the cellulase.The results show that the recombinant lactobacillus pNZ8149-GFP-Cell/NZ3900recombinant bacteria emit green fluorescence can be observed under a fluorescence microscope.the SDS-PAGE analysis showed that the recombnant protein molecular weight is about77KD and it contains the cellulolytic ability.Feed Jilin White Goose by1mL of recombinant lactic acid bacteria whose cell density is109CFU/mL,collect the intestinal canal in different times after oral administration to make frozen sections,then observa the intestinal adhesion and colonization law of the recombinant bacteria in the surface of the intestinal mucosa under fluorescence microscope.The results show that the reorganization of lactic acid bacteria colonization in the jejunum and ileum of geese,most of them colonization in the intestinal chyme and mucus where adjacent to the intestinal villus epithelial cells,in the first72hours after feeding peak for colonization,then showing varying degrees of downward trend. Studies have shown that the recombinant lactic acid bacteria in the goose intestinal colonization of the ileum> jejunum> duodenum> caecum.Select the8weeks of age into a goose,according to the dietary fiber source respectively set as:group A1(corn straw fiber source+recombinant of lactic acid bacteria group),group A2(corn stalks as sources of fiber, the control group),group B1(rice bran fiber sources+recombinant of lactic acid bacteria group),group B2(rice bran fiber sources in the control group),using the received fecal determination of geese on the metabolic rate of the fiber.The results showed that the geese in an oral recombinant lactic acid bacteria on fiber digestibility improved to varying degrees. Compared Group A1with group A2,crude fiber and hemicellulose digestibility of geese were increased by11.26%and3.93%,the difference was extremely significant (P<0.01);compared group B1with group B2,geese’s crude fiber□digestibility of neutral detergent fiber and hemicellulose were increased by5.16%□4.82%and3.09%,the difference was significant (P<0.05).This experiment constructed the reorganization of lactic acid bacteria pNZ8149-GFP-Cell/NZ3900included the cellulase gene labeled by green fluorescent protein gene(GFP),for the system□in-depth study of the recombinant bacteria in vitro and vivo degradation of cellulose and colonization in the digestive tract of livestock and poultry laws,and then provide a theoretical basis and experimental basis for the effective regulation of cellulose micro-organisms to digest.