Construction Expression Cellulase Recombinant Strain Of Lactobacillus Reuteri Without Resistance Marker

There are many enzymes and products supplemented in feeds ,and restrictd factors had some effects on their activities in animal production. Previous studies demonstrated that cellulases gene in vectors were expressed in probiotics (lactic acid bacteria), and those bacteria could live in the gastrointestinal tract of livestock and poultry; Probiotics expressing cellulase in the gastrointestinal tract could improve the digestion of cellulose and hemicellulose. But this method meets problems that vector is not stable in probiotics without pressure such as antibiotic and there are resistant genes in the vectors. In this study, we constructed a marker free cellulase integrated Lactobacillus reuteri strain.1,An integrative plasmids pG-HAF-HAR-C15cassette-loxpCm which containing cellulase Cel15cassette and the Loxp fragments gene was constructed using molecular gene clone technology.2,The pretreatment of Lactobacillus reuteri cells with lithium acetate (LiAc) and dithiothreitol (DTT), more studied optimizing the time of pretreatment and the concentration of cells of electrotransformation.The transformation efficiency was 32.6±7.2×10⁷ transformants /μg DNA, which was more than untreated cells 10⁵ times; The transformation efficiency with the increasing of cell concentration,and the highest cell density was 10¹⁰; This method was repeatable and had high efficiency.3,Using the optimizing transformation method,transfer the integration vector was transforred into the chicken digestive tract of wild-type lactic acid bacteria (Lactobacillus reuteri XNY) genome, and the Cre-Loxp systerm was successfully removed chloramphenicol resistance gene . non-resistant cellulase integration of Lactobacillus L-S2 was obtained. And the cellulase activity of fermentation broth of recombined Lactobacilli L-S2 was 0.426 U/mL.An expression system without resistance marker of Lactobacillus was successfully constructed ,and the expression levels of cellulase was measured.The results provided references for plating cellulose engineering strain of Lactabacillus in gastrointestinal tract of livestock animals, and it will also helps to increase growth performance and improve digestibility of crude fiber in livestock animals...