Construction Of Wild Type Lactobacillus Expression System Integrated With Cellulase Genes

Cellulose is the most abundant renewable resource in the world; it can use in many kinds of areas of people's living and production only when it become glucose hydrolyzed by cellulase. How to improve the utilization rate of feed crude fiber of animals'especially ruminant anmimals is an important problem. Using cellulase is one of the methods of improving the utilization of feed crude fiber. Cellulase widely reside in microorganism, animals and plants, it can degrade cellulose specifically. Lactobacilli are kinds of probiotic widely reside in the nature world, and can adhere to the surface of mucous membranes specifically. The Lactobacilli stick to animal gut can tolerance the gut environment, and can promote the growth and immunity of animals. This research tries to construt a recombined Lactobacillus, and then uses it as a probiotic which can express cellulase. The main objective of this research is using chicken as model animal; establish a completed technique system from genes clone, integrative vector construct, host strain screen and recombine to recombined strain permanent plant and express in chicken gut.1. We isolated a cellulose degrade bacterium from the feces of Qinling Golden Takin(Budorcas Taxicolor Bedfordi) using improved CMC medium based on widely screen. We identified the bacterium belongs to the Bacillus using analysis of morphology and the conserved sequence 16SrDNA, and named it as Bacillus subtilis LN. Two cellulase genes were cloned from the bacterium chromosome DNA by two pairs of special primers. One is 1.5kb encoding 500 amino acid continuously, named Cel15; the other is 741bp, encoding 247 amino acid continuously, named Cel73. They are both the members of the cellulose hydrolysase family, and can be expressed in E.coli. The expression products are 55kDa and 27kDa respectively. Most of the products reside in the form of inclusion body.2. We isolated 6 Lactobacilli from crop, intestine and cecum content and wall of chick, using screen MRS medium. The Lactobacilli were all gram positive strains. They are all rhabditiform excepet one is globular. They then identified and named by 16SrDNA anylysis. The results are: 1 strain is Lactobacillus amylovorus SNY, 2 strains are Lactobacillus reuteri XNY and Lactobacillus reuteri MNY, 1 strain is Lactobacillus crispatus SBY, 1 strain is Lactobacillus brevis XBY, and 1 strain is Enterococcus hirae MBY. The results were reformed by constructing homology tree of these strains and 9 other strains of Lactobacilli from NCBI nucleotide data bank.3. Two integrative plasmids pLEM4153 and pLEM4154 which containing cellulase gene Cel15 and Cel73 respectively was constructed using molecular gene clone technology. The plasmids can integrate to wild type Lactobacillus reuteri XNY, by using an improved electro transformation protocol. And two recombined Lactobacillus reuteri XNY4153 and XNY4154 were screened. The two cellulase genes can express in recombined Lactobacilli detected by Real Time PCR. The two genes'mRNA relative expression amounts are: gene Cel15 is 18849.5 and the relative amount of gene Cel73 is 1388. The cellulase activity of fermentation broth of recombined Lactobacilli was determined, the results are: the highest cellulase activity of recombined Lactobacillus XNY4153 is 0.158U/mL, and the highest cellulase activity of Lactobacillus XNY4154 is 0.15U/mL.4. The recombined Lactobacilli can adhere to the enterocyte, after co-cultured the recombined Lactobacilli and enterocyte. The recombined Lactobacilli were inoculated to chicken gut in 0 day by drench from oral cavity. The cellulase genes were amplified by PCR in 7 days, 14 days 21 days and 30 days in the content of crop, small intestine and cecum of chicken. Planting recombined Lactobacilli can improve the bodyweights of chicken significantly(P<0.01), and at the same time, the digestibility of crude fiber was also influenced significantly by planting recombined Lactobacilli in chicken gut(P<0.01). This showed that the recombined Lactobacillus can permanent plant and express in chicken gut, the expressed cellulase can improve the grow performance and digestibility of feed crude fiber.We have constructed an expression system of Lactobacillus; permanent planted the recombined strain in chicken gut, and improved the growth profomance and the digestibility of feed crude fiber.