.... ..................................................................5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。
目 录
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 *景先生毕设|www.jxszl.com +Q: ^351916072# 
产物的转化 7
2.1.8 重组质粒DNA的提取、鉴定及测序 7
2.1.9 Mhp/FBA基因的SOEPCR突变连接 7
2.1.10 原核表达载体pET28a(+)/FBA的构建 8
2.1.11 连接产物的转化、重组质粒DNA的提取及鉴定 8
2.1.12 重组质粒的诱导表达及SDSPAGE检测 9
2.1.13 蛋白的纯化 9
2.1.14 表达蛋白的Westernblot检测 9
2.1.15 蛋白浓度的测定 9
2.1.16 蛋白的冻干 9
2.1.17 蛋白浓度的测定 10
2.2 实验结果 .10
2.2.1 猪肺炎支原体FBA基因的扩增 10
2.2.2 Mhp/FBA基因的克隆测序 11
2.2.3 Mhp/FBA基因的SOEPCR突变 11
2.2.4 pET 28a（+）/FBA重组质粒的构建与鉴定 12
2.2.5 重组蛋白表达产物的SDSPAGE检测 12
2.2.6纯化重组蛋白及Westernblot鉴定 12
2.2.7 BCA法测定蛋白浓度 13
3 Mhp168 FBA对猪气管上皮细胞的影响的探究 . 13
3.1实验方法 .13
3.1.1 猪气管上皮细胞的解冻 14
3.1.2 猪气管上皮细胞的铺板 14
3.1.3 猪气管上皮细胞的FBA接种 14
3.1.4 流式细胞术 14
3.1.5 Mhp168 FBA对猪气管上皮细胞糖代谢的影响 14
3.2实验结果 .14
3.2.1 流式细胞术 14
3.2.2 Mhp168 FBA对猪气管上皮细胞糖代谢的影响 15
4 测定Mhp168 F113和F333在KM2培养基中的代谢差异 .19
4.1实验方法 .19
4.1.1采集样品 19
4.1.2超氧阴离子浓度测定 19
4.1.3 丙酮酸浓度测定 19
4.1.4 过氧化氢浓度测定 19
4.2 实验结果 .20
4.2.1超氧阴离子浓度测定 20
4.2.2丙酮酸浓度测定 20
4.2.3过氧化氢浓度测定 21
5 讨论 . 21
致谢23
参考文献.24
附录.26
猪肺炎支原体醛缩酶的致病机制研究
动物医学专业学生 马丹夫
指导教师 JUNG YongSam
Study on the pathogenic mechanism of mycoplasma hyopneumoniae aldolase
Student majoring in Veterinary Medicine MA Danfu
Mentor JUNG YongSam
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 coincubated 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.

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