Studies On Rreparation And Observation Of The Immunological Effectiveness Of Recombinant Lactobacillus Plantarum Expressing Rorcine Interleukin-18

Interleukin-18can induce T lymphocyte, NK cells and macrophages to produce IFN-y, IL-18has many biological activities, such as the activity of proinflammatory factor, anti-infection, anti-tumor immunity, resisting all allergic disorder effect and so on. It can regulate immune response and promoted the regulation of various Thl cytokine and played an important role in physiological functions. Lactic acid bacteria can regulated normal flora of gastrointestinal tract, maintains micro ecological balance in vivo, improve the immunity of animal. Lactic acid bacteria has strong tonifying didney growth functions, does not produce endotoxin, expression of heterologous proteins without purification can be directly with the cell together.So it was the ideal conversion or expression system. In view of the IL-18multiple biological activity, as well as a variety of probiotic Lactic acid bacteria function, research and development of the two effective combination of biological agents has significant clinical value and application prospect. The specific research contents and results are follows:Aseptic extracted total RNA from spleen lymphocytes of swine, according to GenBank included pIL-18gene sequences and using Primers5.0design primer with NcoI and XhoI restriction site, amplification579bp pIL-18gene fragment of RT-PCR. Connected pIL-18genes with pMD18-T carrier and transited into the E.coli DH5a, constructing the cloning vector pMD18-T-IL-18, extraction of plasmid DNA, by double enzyme, PCR identification are579bp fragment, and gene sequences homology are the same with sequences form the publication of GenBank. Therefore,we successfully constructs pMD18-T-IL-18cloning vector. The recombinant plasmid pMD18-T-IL-18was digested with restriction enzyme generating IL-18gene, and subcloned into the vector PSIP409,construction of recombinant plasmid pSIP-409-IL-18. Transited recombinant plasmid pSIP-409-IL-18into the E.coli DH5a, constructing the cloning vector pMD18-T-IL-18, extraction of plasmid DNA, by double enzyme, PCR identification are579bp fragment. The recombinant expression vector pSIP-409-IL-18was successfully constructed.The recombinant expression vector pSIP-409-IL-18transformated to Lb.plantarum NC8through electric conversion technical, proceeded double cleavage and identification from PCR, gaining579bp target fragment. Western-blot analysis showed that the recombinant lactic acid bacteria expressing18kDa fusion protein, and matched the expected results. Description that we success prepared of porcine interleukin-18gene engineering strains of Lactic acid bacteria.The recombinant porcine interleukin-18gene engineering strains of Lactic acid bacteria in3.0×1010cfu/ml dose drench of6-8weeks old female BALA/c mice for21days,15,16,17days for three consecutive days gastric inoculation ETEC0149:K88(2×105cfu/ml), recording of mouse body weight changes. After the acquisition of mouse blood, separating the pure serum, detection of IgG and sIgA antibodies in the content used ELISA; taked spleen, lymph node, detection of related cytokines expression evaluated by flow cytometer. Results showed that the weight increase of recombinant Lactic acid bacteria group and vacancy trager were higher than the control group and the difference was significantly, IgG and sIgA antibodies was significantly higher than the other two groups, significant differences. Flow cytometry, the expression of related cytokine were elevated. These results demonstrated that the preparation of the trial of porcine interleukin-18reorganization of Lactic acid bacteria capable of expressing the biological activity of IL-18to provide a new approach for the development of IL-18biological agents.