Studies On Immune Enhancement Of Recombinant Lactobacillus Expressing Porcine Interleukin-2

With the national scale farming technology promotion, raising environmental degradation intensifies. Avian flu, swine flu and variety of infectious diseases, such as farming and even human hazards more and more serious, the prevention and treatment increasingly difficult, the traditional control methods highlights its shortcomings. Novel immunotherapy agents, immunostimulants and engineered vaccine is particularly urgent.Interleukin-2(IL-2), as a unique cytokines, it can suppresse synthesis of cytokines producing Thl cells. Study found that IL-2is produced by activated immune cells. IL-2can restrain the production and release of inflammation factors TNF and IFN-y from Thl cells, which has a strong anti-inflammatory and immune suppression activityln addition, IL-2can stimulate the proliferation, differentiation and producing antibodies of B cells. This study we clone IL-2gene from pig to E.coli-lactic acid bacteria expression vector, and then electrotransformed into the lactic acid bacteria. Therefore, we obtain recombinant lactic acid bacteria with the activity of immune regulation, which provide certain significance for the development of lactic acid bacteria preparations to decrease antibiotics residual and protect animal safety production. Specific research contents and the results are as follows:According to the pig source interleukin-2(pIL-2) gene sequences form the publication of GenBank, designed and synthesized primer combining with the shuttle express vectors site inserted by purpose gene into. We successfully expand pIL-2in vitro by RT-PCR. Connected pIL-2genes with pMD-18T carrier and transited into the E.coli DH5a, constructing the cloning vector pMD-18T-pIL-2, extraction plasmid. We identificated the sequence through the PCR, restriction enzymes and sequencing method, and gene sequences homology are the same with sequences form the publication of GenBank. Therefore, we successfully constructs pMD-18T-pIL-2cloning vector.The recombinant plasmid pMD-18T-pIL-2was digested with restriction enzyme generating IL-2gene, and subcloned into the vector PSIP409. The recombinant plasmid pSIP-409-pIL-2was transferred into the Lb.plantarum NC8by electroporation. The recombinant lactic acid bacteria was identified. The recombinant lactic acid bacteria was induced by Sakacin-P (SppIP) and identified by SDS-PAGE analysis. The recombinant lactic acid bacteria express17.5kDa protein and the results are consistent with the expectations. The Western blotting analysis shows that pIL-2possess immunogenicity. Weaning piglets were inoculated with Lactic acid bacteria continuouslly for three weeks, in the second week, and were inoculated with ETEC O149:K88for one week until the piglets fall ill. The results show that, comparing with the control group, the recombinant lactic acid bacteria pSIP-409-pIL-2can enhance the cell immune level, stimulating the production of antibody and inducing local mucosa reaction, downregulation the level of inflammatory factor TNF-a and at the same time promoting the expression of anti-inflammatory factor IL-4. It can obviously improve the changes of piglets histopathological. The recombinant strains were successfully field planting in the intestinal through the antigen positioning and detection of intestinal content to IL-2and TNF-a.