The Construction Of A Recombinant Lactobacillus Acidophilus Expressing Rat IL-10and The Effect On IBD Induced By TNBS In Rats

Lactobacillus acidophilus is an important probiotic, which colonize in the ileum and thelarge intestine of human and other homoiothermal animals.It play an very important role onhuman health,such as adjusting the immune function, preventing infection, reducing serumcholesterol, promoting lactose digestion and preventing colon cancer. IL-10not only hasstrong anti-inflammatory capabilities,it also can inhibit the production and release of IL-2,IFN-γ and proinflammatory cytokine, meanwhile it can induce peripheral tolerance andimmune suppression. IL-10play an immunosuppressive effect on mononuclear macrophages,T cells and NK cells, moreover, it can also have immune-stimulating function to B cells andmast cells. Lactobacillus acidophilus plays a positive effect on inflammation of gastro-intestinal tract, IL-10is an anti-inflammatory factor. If we build a high expression cloningvector of IL-10and make the cloning vector expressing in Lactobacillus acidophilus,it wouldbe a further exploration of the probiotic transformation.Inflammatory bowel disease (IBD) is a group of unknown etiology, chronic infla-mmatory diseases involving in the gastrointestinal tract, including ulcerative colitis (UC) andCrohn’s disease (CD). The distribution of IBD is world-wide, and it seriously endangershealth, and the incidence rate is increasing recently. The etiology and pathogenesis of IBDare not very clear yet. The majority of researches suggest that IBD is a kind of autoimmunedisease caused by the interaction of many factors. Among all of these, immunity plays a vitalrole in the occurrence and development of IBD. Many datas show that its pathogenesis isauto-immune abnormality referring to a variety of cytokines, eventually resulting in tissuedamage. The imbalance of Th1/Th2immune response and the imbalance between pro-inflammatory cytokines and anti-inflammatory cytokines play an important role in thepathogenesis of IBD. IFN-γ are pro-inflammatory cytokines mainly secreted by Th1cellsand have wide role in immuno-regulation and play a significant role during the regulation ofcytokine network.To construct a recombinant Lactobacillus acidophilus expressing IL-10. Asignal peptide sequence of Lactobacillus acidophilus and a IL-10sequence of rat weresynthesized and connected by PCR and cloned into the lactobacillus shuttle vectorpMG36e.The recombined plasmid pMG36e-sp-IL10was then introduced into Lactobacillusacidophilus LA-5by electroporation. PCR and sequencing were used to appraise therecombination of IL-10and LA-5, SDS-PAGE and Western blot were used to detect the expression of the recombinant protein. The results confirmed that the IL-10gene had beenintegrated into the genome of Lactobacillus acidophilus, and the objective protein waspartially secreted into the supernatant. Therefore, IL-10was expressed and secreted byrecombinant Lactobacillus acidophilus LA-5.Lactobacillus acidophilus expressing IL-10of mouse source will be applied in theinflammatory bowel disease model in rats so as to firstly provide the experiment basis forfurther exploring the occurrence and development of IBD, and detecting the levels of IL-10,IFN-γ mRNA in colon by Real-time PCR,and its the relationship with probiotics and IL-10,and secondly provide new ideas for IBD prevention and treatment.