Research And Immune Experiments Of Multi-Epitope Vaccine Of Mycoplasma Ovipneumoniae

Mycoplasma ovipneumoniae （Mo）is the main pathogen causing contagious pleuropneumonia ofsheep and goat, especially the lamb from one to three months is major object. It takes place in manycountries of world, is responsible for important economic losses in ovine industry worldwide. At present,immunoligicdefence is still the most effective method to control the disease, so the research of vaccineis becoming more and more popular in relative laboratories of all over the world. With the developmentof molecular biology and bioinformatics, recent years, the multi-epitope has been the focus of vaccineresearch. Firstly some T cell epitope and B cell epitope were identified, and then the multi-epitope ofMo was researched by my work.In the present study, EF-Tu of Mycoplasma ovipneumoniae was cloned and expressed, then therecombinant protein was purified to subject to immunoblotting with antiserum of Mo and its immuneexperiment in mice, the results suggested that the EF-Tu protein can react with the antiserum againstMo and can induce humoral immunoreaction in mice, which indicated that the EF-Tu protein can beused as a candidate vaccine protein of M. ovipneumniae. In addition, the potential epitopes in thesequence of amino acids of EF-Tu and PDHA were predicted to identify its immunogenicity in furtherstudy. Finally, the best four B cell epitopes and2T cell epitopes were evaluated by animal experiment.Add to the C-terminus of the HSP70has proved to have the function of enhance the immunity, the fiveepitopes were directly contructed through the GGG and KKK spacer sequence, which has beenidentified by bioinformatics, and the construct was named PEH. The PEH gene was cloned andexpressed, then the recombinant protein was purified to subject to immunoblotting with antiserum ofMo and its immune experiment in mice, including the detection of specific antibodies, the lymphocyteproliferation and the levels of several cytokines. The results showed that the expression of fusionprotein was significant and fusion protein by western-blot analysis found that it has goodreactionogenicity. After immunization to BALB/c mice, the datas indicated that the PEH protein couldinduce mouse high levels of specific antibodies and stimulate lymphocyte proliferation and theproduction of several cytokines in some degree. Above results suggested that the fusion protein not onlycan induce the humoral immunoreactions, but also induce the cellular immunoreactions, which providedbasic data for later multi-epitope vaccine research against Mycoplasma ovipneumoniae.