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摘要：猪支原体肺炎(Mycoplasma al pneumonia of Swine, MPS)是由猪肺炎支原体(Mycoplasma hyopneumoniae, Mhp)感染造成的呼吸系统疾病。这种疾病虽不致死但给世界各国养猪业造成巨大经济损失，受到了国内外学者的广泛关注。猪肺炎支原体毒株的毒力与1,6果糖二磷酸醛缩酶（FBA）的表达量呈正相关。所以研究Mhp/FBA对于该病的诊断与药物研究都有重要意义。本试验使用大肠杆菌原核表达系统表达FBA，测定了FBA与猪气管上皮细胞共孵育产生的糖酵解产物，并且测定了Mhp 168的强弱毒株在KM2培养基中的糖代谢情况。经研究发现，FBA在体外试验中具有对猪气管上皮细胞损伤的作用。并且在FBA浓度为0.953mg/mL时，猪气管上皮细胞培养液中的过氧化氢和丙酮酸浓度达到最高并且和其他浓度均产生显著差异，由此得出FBA浓度为0.953mg/mL时，FBA对活细胞的损伤作用达到巅峰。在KM2培养基中的实验证实，Mhp 168在第12h时生长速度达到最高峰，并且在其生长的第48h时，其毒力达到最高峰。综上所述，本实验为进一步揭示Mhp致病机理、研发新的诊断方法、疫苗和治疗药物的研究奠定了基础。26596
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毕业论文关键词：猪肺炎支原体；醛缩酶；致病机理；蛋白浓度
Study on the pathogenic mechanism of mycoplasma hyopneumoniae aldolase
Abstract： Mycoplasma hyopneumoniae (Mhp) is the pathogen of mycoplasma pneumoniae (Mycoplasma al pneumonia of Swine, MPS). Although this disease is not life threating, it is causing huge economic losses to the animal husbandry. And this disease has been widely concerned by scientists. The expression levels of 1,6 fructose diphosphate aldolase(FBA) are positive correlated with the Mhp strains virulent. Therefore, it is important to study about Mhp/FBA for the diagnosis and medicine research of MPS. In this study, FBA was expressed in E. coli prokaryotic expression system, and the co-incubated glycolysis productions of FBA and swine tracheal epithelial cells were tested. And glycolysis productions of Mhp 168 F113 and F333 strains in KM2 medium were tested. It is indicated that FBA has a significant damaging effect on swine tracheal epithelial cells (STEC) in vitro. When the concentration of FBA was 0.953mg / mL, the concentration of hydrogen peroxide and pyruvic acid in the culture medium of the STEC reached the highest level and were significantly different with the other concentrations. This means that, FBA reached its peak of the damaging effect to living cells, when the concentration of FBA is 0.953mg / mL. Experiments in KM2 medium shows that Mhp 168 reached the highest growing rate at 12h and its virulence reached its peak at the 48th hour. In summary, this study laid a solid foundation for further research on the pathogenesis of Mhp, the development of new diagnostic methods, vaccines and medicine.
Key words: mycoplasma hyopneumoniae；pathogenic mechanism；aldolase；protein concentrations
目 录
摘要 5
关键词5
Abstract .5
Key words .5
引言 .5
1 材料与仪器 5
1.1质粒与菌株 5
1.2 主要试剂与材料 5
1.3 主要实验仪器 .5
2猪肺炎支原体FBA基因的克隆表达及鉴定 .5
2.1实验方法 5
2.1.1 基因获得示意图 5
2.1.2 引物的设计与合成 5
2.1.3 模板的制备 6
2.1.4 扩增Mhp/FBA基因 6
2.1.5 琼脂糖凝胶上基因的回收与纯化 6
2.1.6 猪肺炎支原体醛缩酶基因与载体的连接 6
2.1.7 连接产物的转化 7